Subsequent injections of more virulent organisms had no effect on the bi

rds that had been

previously exposed to the older cultures. In this manner, the rst attenuated vacc
ine was
discovered. Attenuation, or change, may occur through heat, aging, or chemical m
eans, and it
remains the basis for many of the immunizations that are used today. Pasteur app
lied this same
principle of attenuation to the prevention of rabies, a fatal disease at that ti
me. Although he
did not know the causative agent, he recognized that the central nervous system
was affected, and
thus he studied spinal cords from rabid animals. He noted that spinal cords left
to dry for a few
days were less infectious to laboratory animals than fresh spinal cords. In 1885
, when a boy who
was severely bitten by a rabid dog was brought to his home, Pasteur tried out hi
s new procedure. The boy received a series of 12 injections beginning with material from th
e least
infectious cords and progress- ing to the fresher, more infectious material. Mir
aculously, the
boy survived, and a modi cation of this procedure is the standard treatment for ra
bies today.
CELLULAR VERSUS HUMORAL IMMUNITY In the late 1800s, scientists turned to identif
ying the actual
mechanisms that produce immunity in a host. 2 Elie Metchnikoff, a Russia
n scientist,
observed that foreign objects introduced into transparent star sh larvae became
surrounded by
motile cells that attempted to destroy these invaders. He called this process ph
agocytosis,
meaning cells that eat cells. 2 He hypothesized that immunity to disease was bas
ed on the action
of these scavenger cells. Other researchers contended that noncellular elements
in the blood were
responsible for protection from microor- ganisms. The theory of humoral immunity
was thus born,
and this set off a long-lasting dispute over the relative importance of
cellular versus
humoral immunity. CHAPTER OUTLINE IMMUNITY AND IMMUNIZATION CELLULAR VERSUS HUMO
RAL IMMUNITY
EXTERNAL DEFENSE SYSTEM INTERNAL DEFENSE SYSTEM Acute-Phase Reactants Cellular D
efense Mechanisms
Phagocytosis In ammation SUMMARY CASE STUDIES EXERCISE: IN VITRO PHAGOCYTOSIS EXER
CISE: LATEX
AGGLUTINATION TEST FOR C-REACTIVE PROTEIN REVIEW QUESTIONS REFERENCES 1814_Ch01_
001-018.qxd
7/10/09 3:44 PM Page 3 4 SECTION 1 Nature of the Immune System In 1903, an Eng
lish physician
named Almoth Wright linked the two theories by showing that the immune r
esponse involved
both cellular and humoral elements. He observed that certain humoral, or circula
ting, factors
called opsonins acted to coat bacteria so that they became more susceptible to i
nges- tion by
phagocytic cells. 3 These serum factors include speci c proteins known as antibodi
es and
nonspeci c factors called acute-phase reactants that increase nonspeci cally in any

infec- tion.
This chapter will consider the branch of immunity know as natural immunity, whil
e other chapters
will focus on the more speci c response known as acquired immunity. Natural, or in
nate, immunity
is the ability of the indi- vidual to resist infection by means of normally pres
ent body
functions. These are considered nonadaptive or nonspe- cific and are the sam
e for all
pathogens or foreign substances to which one is exposed. No prior exposure is
required, and the
response does not change with subse- quent exposures. Many of these mechanisms a
re subject to
influence by such factors as nutrition, age, fatigue, stress, and genetic determ
inants. Acquired
immunity, in con- trast, is a type of resistance that is characterized
by speci city for
each individual pathogen, or microbial agent, and the ability to remember a prio
r exposure, which
results in an increased response upon repeated exposure. Both sys- tems are esse
ntial to maintain
good health; in fact, they operate in concert and are dependent upon one another
for maximal
effectiveness. The natural defense system is composed of two parts: the external
defense system
and the internal defense system. The external defense system is designed to keep
micro- organisms
from entering the body. If these defenses are overcome, then the internal de
fense system
must clear invaders as quickly as possible. Internal defenses can be cat- egori
zed into cellular
mechanisms and humoral factors. Both of these systems work together to promote p
hagocytosis,
which results in the destruction of foreign cells and organ- isms. The process o
f in ammation
brings cells and humoral factors to the area in need of healing. If the healing
process is begun
and resolved as quickly as possible, the tissues are less likely to be damaged.
EXTERNAL DEFENSE
SYSTEM The external defense system is composed of structural barriers that preve
nt most
infectious agents from entering the body. First and foremost is the unbroken ski
n and the mucosal
membrane surfaces. To understand how important a role these play, one has only t
o consider how
vulnerable victims of severe burns are to infection. Not only does the skin serv
e as a major
structural barrier, but also the pres- ence of several secretions discourage
s the growth
of microorganisms. Lactic acid in sweat, for instance, and fatty acids from seba
ceous glands
maintain the skin at a pH of approximately 5.6. This acid pH keeps most microorg
anisms from
growing. Additionally, each of the various organ systems in the body has its own
unique
mechanisms. In the respiratory tract, mucous secretions and the motion of cilia
lining the
nasopharyngeal passages clear away almost 90 percent of the deposited material.
The flushing
action of urine, plus its slight acidity, helps to remove many potential pathoge

ns from the
genitourinary tract. Lactic acid production in the female genital tract keeps th
e vagina at a pH
of about 5, another means of preventing invasion of pathogens. In the digestive
tract, acidity of
the stomach, which is due to pro- duction of hydrochloric acid, keeps the pH as
low as 1 and
serves to halt microbial growth. Lysozyme is an enzyme found in many secretions
such as tears and
saliva, and it attacks the cell walls of microorganisms, especially those that a
re gram-positive.
In many locations of the body, there is normal ora that often keeps pathogens fro
m establishing
themselves in these areas. This phenomenon is known as competitive exclusion. Th
e signi cance of
the presence of normal ora is readily demonstrated by looking at the side effects
of
antimicrobial therapy. Frequently, yeast infections due to Candida albicans occu
r, the result of
wiping out normal ora that would ordi- narily compete with such opportunists. INT
ERNAL DEFENSE
SYSTEM The second part of natural immunity is the internal defense system, in wh
ich both cells
and soluble factors play essen- tial parts. The internal defense system is
designed to
recognize molecules that are unique to infectious organ- isms. 4 This typically
involves
recognizing a carbohydrate such as mannose that is found in microorganisms and i
s not evident on
human cells. White blood cells seek out and destroy foreign cells by participati
ng in
phagocytosis, which is the engulfment of cells or particulate matter by leuko- c
ytes,
macrophages, and other cells. This process destroys most of the foreign invaders
that enter the
body, and it is the most important function of the internal defense system. Phag
ocytosis is
enhanced by soluble factors called acute- phase reactants. Acute-Phase Reactants
Acute-phase
reactants are normal serum constituents that increase rapidly by at least 25 per
cent due to
infec- tion, injury, or trauma to the tissues. 5 Some of the most important ones
are C-reactive
protein, serum amyloid A, complement components, mannose-binding protein, alp
ha 1
-antitrypsin, haptoglobin, fibrinogen, and cerulo- plasmin. 5,6 They are produce
d primarily by
hepatocytes (liver parenchymal cells) within 12 to 24 hours in response to an in
crease in certain
intercellular signaling polypep- tides called cytokines (see Chapter 5 for
a complete
discussion of cytokines). These cell messengers, most notably interleukin1 (IL-1),
interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-) re m inly produced
by monocytes
nd m croph ges t the sites of infl mm - tion. 7,8 T ble 11 summ rizes ch r cter
istics of the
m in cute-ph se re ct nts. 1814_Ch01_001-018.qxd 7/10/09 3:44 PM P ge 4 CHAP
TER 1
Introduction nd N tur l Immunity 5 C-Re ctive Protein C-re ctive protein (CRP)

is tr ce
constituent of serum origin lly thought to be n ntibody to the c-polys cch rid
e of pneumococci.
It incre ses r pidly within 4 to 6 hours fol- lowing infection, surgery, or
other tr um to
the body. Levels incre se dr m tic lly s much s hundredfold to thous ndf
old, re ching
pe k v lue within 48 hours. 9,10 They lso decline r pidly with cess tion of the
stimulus. CRP
h s pl sm h lf-life of bout 19 hours. 9 Elev ted levels re found in conditi
ons such s
b cteri l infections, rheu- m tic fever, vir l infections, m lign nt dise ses, t
uberculosis, nd
fter he rt tt ck. The medi n CRP v lue for n indi- vidu l incre ses with g
e, re ecting n
incre se in subclinic l in mm tory conditions. 11 C-re ctive protein is
homogen
eous molecule
with molecul r weight of 118,000 d ltons nd
structure th t consists of ve id
entic l
subunits held together by nonco- v lent bonds. It is member of the f mily know
n s the
pentr xins, ll of which re proteins with ve subunits. CRP cts somewh t like n
ntibody, s
it is c p ble of opsoniz - tion (the co ting of foreign p rticles), gglut
in tion,
precipit tion, nd ctiv tion of complement by the cl ssic l p thw y. However, b
inding is
c lcium-dependent nd non- specific, nd the m in substr te is phosphochol
ine,
common
constituent of microbi l membr nes. It lso binds to sm ll ribonucle r proteins;
phospholipids;
peptidogly- c n; nd other constituents of b cteri , fungi, nd p r sites. 9 In
ddition, CRP
binds to speci c receptors found on mono- cytes, m croph ges, nd neutrophils,
which
promotes ph gocytosis. Thus, CRP c n be thought of s primitive, nonspecific f
orm of ntibody
molecule th t is ble to ct s
defense g inst microorg nisms or foreign cell
s until speci c
ntibodies c n be produced. Bec use the levels rise nd then decline so r pidly,
CRP is the most
widely used indic tor of
cute infl mm tion. Although CRP is nonspeci c in
dic tor of
dise se or tr um , monitoring of its levels c n be useful clinic lly to follow
dise se process
nd observe the response to tre tment of in mm tion nd infection. 9 It is lso
noninv sive
me ns of following the course of m lign ncy nd org n tr nspl n- t tion, bec use
rise in the
level m y me n return of the m lign ncy, or in the c se of tr nspl nt tion, th
e beginning of
org n rejection. In ccord with the finding th t therosclerosis, or coro- n ry
rtery dise se,
is the result of
chronic infl mm tory process, 10,12 recent rese rch indic tes
th t n
incre sed level of CRP is signific nt risk f ctor for myoc rdi l inf rc- tion
nd ischemic
stroke in men nd women who h ve no previous history of c rdiov scul r dise se.
1316 A concentr tion of more th n 2 mg/L h s been defined s the threshold for hi

gh c rdiov scul r
risk. 16 Norm l levels in dults r nge from pproxim tely 1.5 mg/L for men to 2.
5 mg/L for women.
17,18 Thus, monitoring CRP m y be n import nt prevent tive me sure in dete
rmining the
potenti l risk of he rt tt ck or stroke, lthough only uto- m ted, high-sensi
tivity tests
for CRP re useful for this purpose. High-sensitivity CRP testing h s low
er level of
detection of 0.01 mg/dL, llowing for me surement of much sm ller incre ses th n
the tr dition l
l tex gglutin tion screening test. 9 Serum Amyloid A Serum myloid A is the oth
er m jor protein
whose concen- tr tion c n incre se lmost
thous ndfold, s is the c se in CRP.
It is n
polipoprotein th t is synthesized in the liver nd h s
molecul r weight of 11
,685 d ltons.
Norm l cir- cul ting levels re pproxim tely 30ug/ml. In pl sm , it is ssoci t
ed with HDL
cholesterol, nd it is thought to pl y role in met bolism of cholesterol. 19 B
y removing
choles- terol from cholesterol- lled m croph ges t the site of tissue injury, ser
um myloid A
contributes to the cle ning up of the re . 19 This lso f cilit tes recycling o
f cell membr ne
cholesterol nd phospholipids for reuse in building mem- br nes of new cells req
uired during
cute infl mm tion. 20 It h s been found to incre se signi c ntly more in b cteri l infections
th n in vir l infections. 21 T ble 1-1. Ch r cteristics of Acute-Ph se Re ct nts
NORMAL RESPONSE
CONCENTRATION PROTEIN TIME (HR) (MG/DL) INCREASE FUNCTION C-re ctive pr
otein 610 0.5
1000 Opsoniz tion, complement ctiv tion Serum myloid A 24 3.0 1000 Remov l of c
holesterol
Alph 1 - ntitrypsin 24 200400 25 Prote se inhibitor Fibrinogen 24 110400 25 Clot form
tion
H ptoglobin 24 40200 210 Binds hemoglobin Cerulopl smin 4872 2040 2 Binds copper nd
oxidizes iron Complement C3 4872 60140 2 Opsoniz tion, lysis M nnose-binding protei
n
?
0.151.0 ? Complement ctiv tion 1814_Ch01_001-018.qxd 7/10/09 3:44 PM P ge 5 6
SECTION 1
N ture of the Immune System Complement Complement refers to
series of serum pr
oteins th t re
norm lly present nd whose over ll function is medi tion of in mm tion. There re
nine such
proteins th t re cti- v ted by bound
ntibodies in
sequence known
s
the cl ssic l
c sc de; n ddition l number
re involved in the ltern te p thw y th t is
triggered by
microorg nisms. The m jor functions of complement re opsoniz tion, chemot
xis, nd lysis
of cells. Complement is discussed more fully in Ch pter 6. M nnose-Binding Prote
in
M nnose-binding protein (MPB), lso c lled m nnose- binding lectin, is
tr
imer th t cts s
n opsonin, which is c lcium-dependent. It is ble to recognize foreign c rbohydr tes such s
m nnose nd sever l other sug rs found prim rily on b cteri , some ye sts,
viruses, nd

sever l p r- sites. 22 It is widely distributed on mucos l surf ces throu

ghout the body.
It h s m ny simil rities to the com- plement component C1q, s binding
cti
v tes the
complement c sc de nd helps to promote ph gocytosis. 22 Norm l concentr tions
re up to 10
ug/ml. L ck of MBP h s been ssoci ted with recurrent ye st infections. 22 Alph
1 -Antitrypsin
Alph 1 - ntitrypsin (AAT) is the m jor component of the lph b nd when serum i
s
electrophoresed. Although the n me implies th t it
cts g inst trypsin,
it is
gener l pl sm inhibitor of prote ses rele sed from leukocytes, espe- ci lly el
st se. 6 El st se
is n endogenous enzyme th t c n degr de el stin nd coll gen. In chronic pulmon
ry in mm tion, lung tissue is d m ged bec use of its ctivity. Thus, lph 1 - ntitryps
in cts to mop
up or counter ct the effects of neutrophil inv sion during n infl mm tory respon
se. It lso
regul tes expression of proinfl mm tory cytokines such s tumor necrosis f ctorlph ,
interleukin-1 , nd interleukin-6, mentioned previously. Alph 1 - ntitrypsin defi
ciency c n
result in prem ture emphysem , especi lly in individu ls who smoke or who re ex
posed to
noxious occup tion l environment. 6,23 In such de ciency, uninhibited prote ses
rem in in the
lower res- pir tory tr ct, le ding to destruction of p renchym l cells in the lu
ngs nd to
development of emphysem or idiop thic pulmon ry fibrosis. It h s been estim ted
th t s m ny s
60,000 Americ ns suffer from this deficiency. 24 There re t le st 17 lleles o
f the gene coding
for AAT th t re sso- ci ted with low production of the enzyme. 6 One p rticul
r v ri nt gene
for lph 1 - ntitrypsin is ssoci ted with l ck of its secretion from the liver
, nd such
individu ls re t risk of developing liver dise se nd emphysem . 6 Homozygous
inherit nce of
this most severe v ri nt gene m y le d to development of cirrhosis, hep titis, o
r hep tom in
e rly childhood. 6 Alph 1 - ntitrypsin c n lso re ct with ny serine pro- te s
e, such s
those gener ted by triggering of the complement c sc de or brinolysis. Once
bound to lph
1 - ntitrypsin, the prote se is completely in ctiv ted nd is subsequently remo
ved from the re
of tissue d m ge nd c t bolized. H ptoglobin H ptoglobin is n lph 2 -globuli
n with
molecul r weight of 100,000 d ltons. Its prim ry function is to bind irre- versi
bly to free
hemoglobin rele sed by intr v scul r hemolysis. Once bound, the complex is cl
e red r pidly by
Kupffer nd p renchym l cells in the liver, thus preventing loss of free hemoglo
bin. 25,26 The
rise in pl sm h ptoglobin is due to de novo synthesis by the liver nd does not
repre- sent
rele se of previously formed h ptoglobin from other sites. 6 A twofold to tenfol
d incre se in

h ptoglobin c n be seen following in mm tion, stress, or tissue necrosis. E rly i

n the
in mm tory response, however, h ptoglobin levels m y drop bec use of intr v scul
r hemolysis,
consequently m sking the proteins beh vior s n cute-ph se re ct nt. Thus, pl s
m levels must
be interpreted in light of other cute-ph se re ct nts. Norm l pl sm concentr t
ions r nge from
40 to 290 mg/dL. 6,25 H ptoglobin pl ys n import nt role in protecting the kidn
ey from d m ge
nd in prevent- ing the loss of iron by urin ry excretion. However, its most imp
ort nt function
m y be to provide protection g inst oxid tive d m ge medi ted by free hemoglobi
n, powerful
oxidizing gent th t c n gener te peroxides nd hydroxyl r dic ls. 26,27 It m y
lso bind to
p rti lly unfolded nd d m- ged proteins to prevent them from ggreg ting in bl
ood vessels.
Fibrinogen Fibrinogen is the most bund nt of the co gul tion f ctors in pl sm ,
nd it forms the
fibrin clot. 6 The molecule is
dimer with molecul r weight of 340,000 d lton
s. Norm l levels
r nge from 100 to 400 mg/dL. 6 A sm ll portion is cle ved by thrombin to form fi
brils th t m ke
up fibrin clot. This incre ses the strength of wound nd stimul tes endothel
i l cell dhesion
nd prolifer tion, which re crit- ic l to the he ling process. 5 Form tion of
clot lso
cre tes b rrier th t helps prevent the spre d of microorg nisms further into t
he body.
Fibrinogen lso serves to promote ggreg tion of red blood cells, nd incre sed
levels contribute to n incre sed risk for developing coron ry rtery dise se, especi lly
in women. 28,29
Cerulopl smin Cerulopl smin consists of single polypeptide ch in with
molecu
l r weight of
132,000 d ltons. 6 It is the princip l copper-tr nsporting protein in hum n
pl sm , binding
90 to 95 percent of the copper found in pl sm by tt ch- ing six cupric ions pe
r molecule.
Addition lly, cerulopl smin cts s
ferroxid se, oxidizing iron from Fe 2+ to
Fe 3+ . This m y
serve s me ns of rele sing iron from ferritin for bind- ing to tr nsferrin. 6
1814_Ch01_001-018.qxd 7/10/09 3:44 PM P ge 6 CHAPTER 1 Introduction nd N tur
l Immunity 7 A
depletion of cerulopl smin is found in Wilsons dis- e se, n utosom l recessive
genetic
disorder ch r cterized by m ssive incre se of copper in the tissues. Norm lly,
cir- cul ting
copper is bsorbed out by the liver nd either combined with cerulopl sm
in nd returned
to the pl sm or excreted into the bile duct. In Wilsons dise se, copper ccumul
tes in the
liver nd subsequently in other tissues such s the br in, corne , kidneys, nd
bones. 30,31
Cellul r Defense Mech nisms There re ve princip l types of leukocytes, or white
blood cells, in
peripher l blood: neutrophils, eosinophils, b sophils, monocytes, nd lymphocyte
s. Some of these
white blood cells p rticip te in the process of ph gocytosis; these re k

nown s the
myeloid line nd rise from common pre- cursor in the m rrow. These c n be fur
ther divided into
gr nulocytes nd monocytes, or mononucle r cells. Neutrophils, eosinophils,
nd b sophils
re considered gr n- ulocytes. E ch of these cell types is described in this ch
pter. Sever l
cell lines th t re found in the tissues, n mely m st cells, m croph ges, nd de
ndritic cells,
will lso be discussed in this ch pter, s they ll contribute to the process of
n t- ur l
immunity. Lymphocytes form the b sis of the cquired immune response nd re dis
cussed in Ch pter
2. Neutrophils The neutrophil, or polymorphonucle r neutrophilic (PMN) leukocyte
, represents
pproxim tely 50 to 70 percent of the tot l peripher l white blood cells. These
re round 10 to
15 m in di meter, with nucleus th t h s between two nd ve lobes (Fig. 11). They
cont in
l rge number of neu- tr l st ining gr nules, which
re cl ssified
s prim
ry, second ry,
nd terti ry gr nules. Prim ry gr nules, lso c lled zurophilic gr nules, con
t in enzymes
such s myeloperoxid se; el st se; protein se 3; lysozyme; c thepsin G; nd def
ensins, sm ll
proteins th t h ve ntib cteri l ctiv- ity. 25,32 Second ry gr nules re ch r c
terized by the
presence of coll gen se, l ctoferrin, lysozyme, reduced nicotin mide denine din
ucleotide
phosph te (NADPH) oxid se, nd other membr ne proteins norm lly ssoci ted
with the pl sm
membr ne. 25,32 Newly discovered terti ry gr n- ules cont in gel tin se nd
pl sminogen
ctiv tor. 25,32 Acid hydrol ses re found in sep r te comp rtments c lled lysos
omes. 32,33
Norm lly, h lf of the tot l neutrophil popul tion is found in
m rgin ting pool
on blood vessel
w lls, while the rest circul te freely for pproxim tely 6 to 10 hours. There is
continuous
interch nge, however, between the m rgin ting nd the circul ting pools. M rgin
ting occurs to
llow neu- trophils to move from the circul ting blood to the tissues through
process known
s di pedesis, or movement through blood vessel w lls. Receptors known s sele
ctins help m ke
neutrophils sticky nd enh nce dherence to endothe- li l cells th t m ke up the
vessel w ll. 34
Neutrophils then form pseudopods, which squeeze through junctions of the endothe
li l cells. They
re ttr cted to specific re by chemot ctic f ctors. Chemot xins re chemic
l messen- gers
th t c use cells to migr te in p rticul r direction. F ctors th t re chemot c
tic for
neutrophils include com- plement components; proteins from the co gul tion
c sc de; products
from b cteri nd viruses; pl telet cti- v ting f ctor; nd secretions from m s
t cells,
lymphocytes, m croph ges, nd other neutrophils. Once in the tissues, neutrophil
s h ve life
sp n of bout 5 d ys. 35 Norm lly, the input of neutrophils from the bone m rrow

equ ls the
output from the blood to the tissues to m int in
ste dy st te. However, in the
c se of cute
infection, n incre se of neutrophils in the circul ting blood c n occur lmost
immedi tely. 25
Eosinophils Eosinophils re pproxim tely 12 to 15 m in di meter, nd they norm l
ly m ke up
between 1 nd 3 percent of the circul ting white blood cells in non llergic pe
rson. Their
number incre ses in n llergic re ction or in response to m ny p r sitic infect
ions. The nucleus
is usu lly bilobed or ellipsoid l nd is often eccentric lly loc ted (Fig. 12). E
osinophils t ke
up the cid eosin dye, nd the cytopl sm is filled with l rge or nge to re
ddish or nge
gr nules. FIGURE 11. Photo of neutrophils. (From H rmening, D. Clinic l Hem tolog
y nd
Fund ment ls of Hemost sis, ed 4. F. A. D vis, Phil delphi , 2002. Color Pl te 1
0.) See Color
Pl te 1. FIGURE 12. Photo of n eosinophil. (From H rmening, D. Clinic l Hem tolo
gy nd
Fund ment ls of Hemost sis, ed 4. F. A. D vis, Phil delphi , 2002. Color Pl te 1
2.) See Color
Pl te 2. 1814_Ch01_001-018.qxd 7/10/09 3:44 PM P ge 7 8 SECTION 1 N ture of t
he Immune System
Prim ry gr nules cont in cid phosph t se nd rylsulf - t se, while eosinophilspecific gr nules
cont in sever l different proteins: m jor b sic protein, eosinophil c tionic pro
tein, eosinophil
peroxid se, nd eosinophil-derived neu- rotoxin. 25 These cells re c p ble of p
h gocytosis but
re much less efficient th n neutrophils bec use of the sm ller numbers present
nd their l ck of
digestive enzymes. 35 Their most import nt role is neutr lizing b sophil nd m s
t cell products
nd killing cert in p r sites (discussed in Ch pter 20). B sophils B sophils re
found in very
sm ll numbers, representing less th n 1 percent of ll circul ting white blood c
ells. The sm llest of the gr nulocytes, they re between 10 to 15 m in di meter nd cont in co r
se, densely
st ining deep-bluish- purple gr nules th t often obscure the nucleus 35 (Fig. 13)
. Constituents
of these gr nules re hist mine, sm ll mount of hep rin, nd eosinophil chemo
t ctic f ctor-A,
ll of which h ve n import nt function in inducing nd m int ining immedi te hy
persensitivity
re ctions. 25,35 Hist mine is v so ctive mine th t contr cts smooth muscle,
nd hep rin is n
ntico gul nt. IgE, the immunoglobulin formed in llergic re ctions, binds re di
ly to b sophil
cell membr nes, nd gr nules rele se their constituents when they cont ct n nt
igen. The
gr nules l ck hydrolytic enzymes, lthough peroxid se is present. B sophils exis
t for only
few
hours in the bloodstre m. M st Cells Tissue m st cells resemble b sophils, but t
hey re connective tissue cells of mesenchym l origin. 36 They re widely distributed through
out the body
nd re l rger th n b sophils, with sm ll round nucleus nd more gr nules (

Fig. 14). Unlike

b sophils, they h ve
long life sp n of between 9 nd 18 months. 36 The enzyme
content of the
gr nules helps to distinguish them from b sophils, s they cont in cid phosph t
se, lk line
phosph t se, nd pro- te se. 25 The m st cell, like the b sophil, pl ys
role in
hypersensitivity re ctions by binding IgE. Monocytes Monocytes, or mononucle r c
ells, re the
l rgest cells in the peripher l blood, with di meter th t c n v ry from 12 to
22 m; they h ve
n ver ge size of 18 m. One distinguish- ing fe ture is n irregul rly folded or
horseshoe-sh ped nucleus th t occupies lmost one-h lf of the entire cells volume
(Fig. 15).
The bund nt cytopl sm st ins
dull gr yish blue nd h s ground-gl ss ppe r
nce due to the
presence of ne dustlike gr nules. These gr nules re ctu- lly of two types, one
of which
cont ins peroxid se, cid phosph t se, nd rylsulf t se; this indic tes th t th
ese gr n- ules
re simil r to the lysosomes of neutrophils. 25 The other type of gr nule m y co
nt in
-glucuronidase, lysozyme, and lipase,
ut no alkaline phosphatase. Digestive vacu
oles may also

e o
served in the cytoplasm. These make up
etween 4 and 10 percent of total ci
rculating white

lood cells; how- ever, they do not remain in the circulation for long. They sta
y in peripheral

lood for up to 70 hours, and then they migrate to the tissues and
ecome known
as macrophages.
FIGURE 14. Photo of a mast cell. (From Harmening, D. Clinical Hematology and Fun
damentals of
Hemostasis, ed 4. F. A. Davis, Philadelphia, 2002. Color Plate 42.) See Color Pl
ate 4. FIGURE
15. Photo of two monocytes. (From Harmening, D. Clinical Hematology and Fundament
als of
Hemostasis, ed 4. F. A. Davis, Philadelphia, 2002. Color Plate 17.) See Color Pl
ate 5. FIGURE
13. Photo of a
asophil. (From Harmening, D. Clinical Hematology and Fundamentals
of Hemostasis,
ed 4. F. A. Davis, Philadelphia, 2002. Color Plate 13.) See Color Plate 3. 1814_
Ch01_001-018.qxd
7/10/09 3:44 PM Page 8 CHAPTER 1 Introduction and Natural Immunity 9 Tissue Ma
crophages All
tissue macrophages arise from monocytes, which can
e thought of as macrophage p
recursors,

ecause additional differentiation and cell division takes place in the tissues.
The transition
from monocyte to macrophage is character- ized
y progressive cellular enlargeme
nt to
etween 25
and 80 m. 25 As the monocyte matures into a macrophage, there is an increase in e
ndoplasmic
reticulum, lysosomes, and mitochondria. Unlike monocytes, macrophages contain no
peroxidase.
25,35 There are no conclusive data to indicate that monocytes are predestined fo
r any particular
tissue, so tissue distri
ution appears to
e a random phenomenon. Macrophages ha
ve speci c names
according to their par- ticular tissue location. Some are immo
ile, while
others progress

through the tissues
y means of amoe
oid action. Macrophages in the lung are alv
eolar
macrophages; in the liver, Kupffer cells; in the
rain, microglial cells; and in
con- nective
tissue, histiocytes. Macrophages may not
e as ef cient as neutrophils in phagocyt
osis,
ecause
their motility is slow compared to that of the neutrophils. However, their life
span appears to

e in the range of months rather than days. The monocytemacrophage system plays a
n important
role in initiating and regulating the immune response. Their functions include m
icro
ial killing,
tumoricidal activity, intracellular parasite eradication, phagocytosis, secretio
n of cell
mediators, and antigen presentation. Killing activity is enhanced when macrophag
es
ecome
activated
y con- tact with microorganisms or with chemical messengers calle
d cytokines,
which are released
y T lymphocytes dur- ing the immune response. (See Chapter 5
for a complete
discussion of cytokines.) Dendritic Cells Dendritic cells are so named
ecause t
hey are covered
with long mem
ranous extensions that make them resem
le nerve cell dendrit
es. Their main
function is to phagocytose antigen and present it to helper T lymphocytes. While
their actual
developmental lineage is not known, they are
elieved to
e descendents of the m
yeloid line. They
are classified according to their tissue location, in a similar manner to macrop
hages.
Langerhans cells are found on skin and mucous mem
ranes; interstitial dend
ritic cells
populate the major organs such as the heart, lungs, liver, kidney, and the gastr
ointestinal
tract; and interdigitating dendritic cells are present in the T lymphocyte areas
of secondary
lymphoid tissue and the thymus. After capturing antigen in the tissue
y phagocy
tosis or
endocytosis, they migrate to the
lood and to lymphoid organs, where they presen
t antigen to T
lymphocytes to initiate the acquired immune response. They are the most potent p
hagocytic cell in
the tissue. Toll-like Receptors While each of the aforementioned cells has
its own unique
receptors to attach to microorganisms, an additional mechanism recently discover
ed on certain
cells is Toll-like receptors. Toll is a protein originally discovered in the fru
it y Drosophila,
where it plays an important role in antifun- gal immunity in the adult fly. Very
similar
molecules are found on human leukocytes and some nonleukocyte cell types, and th
ese are called
Toll-like receptors (TLRs). The highest concentration of these receptors o
ccurs on
monocytes, macrophages, and neutrophils. 37 There are 11 slightly different TLRs
in humans. 37
Each of these recep- tors recognizes a different micro
ial product. For example,
TLR2 recognizes
teichoic acid and peptidoglycan found in gram-positive
acteria, while TLR4 reco
gnizes

lipopolysac- charide found in gram-negative
acteria (Fig. 16). Once a receptor

inds to its
particular su
stance, or ligand, phago- cytosis may
e stimulated, or the cell p
roduces cytokines
that enhance in ammation and eventual destruction of the microorganism. Thus, th
ey play an
important role in enhancing natural immunity. Phagocytosis The process of phag
ocytosis consists
of four main steps: (1) physical contact
etween the white cell and the foreign
particle, (2)
formation of a phagosome, (3) fusion with cyto- plasmic granules to form a phago
lysosome, and (4)
digestion and release of de
ris to the outside (Fig. 17). Physical con- tact occu
rs as
neutrophils roll along until they encounter the site of injury or infection. 33
They adhere to
receptors on the endothelial cell wall of the
lood vessels and pene- trate thro
ugh to the tissue

y means of diapedesis. This process is aided
y chemotaxis, where
y cells are a
ttracted to the
site of inflammation
y chemical su
stances such as solu
le
acterial factors, c
omplement
components, or C-reactive protein. Receptors on neutrophils or monocytes come i
nto contact
with the foreign particle surface, TLR1 Myco
acteria Gram-negative
acteria
Gram-positive

acteria TLR2 TLR4 Neutrophil or Macrophage Cell mem
rane FIGURE 16. Toll-like r
eceptors on a
white cell mem
rane. Each of the 11 different Toll-like receptors recognizes a d
ifferent pathogenic product. A few examples are shown here. TLR1 recognizes lipoprotein found
in myco
acteria;
TLR2
inds to peptidoglycan in gram-positive
acteria; TLR4 recognizes lipopolys
accharide in
gram-negative
acteria. 1814_Ch01_001-018.qxd 7/10/09 3:44 PM Page 9 10 SECTI
ON 1 Nature of
the Immune System enhanced
y opsonins, a term derived from the Greek word meani
ng to prepare
for eating. Opsonins are serum pro- teins that attach to a foreign su
stance and
help prepare it
for phagocytosis. C-reactive protein, complement compo- nents, and anti
odies ar
e all important
opsonins. Phagocytic cells have receptors for immunoglo
ulins and for comple- me
nt components,
which aid in contact and in initiating ingestion. Opsonins may act
y neutralizi
ng the surface
charge on the foreign particle, making it easier for the cells to approach one a
nother. Once
attachment has occurred, the cellular cytoplasm flows around the particle and ev
entually fuses
with it. An increase in oxygen consumption, known as the respiratory, or oxidati
ve,
urst, occurs
within the cell as the pseudopo- dia enclose the particle within a vacuole. The
structure formed
is known as a phagosome. The phagosome is grad- ually moved toward the center of
the cell. Next,
contact with cytoplasmic granules takes place, and fusion
etween gran- ules and
the phagosome
occurs. At this point, the fused elements are known as a phagolysosome. The gran
ules then release

their contents, and digestion occurs. Any undi- gested material is excreted from
the cells
y
exocytosis. The actual process of killing is oxygen-dependent and results from t
he generation of

actericidal meta
olites. Heavily opsonized particles are taken up in as little
as 20 seconds,
and killing is almost immediate. 32 Resting cells derive their energy from anaer
o
ic glycolysis; however, when phagocytosis is triggered, the respiratory
urst produces gr
eater energy via
oxidative meta
olism. The hexose monophosphate shunt is used to change ni
coti- namide
adenine dinucleotide phosphate (NADP) to its reduced form
y adding a hydro
gen. NADPH. NADPH
then donates an electron to oxygen in the presence of NADPH oxidase, a
mem
rane-
ound
enzyme, which is only activated through conformational change triggered
y
micro
es
themselves. 38 A radical known as O 2 (superoxide) is formed. Superoxide is high
ly toxic
ut
can
e rapidly con- verted to more lethal products. By adding hydrogen ions, the
enzyme
superoxide dismutase (SOD) converts superox- ide to hydrogen peroxide or th
e hydroxyl
radical OH. Hydrogen peroxide has long
een considered an important
actericida
l agent, and it
is more sta
le than any of the free radicals. Its effect is potentiated
y
the formation
of hypochlorite ions. This is accomplished through the action of the enzyme myel
operoxidase in
the presence of chloride ions. Hypochlorite ions are powerful oxidizing agents.
All of these
su
stances contri
ute to killing within the phago- cyte (Fig. 18). However, new
evidence
indicates that an electron- transport system that alters the charge across th
e mem
rane of the
phagolysosome is more important than actual forma- tion of oxygen radicals thems
elves. 32 NADPH
oxidase may depolarize the mem
rane, allowing hydrogen and potas- sium ions to e
nter the vacuole.
When hydrogen com
ines with the superoxides, the pH increases, which in turn act
i- vates
proteases that contri
ute to micro
ial killing. NADPH oxidase is known to
e cen
tral to the
killing of micro
es,
ecause its dysfunction causes chronic granulomatous d
isease. Patients
with this disease suffer from recurring, severe
acterial infections. In ammation
The overall
reaction of the
ody to injury or invasion
y an infectious agent is known as in
flammation. Both
cellular and humoral mechanisms are involved in this complex, A B C D E
F Physical
contact Outflowing of cytoplasm Phagosome Phagolysosome Digestion Excretion FIGU
RE 17. Steps
involved in phagocytosis. (A) Adherence: physical contact
etween the phagocytic
cell and the
microorgan- ism occurs, aided
y opsonins. (B) Out owing of cytoplasm to surround
the
microorganism. (C) Formation of phagosome: microor- ganism is completely surroun
ded
y a part of

the cell mem
rane. (D) Formation of the phagolysome: cytoplasmic granules fuse w
ith mem
rane of
phagosome, emptying contents into this mem
rane-
ound space. (E) Digestion of t
he microorganism

y hydrolytic enzymes. (F) Excretion of contents of phagolysosome to the outside

y exocytosis.
NADP oxidase O 2 O 2 - and NADP + H +
Superoxide dismutase H 2 O 2 + O 2 Myelope
roxidase Cl OCl - + H 2 O
NADP NADPH Hexose monophosphate shunt O 2 + OH - + OH
FIGURE 18. Creation of oxygen radicals in the phagocytic cell. The hexose
monophosphate
shunt is used to reduce NADP to NADPH. NADPH can reduce oxygen in the presence o
f NADPH oxidase
to O 2 -, known as superoxide. Superoxide is converted to hydrogen peroxide thro
ugh the action of
the enzyme superoxide dismutase. Myeloperoxidase catalyzes formation of the hypo
chlorite radical,
a very powerful oxydizing agent. Hydroxyl radicals, other powerful oxidizing age
nts, may also
e
formed. 1814_Ch01_001-018.qxd 7/10/09 3:44 PM Page 10 CHAPTER 1 Introduction
and Natural
Immunity 11 highly orchestrated process. Each individual reactant plays a role i
n initiating,
amplifying, or sustaining the reaction, and a delicate
alance must
e maintaine
d for the process
to
e speedily resolved. The four cardinal signs or clinical symptoms are rednes
s, swelling,
heat, and pain. Major events associated with the process of in ammation are (1) in
creased
lood
supply to the infected area; (2) increased capillary per- mea
ility caused
y re
traction of
endothelial cells lining the vessels; (3) migration of white
lood cells, mainly
neutrophils,
from the capillaries to the surrounding tissue; and (4) migra- tion of macrophag
es to the injured
area 25 (Fig. 19). Chemical mediators such as histamine, which are released from
injured mast
cells, cause dilation of the
lood vessels and
ring additional
lood ow to the a
ffected area,
result- ing in redness and heat. The increased permea
ility of the vessels allow
s uids in the
plasma to leak to the tissues. This produces the swelling and pain associated wi
th in amma- tion.
Solu
le mediators, including acute-phase reactants, initiate and control the
response.
Amplification occurs through formation of clots
y the coagulation system and t
hen the
triggering of the
rinolytic system. As the endothelial cells of the vessels cont
ract,
neutrophils move through the endothelial cells of the vessel and out into the ti
ssues. They are
attracted to the site of injury or infection
y the chemotaxins mentioned p
reviously.
Neutrophils, which are mo
ilized within 30 to 60 minutes after the injury, are t
he major type of
cell present in acute in ammation. Neutrophil emigration may last 24 to 48 hours a
nd is
proportional to the level of chemotactic factors pres- ent in the area.
Vasodila
tion

Increased capillary permea
ility Endothelium Red
lood cells Basement mem
rane A

dherence of
neutrophils Diapedesis Chemotaxis Phagocytosis Erythema Edema Tissue Healing
FIG
URE 19.
Events in the in ammatory response. Increased
lood supply to the affected area is
followed
y
increased capillary permea
ility and migration of neu- trophils and macrophages
to the tissues.
Macrophages in the tissue are directed to the area. Acute-phase reactants stimul
ate phagocytosis
of microorganisms. Clinical signs at the site of in ammation include edema and ery
thema. Tissue
healing, with or without scarring, eventually results. Migration of macrophages
from surrounding
tissue and from
lood monocytes occurs several hours later and peaks at 16 to 48
hours.
Macrophages attempt to clear the area through phagocytosis, and in most cases th
e healing process
is completed with a return of normal tissue structure. However, when the
inflammatory
process
ecomes pro- longed, it is said to
e chronic, and tissue damage and los
s of function may
result. Speci c immunity with in ltration of lymphocytes may contri
ute to the damag
e. Thus, a
failure to remove microorganisms or injured tissue may result in continued tissu
e damage. SUMMARY
The study of immunology
egan as an interest in achieving immunity, or resista
nce to disease.
Edward Jenner, an Englishman, performed the first successful vaccination
against smallpox
and ushered in the age of immunologic investigation. Another well-known name in
the early history of immunology is Louis Pasteur, whose discovery of attenuated vaccines form
ed the
asis of
todays vaccination programs. The controversy over cellular versus humoral immunit
y was
responsi
le for spawning much important research in the early years. Elie
Metchnikoff
identified phagocytic cells as a part of cellular immunity, and other researcher
s postulated that
a humoral, or noncellular, fac- tor in the
lood was also involved in immunity.
Both of these
theories were
rought together
y Almuth Wright, an English physician who o
serv
ed that
oth
circulating and cellular factors are necessary to produce immunity. 1814_Ch01_00
1-018.qxd
7/10/09 3:44 PM Page 11 12 SECTION 1 Nature of the Immune System Natural, or i
nnate, immunity
encompasses all the
odys defense mechanisms for resisting disease. These are alw
ays present,
and the response is nonspeci c, in that it is the same for any infectious agent en
countered.
External defenses are structural
arriers such as skin, mucous mem
ranes, and ci
lia that keep
microorganisms from entering the
ody. Internal defenses are centered on the pro
cess of phagocytosis, which is the engulfment of cells or particulate matter
y leukocytes, mac
rophages, and
other cells. This process destroys most of the foreign cells that penetrate the
exter- nal

defenses. Cells that are most active in phagocytosis include neu- trophils, mono
cytes, and
macrophages. Physical contact
etween the phagocytic cell and the foreign partic
le is aided
y
chemotaxis, where
y cells are attracted to the area, and opsonization, or coatin
g of the foreign
particle
y serum proteins such as complement and anti
ody. Once contact occurs,
cytoplasm ows
around the foreign particle to form a phagosome. Fusion of the phagosome w
ith lysosomal
granules creates a phagolysosome. Inside this structure, enzymes are released, a
nd the foreign
particle is digested. The process is oxygen-dependent, and killing results from
the creation of
hypochlorite and hydroxyl ions, which dam- age protein irreversi
ly. Acute-phase
reactants are
serum constituents that increase rapidly in response to infection or injury to
the tis- sues.
They enhance the process of phagocytosis
y attracting leukocytes to the area of
injury and
y
coating the foreign material so that it can
e ingested more easily. Other functions of the
acute-phase reactants include neutralization of mediators and proteolytic enzyme
s generated
during the process of responding to pathogens. CRP is the most widely monitored
of the
acute-phase reactants and is the
est indi- cator of acute in ammation. In ammation
is the
odys
response to injury or invasion
y a pathogen, and it is characterized
y increas
ed
lood supply
to the affected area, increased capillary permea
ility, migration of neutrophils
to the
surrounding tissue, and migration of macrophages to the injured area. In ammation
and the
process of phagocytosis are considered natural immunity in that the respons
e to any injury
or pathogen is nonspeci c. Phagocytosis, however, must occur
efore the speci c immu
ne response
can
e initiated, so this process is essential to
oth natural and acquired immu
nity.
1814_Ch01_001-018.qxd 7/10/09 3:44 PM Page 12 CHAPTER 1 Introduction and Natu
ral Immunity 13
1. A 45-year-old male named Rick went to his physician for an annual checkup. Al
though he was
slightly over- weight, his la
results indicated that
oth his total cho
lesterol and his
HDL cholesterol were within normal limits. His fi
rinogen level was 450 mg/dL, a
nd his CRP level
was 3.5 mg/dL. His physical examination was perfectly normal. The physician caut
ioned Rick that
he might
e at risk for a future heart attack, and he counseled him to
e s
ure to exercise
and eat a healthy, low-fat diet. Ricks wife told him that as long as his chole
sterol level
was normal, he didnt have any- thing to worry a
out. Who is correct? Explain your
answer. 2. A
20-year-old female college student went to the in r- mary with symptoms of malaise
, fatigue, sore
throat, and a slight fever. A complete
lood count was performed, and
oth the r
ed cell and white

cell count were within normal limits. A rapid strep test was performed,
and this was
negative. A slide agglutination test for infectious mononucleosis was indetermin
ate, while a
slide aggluti- nation test for CRP was positive. Results of a semiquanti
tative CRP
determination indicated an increased level of approximately 20 mg/dL. The stud
ent was advised
to return in a few days for a repeat mono test. How does a test result showing a
n increase in CRP
help in a presumptive diagnosis of infectious mononucleosis? CASE STUDIES 1814_C
h01_001-018.qxd
7/10/09 3:44 PM Page 13 14 SECTION 1 Nature of the Immune System IN VITRO PHAG
OCYTOSIS
Principle A drop of whole
lood is mixed with a drop of a
acterial culture and
incu
ated at room
temperature to demonstrate engulfment of
acteria
y leukocytes. Materials Test
tu
es, 12 75 mm
Broth culture of Staphylococcus epidermidis Lancets for nger puncture Heparinized
microhematocrit tu
es Microscope slides Wright stain Procedure 1. Take two 12 75
mm test tu
es
and la
el one 0 minutes and the other 5 minutes. If there is enough
lood availa
le, a
10-minute tu
e can also
e set up. 2. Do a nger puncture and ll a heparinized micr
ohema- tocrit
tu
e a
out three-quarters full of
lood. Note that
lood drawn in an EDTA tu
e w
ithin the last 10
minutes can also
e used. 3. Using a
lack ru

er
ul
, expel one drop of
lood
into each la
eled
test tu
e. 4. Add one drop of Staphylococcus epidermidis culture to each tu
e, u
sing a disposa
le
Pasteur pipette. The culture should
e no more than a 0.5 McFarland standard in
concentration.
Dilute in an additional
roth tu
e or in sterile saline if necessary. 5. Shake t
he tu
es to mix,
and make a
lood lm of the 0 tu
e immediately. Let the other tu
es incu
ate for 5
min- utes and
10 minutes, respectively, at room temperature
efore making
lood lms of them. Me
thod for Making
Blood Smears 1. O
tain two clean glass slides, one of which will
e used as a sp
reader slide. 2.
With a Pasteur pipette, carefully place a small drop of
lood at one end of a mi
croscope slide.
3. Holding the spreader slide with the thum
and fore n- ger, place the spreader s
lide slightly
in front of the drop of
lood on the other slide, maintaining a 25-degree angle

etween the
slides. 4. Move the spreader slide
ack toward the drop of
lood. As soo
n as the slide
comes in contact with the drop of
lood, the
lood will start to spread along th
e edge. 5.
Keeping the spreader slide at a 25-degree angle, push it rapidly over the length
of the slide.
There should
e a feathered edge on the end of the smear. Wright Stain 1. Allow
the
lood smear
to air dry. 2. Stain according to typical la
oratory protocol for stain- ing
lo
od smears. 3.
Blot the
ack of the slides to remove excess stain and let them air dry. 4. Use
immersion oil and
look for engulfment. Comments These
lood smears will
e more watery than usual

due to the
addition of the
roth culture with
acteria; therefore, it may
e difficult to g
et a
lood smear
with a feathered edge. The feathered edge is not essential, as long as the
lood
cells are spread
out on the slide. There should
e a noticea
le difference
etween the 0- and the
5-minute slide.
The 0-minute slide will pro
a
ly not show much engulf- ment,
ut
acteria may
e
seen in contact
with leukocytes. The 5-minute slide should show
acteria within the cell as smal
l purple dots.
Neutrophils will
e the predominant phagocytic cells,
ut an occasional monocyte
may
e seen. If
lymphocytes are the only white
lood cell seen, the
ac- terial suspension was t
oo heavy, and the
phagocytic cells destroyed themselves in attempting to engulf the
acteria prese
nt. EXERCISE
1814_Ch01_001-018.qxd 7/10/09 3:44 PM Page 14 CHTPER 1 Introduction and Natur
al Immunity 15
LATEX AGGLUTINATION TEST FOR C-REACTIVE PROTEIN PRINCIPLE Latex particles coated
with anti
ody to
CRP are reacted with patient serum. In this case, the CRP is acting as the antig
en. If CRP is
present a
ove normal threshold levels, the antigenanti
ody com
ination will resul
t in a visi
le
agglutination reaction. An elevated CRP level is a sensitive, although nonspeci c,
indicator of
in ammation. Reagents (Kit from Wampole, Remel, or other manufacturers) CRP latex
reagent, which
contains a 1 percent suspension of polystyrene latex particles coated with antihu
man CRP
produced in goats or ra

its Positive human serum control with a concentration o
f approximately
20 mg/dL of CRP Negative human serum control Disposa
le sampling pipettes Dispos
a
le test slides
Not in kit
ut needed: Timer; disposa
le stirrers; serological pipettes; test tu

es, 12 75 mm
CAUTION: The human serum used in the preparation of controls is tested
y an FDA
-approved method
for the presence of anti
odies to HIV as well as for hepati- tis B surface antig
en and HCV and
found to
e negative. However,
ecause no test method can offer complete assuran
ce that HIV,
hepatitis B, hepatitis C, or other infectious agents are a
sent, the reagent sho
uld
e han- dled
with the same care as a clinical specimen. Reagents in the kit contain sodium az
ide as a preservative. Sodium azide may form lead or copper azide in la
oratory plum
ing. An ex
plosion may occur
upon per- cussion. Flush drains thoroughly with water after disposing of ui
ds containing
sodium azide. Specimen Collection Collect
lood aseptically
y venipuncture into
a clean, dry,
sterile tu
e and allow it to clot. Separate the serum without transferring any
cellular
elements. Do not use grossly hemolyzed, excessively lipemic, or
acterially
contaminated
specimens. Fresh nonheat inactivated serum is recom- mended for the tes
t. However, if
the test cannot
e performed immediately, serum may
e stored
etween 2C and 8C

for up to 2
days. If there is any additional delay, freeze the serum at 20C or
elow. Procedur
e*
Qualitative Slide Test 1. Be sure reagents and specimens are at room temperature
. 2. Using one of
the pipettes provided, fill it a
out two- thirds full with undiluted serum.
While holding
the pipette perpendicular to the slide, deliver one free-falling drop to the cen
ter of one oval
on the slide. If a cali
rated pipetter is used instead of the pipettes provided,
adjust the
pipetter to deliver 0.05 mL (50 mL). 3. Using the squeeze-dropper vials provided
, add one drop of
positive control and one drop of negative control to separate ovals on the slide
. Note: A
positive and a nega- tive control should
e run with each test. 4. Resuspend the
latex reagent
y
gently mixing the vial until the suspension is homogeneous. Place one drop of CR
P latex reagent
next to each serum specimen and to each control. 5. Using separate stirrers, mix
each specimen
and control until the entire area of each oval is lled. 6. Tilt the slide
ack
and forth,
slowly and evenly, for 2 minutes. Place the slide on a at surface and o
serve
for
agglutination using a direct light source. 7. The CRP positive control serum mus
t show distinct
agglutination, and the negative control must
e nonreac- tive. If the reagent fa
ils to
agglutinate with the positive control, or does agglutinate with the negative con
trol, it should

e discarded. Semiquantitative Slide Test 1. If a positive reaction is o
tained,
the specimen may

e serially diluted with a glycine-saline
uffer in order to o
tain a semiqua
ntitative
estimate of the CRP level. 2. Begin with a 1:2 dilution of patient serum o
ta
ined
y mixing
equal parts specimen and glycine-saline
uffer. Blend the tu
e contents thorough
ly. 3. Add 0.1 mL
of
uffer to the desired num
er of test tu
es. Add 0.1 mL of 1:2 dilution to the
first tu
e; mix
and transfer 0.1 mL to the next additional tu
e. Continue until all tu
es are di
luted. 4. Perform
a slide agglutination test on each dilution
y repeating the procedure (steps 3
through 7) as
a
ove, and look for agglutination. EXERCISE 1814_Ch01_001-018.qxd 7/10/09 3:44
PM Page 15 16
SECTION 1 Nature of the Immune System Results 1. A positive reaction is reported
when the
specimen shows agglutination, indicating the presence of CRP in the serum at a l
evel equal to or
greater than 0.6 mg/dL. 2. The titer is represented
y the last dilution that sh
ows a positive
reaction. 3. A negative reaction is characterized
y a lack of visi
le agglutina
tion in the
undiluted specimen. Comments The latex agglutination test for CRP is a screening
test for
elevated levels of CRP in serum. A level of 0.6 mg/dL or higher gives a positive
result with the
undiluted specimen. Normal levels range from 0.1 mg/dL in new
orns to 0.5 mg/dL

in adults.
Usually, with the onset of a su
stantial in ammatory stimulus, such as infection,
myocardial
infarc- tion, or surgical procedures, the CRP level increases very signi cantly (>
tenfold) a
ove
the value reported for healthy individuals. Following surgery, CRP levels rise s
harply and
usually peak
etween 48 and 72 hours. Levels decrease after the third postoperat
ive day and
should return to near nor- mal
etween the fth and seventh postoperative day. Thu
s, CRP levels
can
e used to monitor the outcome of surgery. CRP testing can also
e used to m
onitor graft
rejection, drug therapy with anti-inflammatory agents, and recur- rence of mal
ignancies. For
patients with rheumatoid arthritis, elevated CRP can
e used as an indicator o
f the active
stage of the disease. In most situations, however, it is desira
le to have more
than one
determination so that
ase levels can
e esta
lished. Limitations of Procedures
1. Reagent,
controls, and test specimens should
e
rought to room temperature and gently mi
xed
efore using.
2. Reagent and control should not
e used after the expi- ration date indicated
on the outside
kit la
el. 3. Do not use the CRP reagent if there is evidence of freezing. 4. Fa
lse-negative
reactions may
e due to high levels of CRP in undiluted specimens. A 1:5 d
ilution should
always
e run for this reason. False-positive reactions may occur with a reactio
n time longer
than 2 minutes or with specimens that are lipemic, hemolyzed, or contam- inated
with
acteria.
Therefore, any visi
ly contaminated, lipemic, or hemolyzed specimen should not

e used. 5.
Discard
uffer if contaminated (evidence of cloudiness or particulate material i
n solution).
WARNING Latex reagent controls and
uffer contain 0.1 percent sodium azide as a
preservative.
Sodium azide may react with lead and copper plum
ing to form highly explosive me
tal azides. On
disposal, ush with a large volume of water to prevent azide
uildup. * From Remel
SeraTest CRP
Latex, Package insert, Remel Incorporated, Lenexa, KS 66215 1814_Ch01_001-018.qx
d 7/10/09 3:44
PM Page 16 CHAPTER 1 Introduction and Natural Immunity 17 1. Enhancement of pha
gocytosis
y
coating of foreign par- ticles with serum proteins is called a. opsonization.
.
agglutination.
c. solu
ilization. d. chemotaxis. 2. Jenners work with cowpox, which provided imm
unity against
smallpox, demonstrates which phenomenon? a. Natural immunity
. Attenuation of v
accines c.
Phagocytosis d. Cross-immunity 3. Which of the following can
e attri
uted to Pa
steur? a.
Discovery of opsonins
. Research on haptens c. First attenuated vaccines d. Dis
covery of the ABO

lood groups 4. Which of the following peripheral
lood cells plays a key role i
n killing of
parasites? a. Neutrophils
. Monocytes c. Lymphocytes d. Eosinophils 5. Which of

the following
plays an important role as an external defense mechanism? a. Phagocytosis
. C-r
eactive protein
c. Lysozyme d. Complement 6. The process of inflammation is characterized
y all
of the following
except a. increased
lood supply to the area.
. migration of white
lood cells.
c. decreased
capillary permea
ility. d. appearance of acute-phase reactants. 7. Skin, lactic
acid
secretions, stomach acidity, and the motion of cilia represent which type of
immunity? a.
Natural
. Acquired c. Adaptive d. Auto 8. The structure formed
y the fusion of
engulfed
material and enzymatic granules within the phagocytic cell is called a a. phagos
ome.
. lysosome.
c. vacuole. d. phagolysosome. 9. Which of the following white
lood cells is cap
a
le of further
differentiation in the tissues? a. Neutrophil
. Eosinophil c. Basophil d. Monoc
yte 10. The
presence of normal ora acts as a defense mecha- nism
y which of the following me
ans? a.
Maintaining an acid environment
. Competing with pathogens for nutrients c. Kee
ping phagocytes
in the area d. Coating mucosal surfaces 11. Measurement of CRP levels can
e use
d for all of the
following except a. monitoring drug therapy with anti-in ammatory agents.
. track
ing the normal
progress of surgery. c. diagnosis of a speci c
acterial infection. d. determining
active phases
of rheumatoid arthritis. 12. Which of the following are characteristics of acute
- phase
reactants? a. Rapid increase following infection
. Enhancement of phagocytosis
c. Nonspeci c
indicators of in ammation d. All of the a
ove 13. A latex agglutination test for C
RP is run on a
12-year- old girl who has
een ill for the past 5 days with an undiagnosed disea
se. The results
o
tained are as fol- lows: weakly reactive with the undiluted serum and negative
for
oth the
positive and negative controls. What should the technologist do next? a. Repeat
the entire test

. Report the results as indeterminate c. Report the result as positive d. O
tai
n a new sample
14. Which is the most significant agent formed in the phagolysosome for
the killing of
microorganisms? a. Proteolytic enzymes
. Hydroxyl radicals c. Hydrogen peroxide
d. Superoxides
15. The action of CRP can
e distinguished from that of an anti
ody in which of
the following
ways? a. CRP acts
efore the anti
ody appears.
. Only the anti
ody triggers the
complement
cascade. c. Binding of the anti
ody is calcium-dependent. d. Only CRP acts as an
opsonin. REVIEW
QUESTIONS 1814_Ch01_001-018.qxd 7/10/09 3:44 PM Page 17 18 SECTION 1 Nature o
f the Immune
System References 1. http://www.keratin.com/am/am003.shtml, accessed Novem
er 16
, 2006. 2.
Silverstein, AM: The history of immunology. In Paul, WE (ed): Fundamental Immuno
logy, ed. 4.
Lippincott Williams & Wilkins, Philadelphia, 1999, pp. 1935. 3. Clark, WR. The

Experimental
Foundation of Modern Immunology, ed. 4. John Wiley & Sons, New York, 1991. 4.
Fearon, DT. The
instructive role of innate immunity in the acquired response. Science 272:50, 19
96. 5. Ga
ay, C,
and Kushner, I. Acute-phase proteins and other sys- temic responses to in ammation
. N Engl J Med
340:448, 1999. 6. McPherson, RA: Specific proteins. In McPherson, RA, and
Pincus, MR
(eds.): Henrys Clinical Diagnosis and Management
y La
oratory Methods, e
d. 21.
Saunders Elsevier, Philadelphia, 2007, pp. 231244. 7. Yudkin, JS, Kumari, M, Hump
hries, SE, and
Mohamed-Ali, V. In ammation, o
esity, stress and coronary heart disease: Is interl
eukin-6 the
link? Atherosclerosis 148:20914, 2000. 8. Wein
erg, MD, Hooper, WC, and Dangas, G
. Cardiac
iomarkers for the prediction and diagnosis of atherosclerotic disease and its comp
lications. Curr
Mol Med 6:557569, 2006. 9. Pepys, MB, and Hirschfield, GM. C-reactive prote
in: A critical
update. J Clin Invest 111:18051812, 2003. 10. Woodhouse, S. C-reactive protein: F
rom acute phase
reactant to cardiovascular disease risk factor. MLO 1220, March 2002. 11. Hutchin
son, WL, et al.
Immunoradiometric assay of circulating C-reactive protein: Age-related values in
the adult
general population. Clin Chem 46:934938, 2000. 12. Ross, R, and Epstein, FH. Athe
rosclerosisAn
in ammatory disease. New Engl J Med 340:11526, 1999. 13. Pai, JK, Pischon, T, Ma, J
, Manson, JE,
et al. Inflammatory markers and the risk of coronary heart disease in men and wo
men. N Engl J Med
321:25992610, 2004. 14. Cushman, M, Arnold, AM, Psaty, BM, et al. C-reactive prot
ein and the
10-year incidence of coronary heart disease in older men and women: The cardiova
scular health
study. Circulation 112:2531, 2005. 15. Boekholdt, SM, Hack, CE, Sandhu, et al. Creactive
protein levels and coronary artery disease incidence and mortality in apparently
healthy men
and women: The epic Norfolk prospective population study 19932003. Athero
sclerosis
187:415422, 2006. 16. Ridker, PM, Cannon, CP, Morrow, D, et al. C-reactive protei
n levels and
outcomes after statin therapy. N Engl J Med 352:2028, 2005. 17. Woloshin,
S, and
Schwartz, LM. Distri
ution of C-reactive protein values in the United State
s. N Engl J
Med 352:16111613, 2005. 18. Lakoski, SG, Cushman, M, Criqui, M, Rundek, T,
et al.
Gender and C-reactive protein: Data from the multiethnic study of atherosclero
sis (MESA)
cohort. Am Heart 152:593598, 2006. 19. Kisilevsky, R, and Tam, S-P. Acute phase
serum amyloid
A, cholesterol meta
olism, and cardiovascular disease. Pediatr Path Mol Med 21:2
91305, 2002. 20.
Manley, PN, Ancsin JB, and Kisilevsky, R. Rapid recycling of cholesterol: The jo
int
iologic role
of C-reactive protein and serum amyloid A. Med Hypotheses 66:784792, 2006. 21. La

nnergard, A,
Larsson, A, Krags
jerg, P, and Friman, G. Correlations
etween serum amyloid A p
rotein and
C-reactive protein in infectious diseases. Scand J Clin La
Invest 63:26
7272, 2003. 22.
Ezekowitz, RA. Role of the mannose-
inding lectin in innate immunity. JID 187(Su
ppl 2) S335S339,
2003. 23. Wulfs
erg, EA, Hoffmann, DE, Cohen, MM. 1- ntitrypsin de ciency: Imp ct o
f genetic
discovery on medicine nd society. JAMA 271:21722, 1994. 24. Stoller, JK, nd Abo
ussou n, LS. 1
ntitrypsin deficiency. L ncet 365:222536, 2005. 25. McKenzie, SB. The Leukocy
te. In
Clinic l L bor tory Hem tology. Prentice H ll, Upper S ddle River, NJ, 2004, pp
. 85121. 26.
B mm, VV, Tsem khovich, VA, Sh kl i, M, nd Sh kl i, N. H ptoglobin types differ
in their bility
to inhibit heme tr nsfer from hemoglobin to LDL. Biochemistry 43:38993906, 2004.
27. Yerbury,
JJ, Rybehyn, MS, Esterbrook-Smith, SB, Henriques, C, et l. The cute ph se prot
ein h ptoglobin
is m mm li n extr cellul r ch perone with n ction simil r to clusterin. Bioc
hemistry
44:1091410925, 2005. 28. Ridker, PM, Hennekens, CH, Buring, JE, et l. C-re ctive
protein nd
other m rkers of in mm tion in the prediction of c rdiov scul r dise se in women.
N Engl J Med
342:836, 2000. 29. Rose, VL, nd Foody, JM. Elev ted brinogen levels incre se ris
k of he rt
dise se in women. Am F m Physici n 59:3158, 1999. 30. Schilsky, MI. Di gnosis n
d tre tment of
Wilsons dise se. Pedi tr Tr nspl nt tion 6:1519, 2002. 31. D s, SK, nd R y, K. Wi
lsons
dise se: An upd te. N t Clin Pr ct Neurol 2:48293, 2006. 32. Seg l, AW. How ne
utrophils kill
microbes. Annu Rev Immunol 23:197223, 2005. 33. P rsons, DD, M rty, J, nd Str
uss, RG: Cell
biology, disor- ders of neutrophils, infectious mononucleosis, nd rel ted lymph
ocytosis. In
H rmening, DM (ed.): Clinic l Hem tology nd Fund ment ls of Hemost sis, ed. 4.
F. A. D vis,
Phil delphi , 2002, pp. 251271. 34. Simon, SI, nd Green, CE. Molecul r mech nics
nd dyn m- ics
of leukocyte recruitment during infl mm tion. Ann Rev Biomed Eng 7:151185, 2005.
35. L wrence,
LW. The ph gocytic leukocytes-morphology, kinet- ics, nd function. In Steine-M
rtin EA,
Lotspeich-Steininger, CA, nd Koepke, JA: Clinic l Hem tology, ed. 2. Lippincott
, Phil delphi ,
1998. 36. M thur, S, Schexneider, K, nd Hutchison, RE. Hem topoiesis. In McPher
son, RA, nd
Pincus, MR (eds): Henrys Clinic l Di gnosis nd M n gement by L bor tory Methods,
ed. 21.
S unders Elsevier, Phil delphi , 2007, pp. 484503. 37. M k, TW,
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ME. The
Immune Response: B sic nd Clinic l Principles. Elsevier, Burlington, MA, 2006,
pp. 6992. 38.
N useef, WM. Assembly of the ph gocyte NADPH oxid se. Histochem Cell Biol 122:27
7291, 2004.
1814_Ch01_001-018.qxd 7/10/09 3:44 PM P ge 18 2 The Lymphoid System LEARNING

OBJECTIVES After
nishing this ch pter, the re der will be ble to: 1. Differenti te between prim r
y nd second ry
lymphoid org ns. 2. Describe the function nd rchitecture of
lymph node. 3. C
omp re prim ry
nd second ry follicle. 4. Discuss the role of the thymus in T cell m tur tion
. 5. Describe
m tur tion of B cell from the pro-B cell to
pl sm cell. 6. Expl in wh t con
stitutes
cluster of differenti tion. 7. Identify nd discuss the function of the followin
g key ntigens on
T cells: CD2, CD3, CD4, nd CD8. 8. Comp re nd contr st the CD3 receptor on
T
cell nd the
surf ce immunoglobulin on B cell. 9. Describe cytokine. 10. Differenti te T
cell subsets on
the b sis of ntigenic structure nd function. 11. Expl in how n tur l killer (N
K) cells
recognize t rget cells. 12. Apply knowledge of T- nd B-cell function to immunol
ogic lly b sed
dise se st tes. KEY TERMS Antibody-dependent cell cytotoxicity Apoptosis Bone m
rrow Cell ow
cytometry Clusters of differenti tion (CD) Cytokine Germin l center Lymph node M
emory cell
Neg tive selection Peri rteriol r lymphoid she th Pl sm cell Positive selection
Prim ry follicle
Rosette technique Second ry follicle Spleen Thymocyte Thymus 19 1814_Ch02_019-03
8.qxd 7/10/09
3:45 PM P ge 19 20 SECTION 1 N ture of the Immune System The key cell involved
in the immune
response is the lym- phocyte. Lymphocytes represent between 20 nd 40 percent of
the circul ting
white blood cells. The typic l sm ll lym- phocyte is between 7 nd 10 m in di met
er nd h s
l rge rounded nucleus th t m y be somewh t indented. The nucle r chrom ti
n is dense nd
tends to st in deep blue (Fig. 21, Color Pl te 6). 1 Cytopl sm is sp rse, cont
ining few
org nelles nd no specific gr nules, nd consists of
n rrow ring surrounding t
he nucleus. 2 The
cytopl sm st ins lighter blue. These cells re unique, bec use they rise from
hem topoietic
stem cell nd then re further dif- ferenti ted in the prim ry lymphoid org ns.
They c n be
sep r ted into two m in cl sses, depending on where this differenti tion t kes p
l ce. The prim ry
lymphoid org ns in hum ns re the bone m rrow nd the thymus. Once lymphocytes m
ture in the
prim ry org ns, they re rele sed nd m ke their w y to second ry org ns, which
include the
spleen, lymph nodes, ppendix, tonsils, nd other mucos l- ssoci ted lymphoid ti
ssue. It is in
the second ry org ns th t the m in cont ct with foreign ntigens t kes pl ce. Th
e spleen serves
s
ltering mech nism for nti- gens in the bloodstre m, nd lymph nodes lter uid
from the
tissues. Mucos l surf ces in the respir tory nd limen- t ry tr cts re b cked
with lymphoid
tissue s n ddition l me ns of cont cting foreign ntigens s they enter the b
ody. Lymphocyte
circul tion is complex nd is regul ted by dif- ferent cell surf ce dhesion

molecules nd
by chemic l messengers c lled cytokines. Lymphocytes re segreg ted within the
second ry org ns
ccording to their p rticul r functions. T lymphocytes re effector cells th t s
erve regul tory
role, nd B lympho- cytes produce
ntibody. Both types of cells recircul
te continuously
from the bloodstre m to the second ry lym- phoid org ns nd b ck, in n ttempt
to incre se
cont ct with foreign ntigens. A third type of lymphocyte, the NK cell, is l rge
, gr nul r, nd
pl ys role in both the inn te nd d ptive immune response. This ch pter descr
ibes the prim ry
lymphoid org ns nd ex mines their role in the m tur tion of lymphocytes.
Second ry org ns
re presented in terms of the clim te pro- duced for development of the immune r
esponse. Specific
ch r cteristics of e ch of the three types of lymphocytes re discussed, s well
s how these
ch r cteristics or m rkers c n be used to identify them in the l bor tory. PRIMA
RY LYMPHOID
ORGANS Bone M rrow All lymphocytes rise from pluripotenti l hem topoietic stem
cells th t ppe r
initi lly in the yolk s c of the developing embryo nd re l ter found in the fe
t l liver. Bone
m rrow ssumes this role when the inf nt is born. It c n be consid- ered the l r
gest tissue of
the body, with tot l weight of CHAPTER OUTLINE PRIMARY LYMPHOID ORGANS Bone M
rrow Thymus
SECONDARY LYMPHOID ORGANS Spleen Lymph Nodes Other Second ry Org ns SURFACE MARK
ERS ON
LYMPHOCYTES STAGES IN B-CELL DIFFERENTIATION Pro-B Cells Pre-B Cells Imm ture B
Cells M ture B
Cells Activ ted B Cells Pl sm Cells T-CELL DIFFERENTIATION Double-Neg tive St g
e Double-Positive
St ge M ture T Cells Antigen Activ tion THIRD POPULATION OR NATURAL KILLER CELLS
Mech nism of
Cytotoxicity Antibody-Dependent Cell Cytotoxicity LABORATORY IDENTIFICATION OF L
YMPHOCYTES M nu l
Method SUMMARY CASE STUDY EXERCISE: ENUMERATION OF T CELLS REVIEW QUESTIONS REFE
RENCES FIGURE
21. Typic l lymphocyte found in peripher l blood. (From H rr, R. Clinic l L bor t
ory Science
Review. F. A. D vis, Phil delphi , 2000, Color Pl te 31.) See Color Pl te 6.
1814_Ch02_019-038.qxd 7/10/09 3:45 PM P ge 20 CHAPTER 2 The Lymphoid System 2
1 1300 to 1500 g
in the dult. 2 Bone m rrow lls the core of ll long bones nd is the m in source
of
hem topoietic stem cells, which develop into erythrocytes, gr nulocytes, mono- c
ytes, pl telets,
nd lymphocytes. E ch of these lines h s specific precursors th t origin te from
the
pluripotenti l stem cells. Most uthorities gree th t T, B, nd NK cells rise
from common
precursor known s the common lym- phoid precursor (CLP) (see Fig. 22). 3 Lymphoc
yte precursors
re further developed in the prim ry lymphoid org ns. The bone m rrow functions
s the center for
ntigen- independent lymphopoiesis. Lymphocyte stem cells re rele sed from the
m rrow nd tr vel

to ddition l prim ry lymphoid org ns where further m tur tion t kes pl ce. One
subset goes to
the thymus nd develops into T cells. In hum ns, B-cell m tur tion t kes pl
ce within the
bone m rrow itself. In peripher l blood, pproxim tely 10 to 20 percent of ll l
ymphocytes re B
cells, 61 to 89 percent re T cells, nd up to 22 percent re NK cells. 4 Thymus
T cells
develop their identifying ch r cteristics in the thymus, which is
sm l
l, fl t,
bilobed org n found in the thor x, or chest c vity, right below the thyroid g
l nd nd
overlying the he rt (Fig. 23). In hum ns, it weighs n HSC CLP T/NK progenitor T
cell NK cell B
cell Neutrophil Dendritic cell B cell progenitor MEP CMP Monocyte Eosinophil B s
ophil
Erythrocytes Pl telets GMP EP BP FIGURE 22. Simpli ed scheme of hem topoiesis. In t
he m rrow,
hem topoietic stem cells (HSC) give rise to two different lines common lymphoid
precursor (CLP)
nd common myeloid precursor (CMP). CLPs give rise to T/NK progenitors, which
differenti te
into T nd NK cells, nd to B-cell progenitors, which become B cells nd dendrit
ic cells. The CMP
differenti tes into neutrophils, monocytes/ m croph ges, eosinophils, b sophils,
erythrocytes,
nd pl telets. Lungs Lymph nodes Spleen Sm ll intestine Peyers p tches Lymph node
s Tissue
lymph tics Thymus Liver Bone m rrow FIGURE 23. Sites of lymphoreticul r tissue. P
rim ry org ns
include the bone m rrow nd the thymus. Second ry org ns re distributed through
out the body nd
include the spleen, lymph nodes, nd mucos l- ssoci ted lymphoid tissue. The spl
een lters
ntigens in the blood, while the lymph tic system lters uid from the tissues. (Fro
m Widm nn,
FK. An Introduction to Clinic l Immunology. F. A. D vis, Phil delphi , 1989, wit
h permission.)
1814_Ch02_019-038.qxd 7/10/09 3:45 PM P ge 21 22 SECTION 1 N ture of the Immu
ne System ver ge
of 30 g t birth, re ches bout 35 g t puberty, nd then gr du lly trophies. 5
It w s first
presumed th t e rly in life, the thymus produces enough virgin T lymphocytes to
seed the entire
immune system, m king it unnecess ry l ter on. However, new evidence indic tes t
h t lthough the
thymus diminishes in size, it is still c p ble of producing T lymphocytes until
t le st the fth
or sixth dec de of life. 6 E ch lobe of the thymus is divided into lobules lled w
ith epitheli l
cells th t pl y centr l role in this differenti tion process. Surf ce ntigens
re cquired s
the lymphocytes tr vel from the cortex to the medull over period of 2 to 3 we
eks. M ture T
lymphocytes re then rele sed from the medull . Progenitors of T cells ppe
r in the fetus
s e rly s 8 weeks in the gest tion l period. 6 Thus, differenti tion of lym
phocytes ppe rs
to t ke pl ce very e rly in fet l develop- ment nd is essenti l to cquisition
of

immunocompetence by the time the inf nt is born. SECONDARY LYMPHOID ORGANS Once
differenti tion
occurs, m ture T nd B lymphocytes re rele sed from the bone m rrow nd the thy
mus. They migr te
to second ry lymphoid org ns nd become p rt of
recircul ting pool. E ch lymph
ocyte spends most
of its life sp n in solid tissue, entering the circul tion only periodic lly to
go from one
second ry org n to nother. The second ry lymphoid org ns include the spleen, ly
mph nodes,
tonsils, ppendix, Peyers p tches in the intestines, nd other mucos l- ssoci ted
lymphoid
tissue (MALT; see Fig. 23). Lymphocytes in these org ns tr vel through the tissue
nd return to
the bloodstre m by w y of the tho- r cic duct. A specific lymphocyte m y m ke th
e journey from
blood to second ry lymphoid org ns nd b ck one to two times per d y. 7 This
continuous
recircul tion incre ses the likelihood of lymphocyte coming into con- t ct wit
h its specific
ntigen. Lymphopoiesis, or reproduction of lymphocytes, occurs in the second ry
tissue, but this
is strictly dependent on nti- genic stimul tion, while form tion of lymphocytes
in the bone
m rrow is ntigen-independent. Most n ve or resting lymphocytes die within few
d ys fter
le ving the prim ry lymphoid org ns unless ctiv ted by the presence of for- e
ign ntigen. It
is this second process th t gives rise to long-lived memory cells nd sh
orter-lived
effector cells th t re responsible for the gener tion of the immune response. S
pleen The spleen
is the l rgest second ry lymphoid org n, h ving
length of pproxim tely 12 cm
nd weighing 150
g in the dult. It is loc ted in the upper-left qu dr nt of the bdomen
, just below the
di phr gm nd surrounded by
thin connective tissue c psule. The org n c n be c
h r cter- ized s
l rge discrimin ting filter, s it removes old nd d m ged cells nd foreign
ntigens from the
blood. Splenic tissue c n be divided into two m in types: red pulp nd white pul
p. The red pulp
m kes up more th n one- h lf of the tot l volume, nd its function is to destroy
old red blood
cells. Blood ows from the rterioles into the red pulp nd then exits by w y of t
he splenic
vein. The white pulp comprises pproxim tely 20 percent of the tot l weight of t
he spleen nd
cont ins the lymphoid tissue, which is rr nged round rterioles in
peri rter
iol r lymphoid
she th (PALS; see Fig. 24). This she th cont ins m inly T cells. Att ched to the
she th re
prim ry follicles, which cont in B cells th t
re not yet stimul ted by
ntigen.
Surrounding the PALS is m rgin l zone cont ining den- dritic cells th t tr p
ntigen.
Lymphocytes enter nd le ve this re by me ns of the m ny c pill ry br nches th
t con- nect to
the rterioles. E ch d y, n dults blood volume p sses through the spleen ppro
xim tely four

times, where lymphocytes nd m croph ges c n const ntly survey for infectious g
ents or other
foreign m tter. 7 Lymph Nodes Lymph nodes re loc ted long lymph tic ducts nd
serve s centr l
collecting points for lymph fluid from dj cent tissues. Lymph uid rises from p
ss ge of uids
nd low- molecul r-weight solutes out of blood vessel w lls nd into the interst
iti l sp ces
between cells. Some of this intersti- ti l uid returns to the bloodstre m through
venules, but
portion ows through the tissues nd is eventu lly collected in thin-w lled vessel
s known s
lymph tic vessels. Prim ry follicle (B cells) M rgin l zone Sinuses in red pulp
Tr becul r vein
C psule Germin l center PALS (T cells) Centr l rtery FIGURE 24. Cross-section of
the spleen
showing org niz tion of the lymphoid tissue. T cells surround rterioles in the
PALS. B cells re
just beyond in follicles. When stimul ted by ntigen, the B cells form germin l
centers. All of
the lymphoid tissue is referred to s the white pulp. 1814_Ch02_019-038.qxd 7/1
0/09 3:45 PM
P ge 22 CHAPTER 2 The Lymphoid System 23 Lymph nodes re especi lly numerous ne
r joints nd
where the rms nd legs join the body. Nodes r nge in size from 1 mm to bout 25
mm in di meter.
Filtr tion is m in function of these org ns. The lymph fluid flows slo
wly through
sp ces c lled sinuses, which re lined with m croph ges, cre ting n ide l
loc tion for
ph gocytosis to t ke pl ce. The tissue is org nized into n outer cortex, p r
cortex, nd n
inner medull (Fig. 25). Lymphocytes nd ny foreign ntigens present enter nodes
vi fferent
lymph tic vessels. Numerous lympho- cytes lso enter the nodes from the bloodstr
e m by me ns of
speci lized venules c lled high endotheli l venules, loc ted in p r cortic l re
s. 5 The
outermost l yer, the cortex, con- t ins m croph ges nd ggreg tions of B cells
in prim ry
follicles simil r to those found in the spleen. These re the m ture, resting B
cells th t h ve
not yet been exposed to ntigen. Speci lized cells c lled follicul r dendritic c
ells re lso
loc ted here. They re found only in lymphoid follicles nd h ve long cytopl smi
c processes th t
r di te out like tent cles. These cells exhibit l rge number of receptors for
ntibody nd
complement nd help to c pture ntigen to present to T nd B cells. Second ry fo
llicles consist
of ntigen-stimul ted prolif- er ting B cells. The interior of second ry folli
cle is known s
the germin l center, bec use it is here th t bl st tr nsfor- m tion of the B cel
ls t kes pl ce.
Pl sm cells, which ctively secrete ntibody, nd memory cells, which re just
step w y from
forming pl sm cells, re present. Gener tion of B-cell memory is
prim ry func
tion of lymph
nodes. 7 T lymphocytes re m inly loc lized in the p r cortex, the region betwee
n the follicles

nd the medull . T lym- phocytes re in close proximity to ntigen-presenting ce

lls c lled
interdigit ting cells. The medull is less densely popu- l ted but cont ins some
T cells (in
ddition to B cells), m croph ges, nd numerous pl sm cells. P rticul te ntige
ns re removed s
the uid tr vels cross the node from cortex to medull . The tr nsit time through
lymph node is
pproxim tely 18 hours. Fluid nd lympho- cytes exit by w y of the efferent lymp
h vessels. Such
vessels form
l rger duct th t eventu lly connects with the tho- r cic duct
nd the venous
system. In this m nner, lymphocytes re ble to recircul te continuously betw
een lymph nodes
nd the peripher l blood. If cont ct with ntigen t kes pl ce, lymphocyte tr ffi
c shuts down
due to the prolifer tion of ctiv ted cells. 7 Accumul tion of lymphocytes
nd other cells
c uses the lymph nodes to become enl rged, condition known s lym- ph denop th
y. Recircul tion
of exp nded numbers of lymphocytes then occurs. Other Second ry Org ns Additi
on l re s of
lymphoid tissue include the MALT, ton- sils, ppendix, nd cut neous- ssoci ted
lymphoid tissue.
MALT, the mucos l ssoci ted lymphoid tissue, is found in the g strointestin l,
respir tory, nd
urogenit l tr cts. Here, m croph ges nd lymphocytes re loc lized t some of th
e m in ports of
entry for foreign org nisms. Peyers p tches represent speci lized type of MALT
nd re loc ted
t the lower ileum of the intestin l tr ct. The tonsils re nother re of lymp
hoid tissue found
in the mucous membr ne lining of the or l nd ph rynge l c v- ities. Their funct
ion is to respond
to p thogens entering the respir tory nd liment ry tr cts. An ddition l loc t
ion of lymphoid
tissue is the ppendix. All of these second ry org ns function s potenti l site
s for cont ct
with foreign ntigen, nd they incre se the prob bility of n immune response. T
he epidermis
cont ins number of intr epiderm l lym- phocytes. Most of these re T cells, wh
ich re uniquely
positioned to comb t ny ntigens th t enter through the skin. This
ssoci tio
n of
lymphocytes is known s the cut neous- ssoci ted lymphoid tissue. Within e c
h of these
second ry org ns, T nd B cells re segreg ted nd perform speci lized functions
. B cells
Afferent lymph tic P r cortex (T cell re ) Cortex (B cell re ) Prim ry follicl
e C psule
Efferent lymph tic Afferent lymph tic Subc psul r sinus Second ry follicle with
germin l center
Sinusoid Medull FIGURE 25. Structure of lymph node. A lymph node is surrounded
by tough
outer c psule. Right underne th is the subc psul r sinus, where lymph uid dr ins
from fferent
lym- ph tic vessels. The outer cortex cont ins collections of B cells in prim ry
follicles. When
stimu- l ted by ntigen, second ry follicles re formed. T cells re found in th
e p r cortic l

re . Fluid dr ins slowly through sinusoids to the medull ry region nd out the
efferent
lymph tic vessel to the thor cic duct. 1814_Ch02_019-038.qxd 7/10/09 3:45 PM
P ge 23 24
SECTION 1 N ture of the Immune System differenti te into memory cells nd pl sm
cells nd re
responsible for humor l immunity or ntibody form tion. T cells pl y role in c
ell-medi ted
immunity, nd s such, they produce sensitized lymphocytes th t secrete cyt
okines. Cytokines
re sm ll polypeptides th t regul te the func- tions of lymphocytes nd other ce
lls involved in
the immune response. The ch r cteristics nd m rkers for e ch type of lymphocyte
re considered
sep r tely. SURFACE MARKERS ON LYMPHOCYTES Proteins th t ppe r on cell surf ces
c n be used s
m rk- ers to differenti te T cells nd B cells. Proteins c n lso be used to dis
tinguish the
development l st ges of the two types of cells ccording to when these proteins
ppe r. Such proteins, or ntigens, h ve been detected by monoclon l ntibodies, which r
e extremely
speci c ntibodies m de by cloning single ntibody-producing cell. (Refer to Ch
pter 4 for
discussion of prep r tion of monoclon l ntibodies.) Sever l l bor tories h ve d
eveloped
monoclon l nti- bodies, nd e ch used its own nomencl ture for the sets of nti
gens found. In n
ttempt to rel te rese rch find- ings nd st nd rdize the nomencl ture, scientis
ts set up the
Intern tion l Workshops on Hum n Leukocyte Antigens, beginning in 1982. 8 P
nels of
ntibodies from different l bor tories were used for n lysis, nd ntibod- ies
re cting
simil rly with st nd rd cell lines were s id to define clusters of differenti ti
on (CD). As e ch
ntigen, or CD, w s found, it w s ssigned number. The n me cluster of differe
nti tion c me
bout bec use the ex ct n ture of the proteins identified by the v rious ntibod
ies w s not
known. This CD cl ssific tion now cts s
reference in st nd rdizing n mes of
membr ne proteins
found on ll hum n white blood cells. The most recent Workshop nd Conference on
Hum n Leukocyte
Differenti tion Antigens, now c lled Hum n Cell Differenti tion Molecules,
w s held in
December of 2004, nd the list of CD design tions currently numbers more th n 35
0. 8 The ntigens
th t re most import nt in ch r- cterizing T lymphocytes nd B lymphocytes re
shown in T ble
21 nd re referred to in the following discus- sion of T-cell nd B-cell develop
ment. T ble
2-1. Surf ce M rkers on T nd B Cells MOLECULAR ANTIGEN WEIGHT (KD) CELL TYPE FU
NCTION CD2 4558
Thymocytes, T cells, NK cells Involved in T-cell ctiv tion CD3 2028 Thymocytes,
T cells
Associ ted with T-cell ntigen receptor; role in TCR sign l tr nsduction CD4 55
Helper T cells,
monocytes, m croph ges Coreceptor for MHC cl ss II; receptor for HIV CD5 58 M tu
re T cells,

thymocytes, subset of Positive or neg tive modul tion of T nd B cell B cells (

B1) receptor
sign ling CD8 6076 Thymocyte subsets, cytotoxic T cells Coreceptor for MHC cl ss
I CD10 100 B
nd T cell precursors, bone m rrow Prote se; m rker for pre-B CALLA strom l cel
ls CD16 5080
M croph ges, NK cells, neutrophils Low f nity Fc receptor, medi tes ph gocytosis
nd ADCC CD19
>120 B cells, follicul r dendritic cells P rt of B cell coreceptor, sign l tr ns
duction molecule
th t regul tes B-cell development nd ctiv tion CD21 145 B cells, follicul r de
ndritic cells,
subset of Receptor for complement component C3d; p rt imm ture thymocytes of Bcell coreceptor
with CD19 CD23 45 B cells, monocytes, follicul r dendritic cells Regul tion of I
gE synthesis;
triggers rele se of Il-1, Il-6, nd GM-CSF from monocytes CD25 55 Activ ted T, B
cells, monocytes
Receptor for IL-2 CD44 85 Most leukocytes Adhesion molecule medi ting homing to
peripher l
lymphoid org ns CD45R 180 Different forms on ll hem topoietic cells Essenti l i
n T nd B cell
ntigen-stimul ted ctiv tion CD 56 175220 NK cells, subsets of T cells Not known
CD 94 70 NK
cells, subsets of T cells Subunit of NKG2-A complex involved in inhibition of NK
cell
cytotoxicity NK N tur l killer; TCR CD3- receptor complex; MHC major histocompati
ili
ty
class; HIV human immunode ciency virus; CALLA common acute lympho
lastic leukemia an
tigen; Fc
Fragment crystalliza
le; ADCC anti
ody-dependent cell cytotoxicity; IgE immunoglo
u
lin E;
GM-CSF granulocyte-macrophage colony-stimulating factor 1814_Ch02_019-038.qxd 7/
10/09 3:45 PM
Page 24 CHAPTER 2 The Lymphoid System 25 STAGES IN B-CELL DIFFERENTIATION Pro-B
Cells B cells are
derived from a multipotential progenitor cell (MMP), a lymphoid-myeloid precurso
r that
differentiates to
ecome either a common myeloid progenitor or an early lymphocy
te progenitor.
9,10 Early lymphocyte progenitors
ecome T cell, B cell, NK cell, or dendritic c
ell precursors,
depending on exposure to different cytokines (see Fig. 22). B cells remain in the
microenvironment provided
y
one marrow stromal cells, and the earliest B-cell
precursor can
e
recognized
y the presence of a surface molecule called CD45R (B220 in mice). 9,
11 B-cell
precursors go through a developmental process that prepares them for their role
in anti
ody
production and, at the same time, restricts the types of antigens to which
any one cell can
respond. This part of B-cell development is known as the antigen- independent ph
ase. Figure 26
depicts the changes that occur as B cells mature from the pro-B stage to
ecome
memory cells or
plasma cells. Several transcription, or growth, factors are necessary to differe
ntiate common
lymphoid precursors into pro-B cells. Some of these factors are E2A, EBF (early
B-cell factor),
and paired
ox protein 5 (PAX). 9,10 In addition, a cytokine called interleukin-

7 (IL-7) is also
necessary at this early devel- opment stage. All of these factors are pro
duced in the
microenvironment of the
one marrow. During this maturation process, the fi
rst step is
the rearrangement of genes that code for the heavy and light chains of a
n anti
ody
molecule. The end result is a B lymphocyte programmed to produce a unique anti
o
dy molecule,
which consists of two identical light chains and two identical heavy chain
s (see Chapter 4
for details). Although portions of each chain are identical for every anti-
ody
molecule, it is
the so-called varia
le regions that make each anti
ody molecule specific for a c
ertain antigen or
group of antigens. Heavy chains are coded for on chromo- some 14, and light chai
ns are coded for
on chromosomes 2 and 22. The pro-B cell has distinctive markers that include sur
- face antigens
CD19, CD45R, CD43, CD24, and c-Kit. Intracellular proteins found at this st
age are terminal
deoxyri-
onucleotide transferase (TdT) and recom
ination-activating genes RAG-1
and RAG-2, which
code for enzymes involved in gene rearrangement (Fig. 26A). 11,12,13 Gene rearran
ge- ment of the
DNA that codes for anti
ody production occurs in a strict developmental sequence
. 13
Rearrangement of genes on chromosome 14, which code for the heavy-chain part of
the anti
ody
molecule, takes place rst in a random fashion (see Chapter 4 for details). C-Kit
on the pro-B
cell interacts with a cell surface molecule called stem cell factor, which is fo
und on stromal
cells. This interaction triggers the activa- tion process. Recom
inase enzymes R
AG-1 and RAG-2
cleave the DNA at certain possi
le recom
ination sites, and TdT helps to join th
e pieces
ack
together
y incorporating additional nucleotides in the joining areas. 14 S
uccessful
rearrangement commits a B cell to further development. CD19 acts as a coreceptor
that helps to
regulate further B-cell development and activation. CD45 is a mem
rane glycoprot
ein found on all
hematopoietic cells,
ut the type found on B cells is the largest form, and it i
s designated
CD45R. This is a tyrosine-specific phosphatase that is involved in signal
ing during
B-cell activation. CD19, CD24, and CD45R remain on the cell surface throughout s
u
se- quent
developmental stages. Differentiation of pro-B cells into pre-B cells occurs upo
n successful
rearrangement of heavy-chain genes. Stromal cells interact directly with pro-B c
ells, and they
secrete cytokines, hormones, chemokines, and adhesion molecules, all of which ar
e necessary for
this developmental process. 14,15 Pre-B Cells When synthesis of the heavy chain
part of the
anti
ody mol- ecule occurs, the pre-B stage
egins. 14,15 The rst heavy chains sy
nthesized are
the chains, which
elong to the class of immunoglo
ulins called IgM. The chains

accumulate in
the cytoplasm. Pre-B cells also lose the CD43 marker as well as c-Kit and TdT. 3
Pre-B cells may
also express chains on the cell surface, accompanied
y an unusual light chain m
olecule called
a surrogate light chain. 14,15 Surrogate light chains consist of two short polyp
eptide chains
that are non- covalently associated with each other (Fig. 26B). They are not immu
noglo
ulin
proteins
ut are thought to
e essen- tial for regulating B-cell development. Th
e com
ination of
the two heavy chains with the surrogate light chains plus two very short
chains,
Ig- /Ig- form the pre-B cell receptor. This receptor adheres to
one marrow stromal
cell
mem
ranes and transmits a signal to prevent rearrangement of any other heavy-cha
in genes. 9,10 It
appears that only pre- B cells expressing the heavy chains in association with s
urrogate light
chains survive and proceed to further differentiation. 15 Once the pre-B
receptor
(preBCR) is expressed, neigh
oring pre-B cells may send signals for further ma
turation. 15 This
stimulates a
urst of clonal expansion. 9 Immature B Cells Immature B cells
are
distinguished
y the appearance of complete IgM molecules on the cell surface 9,
12 (Fig. 26C).
This indicates that rearrangement of the genetic sequence coding for light chain
s on either
chromosome 2 or 22 has taken place
y this time. Completion of light cha
in rearrangement
commits a cell to produce an anti
ody mol- ecule with specificity for a particul
ar antigen or
group of related antigens. Varia
le regions, which occur on
oth the light and h
eavy chains,
determine this speci city. Once sur- face immunoglo
ulins appear, chains are
no longer
detecta
le in the cytoplasm. Other surface proteins that appear on the im
mature B cell
include CD21, CD 40, and major histocompati
ility complex (MHC) class II molecul
es (see Chapter
3). 12 CD21 1814_Ch02_019-038.qxd 7/10/09 3:45 PM Page 25 26 SECTION 1 Nature
of the Immune
System acts as a receptor for a
reakdown product of the comple- ment component
C3, known as C3d
(see Chapter 6 for details on complement). This enhances the likelihood of conta
ct
etween B
cells and antigen,
ecause antigen fre- quently
ecomes coated with complement f
ragments during
the immune response. CD40 and MHC class II are impor- tant for interaction of B
cells with T
cells. At this stage, there is evidence that self-antigens give a negative signa
l to immature B
cells, resulting in arrested maturation and cell death. 9,12 Immature B cells th
at tightly
ind
self-antigens through cross-linking of surface IgM molecules receive a signal to
halt
development, and they are eliminated or inactivated. 12 Thus, many B cells capa
le of producing
anti
ody to self-antigens are deleted from the marrow
y the process of programm

ed cell death, or
apoptosis. It is estimated that more than 90 percent of B cells die in this mann
er without
leaving the
one mar- row. 14 Immature B cells leave the
one marrow and
proceed to seed
the spleen and other secondary lymphoid organs. Mature B Cells In the spleen, im
mature B cells
develop into mature cells known as marginal zone B cells. 9 These B cells remain
in the spleen
in order to respond quickly to any
lood-
orne pathogens they may come in
to contact with.
Other imma- ture B cells
ecome follicular B cells, which are found in lymph nod
es and other
secondary organs. Unlike marginal B cells that remain in the spleen, follicular
B cells are constantly recirculating throughout the secondary lymphoid organs. 9 In addition
to IgM, all
mature B cells exhi
it IgD, another class of anti
ody molecule, on their surf
ace (Fig. 26D).
Both IgM and IgD have the same speci city for a particular antigen or group of ant
igens. These
surface immunoglo
ulins provide the primary activating signal to B cells when co
ntact with
antigen takes place. 14 IgD is not required for B-cell function,
ut it may prol
ong the life span
of mature B cells in the periphery. 12 Unless contact with antigen occurs, th
e life span of
a mature B cell is typically only a few days. 12,14 If, however, a B cell i
s stimu- lated
y
antigen, it undergoes transformation to a
last stage, which eventually forms me
mory cells and
anti
ody- secreting plasma cells. This process is known as the antigen-d
ependent phase of
B-cell development. These B cells have a half-life of more than 6 weeks. 12 Acti
vated B Cells
Antigen-dependent activation of B cells takes place in the primary follicles of
peripheral
lymphoid tissue. This ocurs when antigens cross-link several surface immunoglo

u- lins on select
B cells (Fig. 27). Activated B cells exhi
it identifying markers that include CD2
5, which is
found on
oth activated T and B cells and acts as a receptor for inter- leukin-2
(IL-2), a
growth factor produced
y T cells. Additional receptors that appear at this
time are
specific for other growth factors produced
y T cells. When B cells are activate
d in this manner,
they transform into
lasts that will give rise to
oth plasma cells and so-calle
d memory cells.
Plasma Cells Plasma cells are spherical or ellipsoidal cells
etween 10 and 20 m
in size and are
characterized
y the presence of a
undant cytoplasmic immunoglo
ulin and little
to no sur- face
immunoglo
ulin (Fig. 28, Color Plate 7). 1 The nucleus is eccentric or oval w
ith heavily
clumped chro- matin that stains darkly. An a
undant endoplasmic reticulum
and a clear
well-defined Golgi zone are present in the cytoplasm. This represents the most f
ully differentiated lymphocyte, and its main function is anti
ody production. Plasma c

ells are not

normally found in the
lood
ut are located in germinal centers in the periph- e
ral lymphoid
organs. Plasma cells are nondividing, and after several days of anti
ody product
ion, they die
with- out further proliferation. 9 Memory cells (see Fig. 2-7) are also found in
germinal centers
and have a much longer life span than a resting CD 19 A B C D CD 45R CD 24
C-Kit Bone
marrow stromal cell CD 19 CD 45R Rearrangement of heavy chains Rearrangement of
light chains m
chains in cytoplasm m and surrogate light chains CD 19 CD 45R CD 19 IgM IgD IgM C
D 45R CD 24 CD
24 CD 24 FIGURE 26. B-cell development in the
one marrow. Selected markers are s
hown for the
various stages in the differentiation of B cells. Stages up to the formation of
mature B cells
occur in the
one marrow. (A) Pro-B cell. (B) Pre-B cell. (C) Immature B cell. (
D) Mature B cell.
1814_Ch02_019-038.qxd 7/10/09 3:45 PM Page 26 CHAPTER 2 The Lymphoid System 2
7 T-CELL
DIFFERENTIATION Sixty to 80 percent of circulating lymphocytes in the periph- er
al
lood are T
cells, and these
ecome differentiated in the thymus. Lymphocyte precursors call
ed thymocytes
enter the thymus from the
one marrow. Within the lo
ules of the thymus
are two main
zones, the outer cortex and the inner medulla. Early precursors enter the thymus
at the
cortico-medullary junction and migrate to the outer cortex. Migration occurs in
waves and is
driven
y chemi- cal messengers called chemokines. 1618 Early surface markers on
thymocytes that
are committed to
ecoming T cells include CD44 and CD25. 6,1618 As thymocytes t
ravel through
the thymus, they go through a process similar to that of B cells in the marro
w: There is an
orderly rearrangement of the genes coding for the antigen receptor. At the same
time, distinct
surface markers appear during specific stages of development. Maturation is an
ela
orate process
that takes place over a 3-week period as cells filter through the cortex
to the medulla.
Thymic stromal cells include epithelial cells, macrophages, fi
ro
lasts, a
nd dendritic
cells, all of which play a role in T-cell development. Interaction with stro- ma
l cells under
the in uence of cytokines, especially CD 19 IgM IgD CD 45R CD 19 IgM (capping)
IgD CD 45R
Antigen Plasma cells Immunoglo
ulins Memory cells CD 24 CD 25 Cytokines CD 24 FI
GURE 27. B-cell
activation in peripheral lymphoid organs. FIGURE 28. A typical plasma cell. (From
Harr, R.
Clinical La
oratory Science Review. F. A. Davis, Philadelphia, 2000, Color Plate
28.) See Color
Plate 7. B cell. 5 These represent progeny of antigen-stimulated B cells that
are capa
le of
responding to antigen with increased speed and intensity. They are similar i
n appear- ance
to unstimulated B cells,
ut they remain in an activated state for month

s or years, ready
to respond to the initial antigen. 1814_Ch02_019-038.qxd 7/10/09 3:45 PM Page
27 28 SECTION 1
Nature of the Immune System interleukin-7, is critical for growth and differenti
ation. 3,13 A
significant selection process occurs as maturation takes place,
ecause it is es
timated that
approximately 97 per- cent of the cortical cells die intrathymically
efo
re
ecoming
mature T cells. 17 Dou
le-Negative Stage Early thymocytes lack CD4 and CD8
markers, which
are important to their later function; hence they are known as dou
lenegative
thymocytes (DN) (Fig. 29). These large dou
le-negative thymocytes actively prol
if- erate in
the outer cortex under the influence of interleukin-7. Rearrangement of th
e genes that code
for the antigen receptor known as TCR
egins at this stage (Fig. 29). 14 CD3, the
complex that
serves as the main part of the T-cell antigen receptor, consists of eight noncov
alently
associated chains, six of which are common to all T cells. 4,20 However, two cha
ins, the alpha
() nd bet () chains, contain vari- a
le regions that recognize specific antigens
(Fig. 210).
These are coded for
y selecting gene segments and delet- ing others, as is the
case with B
cells. Rearrangement of the chain occurs rst. 16 The appearance of a functional c
hain on the
cell surface sends a signal to suppress any further chain gene rearrangements
. The
com
ination of the chain with the rest of CD3 forms the pre-TRC receptor. Sign
aling
y the
chain also triggers the thymocyte to
ecome CD4-positive (CD4+) and CD8
-positive
(CD8+). 16,17 Early on, some thymocytes, representing 10 percent or less of the
total num
er,
rearrange and express two other chains, gamma () and delta (). It is not known how
this process
is controlle, but these cells procee own a iffer- ent evelopmental pathway.
Cells expressing
the receptor typically remain negative for both CD4 an CD8. However, as mature T
cells, they
appear to represent the ominant T-cell population in the skin, intestinal epith
elium, an pulmonary epithelium. New evience inicates that they may act like natural killer
(NK) cells,
because they can bin to a broa range of cell surface molecules in their
natural,
unprocesse form. 19 They are capable of recognizing anti- gens without being pr
esente by MHC
proteins, so they may represent an important brige between natural an aaptive
immunity.
Double-Positive Stage At this secon stage, when thymocytes express both CD4 an
CD8 antigens,
they are calle ouble-positive. Double- positive thymocytes proliferate an
then begin to
rearrange the genes coing for the alpha chain. 17,18 When the CD3- receptor compl
ex (TCR) is
expressed on the cell surface, a positive selection process takes place that all

ows only
dou
le-positive cells with functional TCR receptors to survive. T cells must rec
onize forein
anti- en in association with class I or class II MHC molecules (Fi. 29). Any th
ymocytes that
are una
le to reconize self-MHC antiens die without leavin the thymus. This w
eedin out is
important,
ecause functionin T cells must
e a
le to reconize forein a
ntien alon
with MHC molecules. Those cells expressin moderate levels of TCR receptor
ind

y means of the
chains to MHC antiens on cells within the thymus. 18,19 In fact, this reconitio
n of MHC protein associated with small peptides has
een found to
e essential for positive
selection. 18,21
TCR
indin transmits a sinal that results in activation of several enzymes cal
led kinases that
are necessary for further development and sur- vival. 18 This process appears to
take place in
the cortex, althouh there are indications that interaction with epithe- lial ce
lls in the
medulla may
e necessary to complete the process. 16 A second selection process,
known as
neative selection, takes place amon the survivin dou
le-positive T cells. Str
on reactions
with self-peptides send a sinal to delete the developin T cell
y means
of apopotosis,
or pro- rammed cell death. . 18,20 Most T cells that would
e capa
le of an au
toimmune response
are eliminated in this manner. This selection process is very riorous,
ecause
only 1 to 3
percent of the dou
le-positive thymocytes in the cortex survive. 16 Blood supply
Macrophae or
dendritic cell (further neative selection) CD4 CD8 SP DP Capsule Cortex Medulla
Survivin mature
T cells exit to periphery Thymic epithelial cell (positive and neative selectio
n)
Corticomedullary junction DN TP FIGURE 29. T-cell maturation in the thymus. T lymp
hocyte
precur- sors (TP) enter the thymus at the cortico-meullary junction. They migra
te upwar in the
cortex an begin evelopment of the T-cell receptor. A small percent of precurso
rs evelop
gamma-elta chains, while the majority evelop alpha-beta chains an become oub
le- positive (DP)
(both CD4 an CD8 are present). Positive an negative selection take place throu
gh the CD3/T cell
receptor for antigen. If positively selecte, the T cell becomes single-positive
(SP); that is,
either CD4 or CD8 . Further interaction with macrophages or enritic cells take pl
ace to wee
out any T cells able to respon to self-antigen. Surviving CD4 an CD8 cells exit
the thymus to
the peripheral bloo. 1814_Ch02_019-038.qx 7/10/09 3:45 PM Page 28 CHAPTER 2
The Lymphoi
System 29 Mature T Cells Survivors of selection exhibit only one type of marker,
either CD4 or
CD8, an they migrate to the meulla. It is not cer- tain why one marker is own
-regulate, but
it may epen on which MHC protein the cell interacts with, an expo- sure to ce

rtain cytokines.
19 CD4 T cells recognize antigen along with MHC class II protein, while CD8+ T cel
ls interact
with antigen an MHC class I proteins. The two separate mature T-cell population
s create iffer
greatly in function. T cells bearing the CD4 receptor are terme helper,
or inucer,
cells, while the CD8-positive (CD8 ) population consists of cytotoxic T cells. App
roximately
two-thirs of peripheral T cells express CD4 antigen, while the remain- ing onethir express CD8
antigen. These mature T cells are release from the thymus an see peripheral l
ymphoi organs.
Resting T cells have a life span of up to several years in these peripheral orga
ns. 5 T helper
cells consist of two subsets, terme Th1 an Th2 cells. They each have a iffere
nt role to play
in the immune response. Th1 cells prouce interferon gamma (IFN-) and tumor necro
sis factor-
eta
(TNF-), which protect cells aainst intracellular pathoens. 22 Th2 cells produce
a variety of
interleukins, includin IL-4, IL-5, IL-10, and IL-13. The essential role of the
Th2 cells is to
help B cells produce anti
ody aainst extracellular pathoens. 22 Recently
, an additional
T-cell su
population has
een descri
ed. These are called T reulatory cells (T
re), and they
possess the CD4 antien and CD25. 5 These cells com- prise approximately 5 to 10
percent of all
CD4-positive T cells. 23,24 T res play an important role in suppressin the imm
une response to
self-antiens. They may do so
y producin interleukins such as IL-10 and
transformin
rowth factor B, which switch off the immune response. 25 Sinle positive T cell
s spend
approximately 12 days in the medulla. 16 If any of the survivin cells are react
ive to tissue
antiens, a further deletion process takes place. 16 This ensures that most T ce
lls leavin the
thymus will not react to self-antiens. However, the process does not wipe out a
ll
self-reactivity, so the presence of T reulatory cells is necessary to prevent a
utoimmune
responses. 16 Additional proliferation of these carefully screened T cells occur
s, and then they
leave the thymus to seed secondary lymphoid orans. They recirculate throuh
the
loodstream
and peripheral orans approximately once every 12 to 24 hours. 19 This is
important for T
cells to
e a
le to make contact with antien. 22 T cells remain meta
olically a
ctive throuh
continuous contact with self-peptide/MHC com- plexes on antien-presentin ce
lls and the
action of interleukin-7 (IL-7). Antien Activation Antien must
e transported
to the T-cell
zones of the sec- ondary lymphoid tissue. 22 When antien reconition occurs, T
lymphocytes are
transformed into lare activated cells that are characterized
y polyri
osome-fi
lled cytoplasm.
Activated T lymphocytes express receptors for IL-2, just as activated B cells do

(Fi. 211). T
lympho
lasts differen- tiate into functionally active small lymphocytes that pro
duce cytokines.
Activities of speci c cytokines include assistin B cells in commencin anti
ody p
roduction,
killin tumor and other taret cells, rejectin rafts, stimulatin hemato
poiesis in the

one marrow, and initiatin delayed hypersensitivity alleric reactions. This ty
pe of immune
TCR S S
}
S S S S S S S S S S S S S S FIGURE 210. Th CD3:T-cll rcptor com- plx
. Th T-cll
rcptor that rcognis antign consists of two chains, nd , which hav constan
t an
varia
l rgions. Four othr typs of chains ar collctivly known as CD3. Ths
 ar , , ,
an . Thy tak part in signaling to th intrior of th cll whn antign
inin
g occurs.
1814_Ch02_019-038.qx 7/10/09 3:45 PM Pag 29 30 SECTION 1 Natur of th Immu
n Systm
rspons is known as cll-miat immunity (s Chaptr 5 for a full scriptio
n of
cll-miat immunity). In ai- tion to ffctor clls, T mmory clls ar als
o gnrat. Thy
hav a highr af nity for antign than unstimulat T clls, an thy may rprsn
t prcursors in
th latr stag of th primary rspons. 22 In a similar mannr to mmory B cll
s, thy ar a
l
to prolifrat soonr than nav T clls. Thy also xprss a
roar array of cyt
okins an
appar to prsist for yars. 26 Ta
l 22 summaris th if- frncs
twn T
clls an B
clls in structur an function. THIRD POPULATION OR NATURAL KILLER CELLS A
small prcntag
of lymphocyts o not xprss th markrs of ithr T clls or B clls. Ths ly
mphocyts ar
gnrally largr than T clls an B clls at approxi- matly 15 m in iamtr, an
 thy contain
kiny-shap nucli with conns chromatin an prominnt nucl- oli. Thy hav
 a highr
cytoplasmic-nuclar ratio, an th cytoplasm contains a num
r of aurophilic gr
anuls. 27 Ths
larg granular lymphocyts mak up 5 to 10 pr- cnt of th circulating lymp
hoi pool 5 an
ar foun mainly in th spln an priphral
loo. Thy hav
n nam natur
al killr, or NK,
clls
caus thy hav th a
ility to miat cytolytic ractions an kill targ
t clls without
prior xposur to thm. Thy play an important rol as a transitional cll

riging th
innat an th acquir rspons to pathogns. 3,23 Th fact that thy lack sp
cificity in
thir rspons is ssntial to thir function as arly fnrs against pathog
ns. 28,29 This
givs tim for th acquir rspons of spcific T an B clls to
 activat.
NK clls aris
from th common lymphocyt prcursor (CLP) an iffrntiat into a T/NK cll th
at can
com
ithr a T cll or an NK cll. 28,29 Prcursors that go to th thymus
com T c

lls, whil th

in unc of
on marrow stromal clls is ncssary for NK cll iffrntiation. 13
,28,32 Within
th
on marrow, intrlukin-15 plays a critical rol in NK cll vlopmnt. 3
Thr ar no
surfac antigns that ar uniqu to NK clls,
ut thy xprss a spci c com
inati
on of antigns
that can
 us for inti cation. On such antign is CD16, which is a rcptor
for th
fragmnt crystallia
l portion, or non- spcific n, of th immunoglo
ulin mol
cul IgG. This
allows NK clls to attach to an lys any clls that ar coat with anti
oy. 3
0 Th othr main
surfac antign is CD56. Two su
sts of NK clls xistthos that hav a high lv
l of CD56 an
low or no CD16, an thos with som CD56 an high lvls of CD16. Th form
r prouc mor
cytokins an hlp support anti
oy prouction, whil th Activat T H cll Ac
tivat cytotoxic
T cll CD8 CD8 CD8 CD8 CD4 CD4 CD4 CD4 IL-2 rcptor IL-2 rcptor Viral a
ntign MHC Class
I Macrophag Digst
actrial antign MHC Class II TCR Viral antign Bactria
l antign
Virally infct host cll Cytokins Killing of virally infct clls Hlp to
B clls FIGURE
211. Activation of T cllsxposur to antign causs prouction of CD25 rcptors
for
intrlukin-2 (IL-2). IL-2 causs snsiti CD4 T clls to scrt cytokins tha
t nhanc
anti
oy prouction
y B clls. CD8 T clls hav incras cytotoxicity to rcogn
i an kill
virally infct clls. 1814_Ch02_019-038.qx 7/10/09 3:45 PM Pag 30 CHAPTER
2 Th Lymphoi
Systm 31 lattr hav a highr cytotoxic activity. 3,5, 31,32 Th cytokins pro
uc inclu
intrfron gamma an tumor ncrosis factor-alpha, so NK clls may also play a ro
l in amplifying
th immun rspons in aition to thir cytolytic activity. 3,32,33 Du to th
fact that NK
clls ar capa
l of rcogniing any forign cll an stroying it without rga
r to MHC
rstriction, thy rprsnt th first lin of fns against virally infct a
n tumor clls.
13,31 Thy accumulat at th matrnal-ftal intrfac, so thy may also play an
ssntial rol in
maintaining prgnancy. 28,32 Matur NK clls hav a half-lif of 7 to 10 ays, a
n in aition to
th utrus, thy ar mainly foun in priphral
loo, th spln, an th livr
. 28,32 NK cll
activity is stimulat
y xposur to cytokins such as intrlukin-12, intrfr
on gamma, an
intrfron
ta. 31 Sinc ths ris rapily uring a viral infction, NK clls
ar a
l to
rspon arly on uring an infction, an thir activity paks in a
out 3 ays,
wll
for
anti
oy prouction or a cytotoxic T cll rspons. Mchanism of Cytotoxicity Fo
r yars it ha

n a mystry how NK clls tll th iffrnc
twn normal an a
nor
mal clls.
Howvr, th mchanism is now
ginning to
 unrstoo. It appars th

at thr is a

alanc
twn activating an inhi
itory signals that na
ls NK clls
to istinguish
halthy clls from infct or cancrous ons. 31 Thr ar two main classs of
rcptors on NK
clls that govrn this rspons: inhi
itory rcptors, which livr inhi
itory
sig- nals, an
activatory rcptors, which livr signals to activat th cytotoxic mcha
nisms. 31 Th
inhi
itory signal is
as on rcognition of MHC class I protin, which is xpr
ss on all
halthy clls. If NK clls ract with MHC class I protins, thn inhi
ition of n
atural killing
occurs. On spcific typ of rcptor on NK clls rsponsi
l for this
ining i
nclu killr
cll immunoglo
ulin-lik rcp- tors (KIRs). 28,33 Othr inhi
itory rcptors in
clu ILT/LIR an
CD94/NKG2A rcptors, which also
in MHC class I molculs. All thr rcptors
can
 foun on
NK clls at th sam tim. 29 Disas an cancrous clls tn to los thir a

ility to prouc
MHC protins. NK clls ar thus triggr
y a lack of MHC antigns, somtims r
frr to as
rcogni- tion of missing slf. 31,33,34 This lack of inhi
ition appars to
 com

in with an
activating signal switch on
y th prsnc of protins prouc
y clls un
r strss, namly
thos clls that ar infct or cancrous. Ths protins ar nam MICA an
MICB.
Rcptors call CD94/ NKG2C an CD94/NKG2D on NK clls
in MICA or MICB an
sn a signal
to stroy th cll. 29,32,34 If an inhi
itory signal is not rciv at th
sam tim, thn
NK clls rlas su
stancs call prforins an granyms (Fig. 212). Prforins
ar
por-forming protins that poly- mri in th prsnc of Ca2 an form channls
in th targt
cll mm
ran. 29 Granyms ar packts of srin stras nyms that may nt
r through th
channls an miat cll lysis. Thus, NK clls ar constantly monitor- ing pot
ntial targt
clls through
ining to ithr activating or inhi
itory rcptors, an th NK c
ll ithr
tachs an movs on or
ins an activats cll struction. Anti
oy-Dpnn
t Cll
Cytotoxicity A scon mtho of stroying targt clls is also avail- a
l to N
K clls. Thy
rcogni an lys anti
oy-coat clls through a procss call anti
oy-pn
nt cll
cytotoxicity. Bining occurs through th CD16 rcptor for IgG. Any targt cll
coat with IgG
can

oun Ta
l 55-2 Comparison of Tsts Us T CELLS B CELLS Dvlop in th
thymus Dvlop in
th
on marrow Foun in
loo (60%80% Foun in
on marrow, spln, of circulat
ing
lymphocyts), lymph nos thoracic uct ui, lymph nos Inti 
y rostt form
ation
Inti 
y surfac with SRBCs immunoglo
ulin En proucts of activation ar En
prouct of
activation is cytokins anti
oy Antigns inclu CD2, CD3, Antigns inclu CD

19, CD20, CD4,

CD8 CD21, CD40, MHC class II Locat in paracortical Locat in cortical rgion
of rgion of
lymph nos lymph nos SRBC = Shp r
loo clls Ta
l 2-2. Comparison of T
an B Clls No
killing NK cll Normal cll NK cll Class I MHC Inhi
itory rcptor Inhi
itory
rcptor
Activating rcptor Ligan Activating rcptor Ligan Virus-infct cll (
c
lass I MHC)
Killing A B FIGURE 212. Mchanism of NK cll cytotoxicity. (From Kint, TJ, Gols

y, RA, an
Os
orn, BA. Ku
y Immunology, . 6. WH Frman, Nw York, 2007, p. 363, with p
rmission.)
1814_Ch02_019-038.qx 7/10/09 3:45 PM Pag 31 32 SECTION 1 Natur of th Immu
n Systm an
stroy. This mtho is not uniqu to NK clls, as monocyts, macrophags, an
nutrophils also
xhi
it such a rcptor an act in a similar mannr. Nonthlss, th ovrall im
portanc of NK
clls as a fns mcha- nism is monstrat
y th fact that patints who lac
k ths clls
hav rcurring, srious viral infctions. 28 LABORATORY IDENTIFICATION OF LYMPHO
CYTES
Intification of lymphocyts as ithr T clls or B clls may
 usful in iag
nosis of any of
th following stats: lymphoprolifrativ malignancis, immuno cincy is- ass
, unxplain
infctious isass, monitoring of transplants, an acquir immunologic a
no
rmalitis such as
AIDS. Most mthos ar
as on sparation of mononuclar clls from who
l
loo an
tction of sp- ci c cll surfac markrs. On of th most frquntly us mtho
s for
o
taining lymphocyts is nsity graint cntrifugation with Ficoll- Hypaqu. 3
5 Ficoll-Hypaqu
is availa
l commrcially an has a spci c gravity that varis
twn 1.077 an
1.114,
pning on th manufacturr. Dilut fi
rinat or hparini
loo is car
fully layr on
top of th solu- tion, an th tu
 is cntrifug. Cntrifugation proucs thr
 istinct
layrs: plasma at th top of th tu
, a mononuclar layr
aning on to
p of th nsity
graint solution, an rythrocyts an granulocyts at th
ottom of th tu
 (
Fig. 213). Onc
a lymphocyt population has
n o
tain, sg- rgation into su
sts is accompl
ish via flow
cytomtry. This tchniqu rlis on th us of la
l monoclonal anti
ois aga
inst spcific
surfac antigns. Som of th mor common antigns tst for inclu CD2, CD3,
CD4, CD7, an
CD8 on T clls an CD19, CD20, CD22, an surfac immunoglo
ulin on B clls
. Cll flow
cytomtry is an automat systm for intifying clls
as on th scattring o
f light as clls
flow in singl fil through a lasr
am. Fluorscnt anti
ois ar us to scr
n for
su
populations, such as B clls, T hlpr clls, an T cytotoxic clls. Th anti

ois us ar
mono- clonal, an ach has a iffrnt fluorscnt tag. Th principls o

f flow cytomtry
ar iscuss mor fully in in Chaptr 12. Manual Mtho Th rostt tchniqu u
sing r
loo
clls from shp has
n a historical mtho for numration of T lym- phocyts
. Lymphocyts ar
sparat from whol
loo an thn mix with a suspnsion of shp r
loo c
lls. If thr or
mor r
loo clls ar attach to a lympho- cyt, it is consir a rostt
(Fig. 214).
Shp clls attach to th CD2 antign, foun only on T clls. Using a counting c
ham
r, 200 clls
ar count an th pr- cnt forming rostts is calculat. This rprsnts th
 prcntag of T
clls, an th prcnt of B clls is calcu- lat
y su
tracting this num
r fro
m 100. Thr
shoul
 approximatly twic as many T clls as B clls. This tchniqu is not
as prcis as
thos mntion prviously,
caus rostting can
 influnc
y col-racting
anti- lymphocyt
anti
ois that ar form in isass such as rhumatoi arthritis an infc
tious
mononuclosis. Thrfor, this mtho is no longr us in clinical la
o
ratoris.
Ficoll-Hypaqu R
loo clls an granulocyts Mononuclar clls Cntrifug 400
x g 30 min
Whol
loo Plasma
FIGURE 213. Ficoll-Hypaqu sparation of clls in priphral

loo. Whol

loo ilut with
uffr is layr onto Ficoll- Hypaqu mium in a plastic c
ntrifug tu
.
Tu
s ar spun at 4003 g for 30 minuts. R
loo clls an granulocyts sttl
to th
ottom of
th tu
, whil mononuclar clls (monocyts an lymphocyts) form a
an at th
intrfac of th
Ficoll-Hypaqu an plasma. FIGURE 214. T-cll rostt. T cll surroun
y shp
r
loo
clls that attach to CD2 rcptors on T cll. 1814_Ch02_019-038.qx 7/10/09 3:
45 PM Pag 32
SUMMARY All uniffrntiat lymphocyts aris in th
on marrow from hmatopoi
tic stm clls.
Thy matur in th primary lymphoi organs. For B clls, this taks plac in th

on marrow,
whil T clls acquir thir spcificity in th thy- mus. B an T clls can
 r
cogni
y th
prsnc of surfac antigns, or CDs, that ar tct
y monoclonal anti
ois
. B cll markrs
inclu CD19, MHC class II protins, an surfac immunoglo
ulins. Th surfa
c
immunoglo
ulins act as rcptors for antign. MHC class II protins allow B cll
s to intract
with T hlpr clls in th prouction of anti
oy. Whn contact with spci c antign occurs, B
clls iffrntiat into plasma clls, which prouc anti
oy. In th procss, m
mory clls ar
also crat. Ths can rapily rspon th nxt tim that sam antign is sn.
Prouction of
anti
oy is known as humoral immunity T clls ar istinguish
y th prsnc
of CD3, CD2, an
ithr CD4 or CD8. CD2 is th rcptor that intracts with shp r
loo clls
to form
rostts, a simpl tst for th numration of T clls. Clls that xprss CD4

long to a T-cll
su
st that inclus hlpr/inucr clls, whil CD8-carrying T clls ar cytoto
xic/supprssor
clls. Th CD3 markr srvs as th rcptor for antign. Th major portion of i
t is common to
all T clls,
ut two chainsalpha an
tacontain varia
l rgions that can
in to
only CHAPTER
2 Th Lymphoi Systm 33 crtain antigns. T clls go through a positiv an th
n a ngativ
slction procss, whr
y th surviving clls rcogni MHC trminants a
long with forign
antign. Th T clls ar rsponsi
l for cll-miat immunity, which involvs
prouction of
cytokins that srv as rgula- tory factors for th immun rspons. A thir cl
ass of
lymphocyts, known as NK clls, ar foun in th priphral
loo an rprsnt
5 to 15 prcnt
of th total lymphocyt population. Ths ar largr an contain mor cytoplasm
an granuls than
T or B clls. Thy ar rsponsi
l for killing targt clls, incluing thos tha
t ar virally
infct or cancrous, without prvious xpo- sur or snsitiation to thm. Th
y o this
y
rcogniing missing slf-MHC antigns, in aition to tcting th prsnc of
strss protins
on infct an cancrous clls. This is an important rst lin of fns against
invasion
y
such clls. La
oratory trmination of iniviual lymphocyt pop- ulations is
ssntial in
iagnosis of such conitions as lymphomas, immunoficincy isass, un
xplain
infctions, or acquir immun isass such as AIDS. Lymphocyts ar i
ntifi using
monoclonal anti
ois irct against spci c surfac antigns. Thy ar numrat through
th us of cll ow cytomtry, which catgoris clls on th
asis of light scatt
ring.
Automat mthos liminat su
jctivity an ar mor prcis, although mor cos
tly.
1814_Ch02_019-038.qx 7/10/09 3:45 PM Pag 33 34 SECTION 1 Natur of th Immu
n Systm A
2-yar-ol
oy is snt for immunologic tsting
caus of rcurring rspiratory
infctions,
incluing svral
outs of pnumonia. Th rsults show cras immunoglo
- uli
n lvls,
spcially of IgG. Although his whit
loo cll count was within th normal ran
g, th
lymphocyt count was low. Flow cytomtry was prform to trmin if a particu
lar su
st of
lymphocyts was low or missing. It was trmin that h ha a low num
r of CD
3 lympho- cyts,
although th CD19 lymphocyt population was normal. How can this
 intrpr
t? How can
this account for his rcurring infctions? CASE STUDY 1814_Ch02_019-038.qx 7/1
0/09 3:45 PM
Pag 34 CHAPTER 2 Th Lymphoi Systm 35 ENUMERATION OF T CELLS Principl Lympho
cyts ar
sparat from whol
loo
y using a coll nsity graint, which causs rythro
cyts an granulocyts to sttl at th
ottom of a conical cntrifug tu
 whil mononuclar

clls form a
layr at th graints top. R
loo clls from shp ar us to inti
fy th T-cll
population of lymphocyts,
caus ths sponta- nously complx with T clls th
rough th CD2
rcptor. If thr or mor shp clls surroun a T cll, this is known as a ros
tt.
Quantitation of T clls provis information a
out cll-miat immunity, which
plays an
important rol in rgulation of th immun rspons an fns against
virally infct
or cancrous clls. Spcimn Collction Collct
loo
y vnipunctur using str
il tchniqu. A
frshly rawn hparini
loo spcimn is n for this procur,
caus u
s of
thylniaminttraactic aci (EDTA) will rsult in poor rcovry of mononucl
ar clls. An
aitional EDTA tu
 may
 rawn for a complt
loo cll count an a iffrn
tial. Ragnts
Matrials, an Equipmnt Cntrifug Microscop slis Covrslips, 22
22 mm Past
ur piptts
Disposa
l srologic piptts: 1 mL, 5 mL, 10 mL Conical plastic cntrifug tu

s Glass tst
tu
s, 12
75 mm Histopaqu-1077 (Sigma Diagnostics, St. Louis, Missouri) or oth
r
Ficoll-Hypaqu solution Shp r
loo clls (Bcton-Dickinson) Stril phospha
t
uffr
salin (PBS) Procur 1. A 3 mL of Histopaqu-1077 to a 15 mL plastic coni- c
al cntrifug
tu
. 2. Dilut 3 mL of hparini
loo with an qual amount of stril phosph
at
uffr
salin (PBS). Mix an car- fully layr on top of th Histopaqu-1077. 3. Cntri
fug at 400 g at
room tmpratur for 30 min- uts. Allow th cntrifug to coast to a stop. Do n
ot us th
rak.
Th opaqu intrfac that vlops is th mononuclar layr. 4. Aspirat an is
car th uppr
layr to within 0.5 cm of th mononuclar layr. 5. Carfully rmov th opaqu
intrfac with a
capillary piptt an ispns into a 15 mL cntrifug tu
. Do not xc 1.5 m
L total volum in
th transfr. A 10 mL of PBS, mix wll, an cntrifug at 400 g for 10 minuts.
6. Dcant th
suprnatant, an rain th last rops
y invrting th tu
 on top of a papr to
wl. 7. A 1 mL
of PBS, an rsuspn th clls
y aspirat- ing carfully with a Pastur piptt
. 8. O
tain a
final cll count of 6 10 6 clls/mL with a hmacytomtr. Fill th counting cham

r, an count
th clls in th four cornr squars of th cham
r. Multiply
y 20 to o
tain th
 total num
r of
mononu- clar clls harvst. A mor
uffr if furthr ilution is ncssary.
NOTE: Th
instructor can prpar th cll suspnsion
for th la
oratory
gins if tim
is short. 9.
Prpar shp r
loo cll suspnsion
y placing a
out 3 mL of shp r
loo
clls in a 15 mL
cntrifug tu
 an filling with
uffr. Cntrifug for 10 minuts at 400 g. Rsu
spn 0.1 mL of
pack clls in 19.9 mL
uffr to achiv a concntration of 0.5 prcnt. 10. Pi

ptt 0.5 mL of
th lymphocyt suspnsion into a 12 75 mm tst tu
. Piptt 0.5 mL of shp r

loo cll
suspnsion into th sam tu
, an mix wll. 11. Cntrifug mix at 250 g for 5 m
inuts. Rmov
th cntrifug tu
 carfully so th clls ar not istur
. Incu
at at room t
mpratur for
1.5 to 2 hours. Clls can also
 incu
at ovrnight at 4C, if prfrr. 12. Af
tr incu
ation,
carfully rsuspn th clls
y hol- ing th tu
 almost horiontal an carfu
lly twisting
aroun th long axis. 13. With a capillary piptt an no
ul
, gntly transfr
th rostts to a
clan microscop sli an a a covr slip. Plac th covr slip carfully so t
hat no air
u

ls ar form. O
srv th rostts using a 40 o
jctiv. 14. Count onl
y thos
lymphocyts that ar surroun
y thr or mor shp r clls. Count a total
of 200 lymphocyts, incluing thos that o not form rostts. Th prcnt of T lympho
cyts can

o
tain
y iviing th num
r of lymphocyts in rostts
y th total num
r
of lymphocyts
count. EXERCISE 1814_Ch02_019-038.qx 7/10/09 3:45 PM Pag 35 36 SECTION 1
Natur of th
Immun Systm Intrprtation of Rsults In a normal ault, T lymphocyts c
ompos 52 to
81 prcnt of th total num
r of lymphocyts. A low prcnt of T lymphocyts ma
y inicat a
T-cll isorr. Som of ths conitions inclu AIDS, Hogkins isas, c
hronic
lymphocytic lukmia, an immunoficincy isass. Car must
 takn whn
prforming this
procur. Rostts can
 isrupt, so rsuspnsion aftr cntrifuga- tion mus
t
 on vry
gntly; othrwis th prcnt of T lymphocyts will
 falsly lowr. In ait
ion, lymphocyts must
 istinguish from shp r
loo clls,
caus
oth clls ar a

out th sam
si. Lymphocyts, howvr, ar mor rfractil
caus of th prsnc of a nuc
lus. Th
mononuclar layr also contains monocyts. Ths shoul not
 count as nonros
tting
lymphocyts,
caus this, too, will falsly lowr th prcnt of T clls foun.
Monocyts ar
consira
ly largr than lymphocyts, an thy contain mor cytoplasm, which
has a granular
apparanc. This tchniqu is not as accurat a tction of spci c antigns with
monoclonal
anti
oy
caus of th fragil natur of th rostts form. It is, howvr, l
ss xpnsiv to
prform an is of historic signi canc,
caus this rp- rsnts th rst mans of
T-cll
inti cation. 1814_Ch02_019-038.qx 7/10/09 3:45 PM Pag 36 CHAPTER 2 Th Lymp
hoi Systm 37
1. Which of th following is a primary lymphoi organ? a. Lymph no
. Spln c
. Thymus . MALT
2. What typ of clls woul
 foun in a primary follicl? a. Unstimulat B c
lls
. Grminal
cntrs c. Plasma clls . Mmory clls 3. Which of th following is tru of NK

clls? a. Thy
rly on mmory for antign rcognition.
. Thy shar antigns with B clls. c.
Thy ar foun
mainly in lymph nos. . Thy rcogni a lack of MHC protins. 4. Whr ar al
l
uniffrntiat lymphocyts ma? a. Bon marrow
. Thymus c. Spln . Lymph n
os 5. In th
thymus, positiv slction of immatur T clls is
as upon rcognition of whic
h of th
following? a. Slf-antigns
. Strss protins c. MHC antigns . chains 6. Whic
h of ths ar
foun on a matur B cll? a. IgG an IgD
. IgM an IgD c. Alpha an
ta chains
. CD3 7. Which
rcptor on T clls is rsponsi
l for rostting with shp r
loo clls? a.
CD2
. CD3 c. CD4
. CD8 8. Which of th following can
 attri
ut to antign- stimulat T cll
s? a. Humoral
rspons
. Plasma clls c. Cytokins . Anti
oy 9. Which is a istinguishing f
atur of a pr-B
cll? a. chains in th cytoplasm
. Complt IgM on th surfac c. Prsnc of C
D21 antign .
Prsnc of CD25 antign 10. Whn os gntic rarrangmnt for coing of light
chains tak
plac? a. Bfor th pr-B cll stag
. As th cll
coms an immatur B cll
c. Not until th
cll
coms a matur B cll . Whn th B cll
coms a plasma cll 11. Which
of th
following antigns ar foun on th T cll su
st known as hlpr/inucrs?
a. CD3
CD4 c.
CD8 . CD11 12. Whr os th major portion of anti
oy prouction occur? a. P
riphral
loo
.
Bon marrow c. Thymus . Lymph nos 13. Which of th following woul rpr
snt a ou
lngativ thymocyt? a. CD2CD3 CD4CD8
. CD2 CD3CD4CD8 c. CD2CD3 CD4 CD8 .
CD2 CD3 CD4 CD8 14. Which of th following
st scri
s th T-cll rcp- tor for ant
ign? a.
It consists of IgM an IgD molculs.
. It is th sam for all T clls. c. It i
s prsnt in th
ou
l-ngativ stag. . Alpha an
ta chains ar uniqu for ach antign. REV
IEW QUESTIONS
1814_Ch02_019-038.qx 7/10/09 3:45 PM Pag 37 38 SECTION 1 Natur of th Immu
n Systm
Rfrncs 1. Bll, A. Morphology of human
loo an marrow clls. In Harmning,
DM: Clinical
Hmatology an Funamntals of Hmostasis, . 4. F. A. Davis, Philalphia, 200
2, pp. 138. 2.
Vajpay, N, Graham, SS, an Brn, S. Basic xamination of
loo an
on marrow
. In McPhrson,
RA, an Pincus, MR (s): Hnrys Clinical Diagnosis an Managmnt
y La
or
atory Mthos,
. 21. WB Saunrs, Philalphia, 2007, pp. 457483. 3. Blom, B, an Spits, H. D
vlopmnt of
human lymphoi clls. Annu Rv Immunol 24:287320, 2006. 4. Holmr, LD, Hamoui, W
, an
Buso-Ramos, CE. Chronic lukmia an rlat lymphoprolifrativ isorrs.
In Harmning,
DM: Clinical Hmatology an Funamntals of Hmostasis, . 4. F. A. Davis
, Philalphia,
2002, pp. 301330. 5. Kint, TJ, Gols
y, RA, an Os
orn, BA. Ku
y Immunology, 
. 6. WH Frman

an Co, Nw York, 2007, pp. 2375. 6. Ro
rtson, P, an Ponansky, MC. T-lymphocyt
 vlop- mnt
an mols of thymopoitic rconstitution. Transpl Infct Dis 5:3842, 2003. 7. Ma
k, TW, an
Saunrs, ME. Th Immun Rspons: Basic an Clinical Principls. Elsvir Inc,
Burlington, MA,
2006, pp. 3567. 8. http://
loojournal.hmatologyli
rary.org/cgi/contnt/full/ 10
6/9/3123. Zola,
H, Swart, B, Nicholson, I, Aast, B. t al. CD molculs 2005: Human cll iff
rntiation
molculs. Accss May 29, 2007. 9. Matthias, P, an Rolink, AG. Transcriptiona
l ntworks in
vloping an matur B clls. Nat Rv/Immunol 5:497508, 2005. 10. Allman, D, an
Millr, JP.
Th aging of arly B-cll prcur- sors. Immunol Rv 205:1829, 2005. 11. Nagasawa,
T.
Mironvironmntal nichs in th
on marrow rquir for B-cll vlopmnt.
Nat Rv/Immunol
6:107116, 2006. 12. Mak, TW, an Saunrs, ME. Th Immun Rspons: Basic an Cli
nical
Principls. Elsvir, Burlington, MA, 2006, pp. 209245. 13. Mathur, S, Schxni
r, K, an
Hutchison, RE. Hmatopoisis. In McPhrson, RA, an Pincus, MR (s): Hnrys Cli
nical Diagnosis
an Managmnt
y La
oratory Mtho, . 21. WB Saunrs, Philalphia, 20
07, pp. 484503.
14. Kint, TJ, Gols
y, RA, an Os
orn, BA. Ku
y Immunology, . 6. WH Frman
an Co, Nw York,
2007, pp. 271301. 15. Miln, CD, Flming, HE, Zhang, Y, an Paig, CJ. M
chanisms of
slction miat
y intrlukin-7, th prBCR, an hmokinin-1 uring
B-cll
vlopmnt. Immunol Rv 197:7588, 2004. 16. Takahama, Y. Journy through th thy
mus: Stromal
guis for T-cll vlopmnt an slction. Nat Rv/Immunol 6:127135, 2006.
17. Kint, TJ,
Gols
y, RA, an Os
orn, BA. Ku
y Immunology, . 6. WH Frman an Co, Nw Yor
k, 2007, pp.
245270. 18. Starr, TK, Jamson, SC, an Hogquist, KA. Positiv an ng- ativ sl
ction of T
clls. Annu Rv Immunol 21:139176, 2003. 19. Mak, TW, an Saunrs, ME. Th Immun
 Rspons:
Basic an Clinical Principls. Elsvir, Burlington, MA, 2006, pp. 311337.
20. Sprnt, J,
an Kishimoto, H. Th thymus an ngativ slc- tion. Immunol Rv 185:126135, 20
02. 21. Barton,
GM, an Runsky, AY: Rquirmnts for ivrs, low-a
unanc pptis in positi
v slction of T
clls. Scinc 283:67, 1999. 22. Sprnt, J, an Surh, CD. T cll mmory. Annu R
v Immunol
20:551579, 2002. 23. OGarra, A, an Vira, P. Rgulatory T clls an mchanisms of
immun
systm control. Nat M 10: 801805, 2004. 24. Bckr, C, Stoll, S, Bopp, T, Schmi
tt, E, t al.
Rgulatory T clls: Prsnt an futur hops. M Micro
iol Immunol 195:113124, 2
006. 25.
Frank, A, Hungr, JK, Dittmar, KEJ, Gansr, A, t al. Rgulatory T-c
lls in th
control of immunological isass. Ann Hmatol 85:747758, 2006. 26. Hagmann, M. H
ow th immun

systm walks mmory lan. Scinc Now July 12:4, 2000. 27. Yokoyama, WM. Natur
al killr
clls. In Paul, WE (): Funamntal Immunology, . 6 Lippincott Willi
ams & Wilkins,
Philalphia, 2008, pp. 483517. 28. Yokoyama, WM, Kim, S, an Frnch, AR. Th yn
amic lif of
natural killr clls. Annu Rv Immunol 22:40529, 2004. 29. Mak, TW, an Saunrs,
ME. Th Immun
Rspons: Basic an Clinical Principls. Elsvir, Burlington, MA, 2006, p
p. 518552. 30.
McPhrson, RA, an Massy, D. Ovrviw of th Immun Systm an Immunologic Diso
rrs. In
McPhrson, RA, an Pincus, MR (s): Hnrys Clinical Diagnosis an Managmnt
y
La
oratory
Mtho, . 21. WB Saunrs, Philalphia, 2007, pp. 789792. 31. Kint, TJ, Gols

y, RA, an
Os
orn, BA. Ku
y Immunology, . 6. WH Frman an Co, Nw York, 2007, pp. 35137
0. 32. Di
Santo, JP. Natural killr cll vlopmntal pathways: A qustion of
alanc. An
nu Rv Imminol
24:257286, 2006. 33. Lanir, L. NK cll rcognition. Annu Rv Immunol 23:2
25274, 2005.
34. Baur, S, Groh, V, an Wu, J, t al. Activation of NK clls an T clls
y N
KG2D, a rcptor
for strss-inuci
l MICA. Scinc 285:727, 1999. 35. Stvns, RA, Lmpicki, RA,
Natarajan, V,
Higgins, J, t al. Gnral immunologic valuation of patints with human i
mmuno cincy
virus infction. In Dtrick, B, Hamilton, RG, an Fols, JD (s): Manual of Mol
cular an
Clinical La
oratory Immunology, . 7. ASM Prss, Washington, DC, 2006, pp. 84786
1.
1814_Ch02_019-038.qx 7/10/09 3:45 PM Pag 38 3 Natur of Antigns an th Ma
jor
Histocompati
ility Complx LEARNING OBJECTIVES Aftr nishing this chaptr, th r
ar will

a
l to: 1. D n an charactri th natur of immunogns. 2. Diffrntiat an i
mmunogn from
an antign. 3. Intify th charactristics of a haptn. 4. Dscri
 how an pit
op rlats to an
immunogn. 5. Discuss th rol of ajuvants. 6. Diffrntiat htrophil antig
ns from
alloantigns an autoantigns. 7. Explain what a haplotyp is in rgar to inhr
itanc of major
histocom- pati
ility complx (MHC) antigns. 8. Dscri
 iffrncs in structur
 of class I an
class II protins. 9. Compar th transport of antign to cllular surfacs
y c
lass I an class
II protins. 10. Dscri
 th rol of transportrs associat with antign proc
ssing (TAP) in
slcting pptis for
ining to class I molculs. 11. Discuss th iffrncs
in th sourc
an typs of antign procss
y class I an class II molculs. 12. Explain th
 clinical
signi canc of th class I an class II molculs. KEY TERMS Ajuvant Alll Alloa
ntign
Autoantign Class I MHC (HLA) molcul Class II MHC (HLA) molcul Conformationa
l pitop Epitop
Haplotyp Haptn Htroantign Htrophil antign Major histocompati
ility comp
lx (MHC)

Immunogn Invariant chain (Ii) Linar pitop Transportrs associat with antig
n procssing
(TAP) 39 1814_Ch03_039-053.qx 7/10/09 3:46 PM Pag 39 40 SECTION 1 Natur of
th Immun
Systm Th immun rspons of lymphocyts is triggr
y mat- rials call i
mmunogns,
macromolculs capa
l of triggring an aaptiv immun rspons
y inucing t
h formation of
anti
ois or snsiti T clls in an immuno- comptnt host. Immunogns can th
n spcifically
ract with such anti
ois or snsiti T clls. Th trm antign rfrs to a s
u
stanc that
racts with anti
oy or snsiti T clls
ut may not
 a
l to vok an immun
 rspons in th
first plac. Thus, all immunogns ar antigns,
ut th convrs is not tru. Ho
wvr, many tims
th trms ar us synonymously, an th istinction
twn thm is not ma. I
n iscussing
srological ractions or particu- lar nams of su
stancs such as
loo groups,
th trm antign
is still mor commonly us; hnc
oth trms ar us in this chaptr. On of t
h most xciting
aras of rsarch focuss on how an why w rspon to particular immunogns. Th
is rspons is
actually caus
y a com
ination of factors: th natur of th immunogn itslf,
gntic coing
of MHC molculs that must com
in with an immunogn
for T clls ar a
l to
rspon, an
immunogn procssing an prsntation. This chaptr focuss on all thr aras a
n iscusss
futur clinical implications of som rcnt finings. FACTORS INFLUENCING THE IM
MUNE RESPONSE
Svral factors such as ag, ovrall halth, os, rout of inoculation, an gn
tic capacity
in unc th natur of this rspons. In gnral, olr iniviuals ar mor likl
y to hav a
cras rspons to antignic stimulation. At th othr n of th ag scal,
nonats o not
fully rspon to immunogns,
caus thir immun systms ar not com- pltly 
vlop. Ovrall
halth plays a rol, as iniviuals who ar malnourish, fatigu, or strss
ar lss likly
to mount a succssful immun rspons. Thr appars to
 a thrshol os for
ach iniviual
immunogn. This allows th innat immun rspons to tak car of small amounts
of pathogns an
lav th aap- tiv rspons for pathogns that ar prsnt in larg nu
m
rs. Gnrally,
th largr th os of an immungn on is xpos to, th gratr th imm
un rspons
is. Howvr, vry larg oss can rsult in T- an B-cll tolr- anc, a phno
mnon that is
not wll unrstoo. It is possi
l that mmory clls
com ovrwhlm an
thr- for
nonrsponsiv. 1 Th actual amount of immunogn n to gnrat an immun rs
pons iffrs
with th rout of inoculation. Such routs inclu intravnous (into a vin), in
trarmal (into
th skin), su
cutanous (
nath th skin), an oral aministration. Whr th i
mmunogn ntrs

th
oy trmins which cll populations will
 involv in th rspons an
how much is
n to trig- gr a rspons. Lastly, a gntic prisposition may
 involv
that allows
iniviuals to rspon to particular immunogns. This prisposition is link t
o th MHC
(iscuss in th sction on th clinical signi canc of th MHC molculs) an to
th rcptors
gnrat uring T an B lymphocyt vlopmnt. TRAITS OF IMMUNOGENS In gnral
, th a
ility of
an immunogn to stimulat a host rspons pns on th following charact
ristics: (1)
macromolcular si, (2) chmical composition an molc- ular complxity, (3) fo
rignnss, an
(4) th a
ility to
 procss an prsnt with MHC molculs. 1,2 Usually an
immunogn must
hav a molcular wight of at last 10,000 to
 rcogni
y th immun systm
, an th
st
immunogns typically hav a molcular wight of ovr 100,000 altons. 1,2
Howvr, thr
ar xcptions,
caus a fw su
stancs with a molcular wight of lss than 10
00 hav
n
known to inuc an immun rspons. For th most part, th rul of thum
is that
th gratr th
molc- ular wight, th mor potnt th molcul is as an immunogn. Im
munognicity is
also trmin
y a su
stancs chmical composition an molcular complxity
. Protins an
polysaccharis ar th
st immunogns. Protins ar powrful immunogns,
cau
s thy ar ma
up of a varity of units known as amino acis. Th particular squntial CHAPTER
OUTLINE FACTORS
INFLUENCING THE IMMUNE RESPONSE TRAITS OF IMMUNOGENS NATURE OF EPITOPES HAPTENS
RELATIONSHIP OF
ANTIGENS TO THE HOST ADJUVANTS MAJOR HISTOCOMPATIBILITY COMPLEX Gns Coing for
MHC Molculs
(HLA Antigns) Structur of Class I Molculs Structur of Class II Molculs Ro
l of Class I
Molculs Rol of Class II Molculs Clinical Signi canc of MHC SUMMARY CASE STUD
Y EXERCISE:
SPECIFICITY OF ANTIGENANTIBODY REACTIONS REVIEW QUESTIONS REFERENCES 1814_Ch03_03
9-053.qx
7/10/09 3:46 PM Pag 40 CHAPTER 3 Natur of Antigns an th Major Histocompat
i
ility Complx
41 arrangmnt of amino acis, th primary structur, tr- mins th scona
ry structur,
which is th rlativ orintation of amino acis within th chain. Th trti
ary structur
m
ois th spatial or thr-imnsional orin- tation of th ntir molcul;
an th
quatrnary structur is
as on th association of two or mor chains into a si
ngl polymric
unit. Bcaus of th variations in su
units, protins may hav an normous vari
ty of
thr-imnsional shaps. B clls rcogni structurs that projct from th xt
rnal surfacs of
macromolculs, an th mor com- plxity or
ranching thr is, th asir it i
s for B clls to
rspon. Protins hav pitops that also stimulat T clls, which is ssntial
to gnrating

T-cll hlp in anti
oy pro- uction. 1 In contrast, synthtic polymrs such as

nylon or T on
ar ma up of a fw simpl rpating units with no
ning or foling within th
 molcul, an
ths matri- als ar nonimmunognic. For this rason, thy ar us in making a
rti cial hart
valvs, l
ow rplacmnts, an othr mical appliancs. Car
ohyrats ar som
what lss
immunognic than pro- tin,
caus th units of sugars ar mor limit than th
 num
r of amino
acis in protin. As immunogns, car
o- hyrats most oftn occur in th form of
glycolipis or
glycoprotins. Many of th
loo group antigns ar com- pris of such car
ohy
rat complxs.
For xampl, th A, B, an H
loo group antigns ar glycolipis, an th Rh an
 Lwis antigns
ar glycoprotins. 3 Pur nuclic acis an lipis ar not immunognic
y thms
lvs, although a
rspons can
 gnrat whn thy ar attach to a suit- a
l carrir molcul
. 4 This is th
cas for autoanti
ois to DNA that ar form in systmic lupus rythmatosus.
Ths
autoanti
ois ar actually stimulat
y a DNA- protin complx rathr than
y
DNA itslf.
Anothr charactristic that all immunogns shar is for- ignnss. Th immun sy
stm is normally
a
l to istinguish
twn slf an nonslf, an thos su
stancs rcogni as
nonslf ar
immunognic. This a
ility is acquir as lym- phocyts matur in th primary
lymphoi
organs. Any lymphocyt capa
l of racting with slf-antign is normally limin
at. Typically,
th mor istant taxonomically th sourc of th immunogn is from th host, th

ttr it is as
a stimulus. For xampl, plant protin is a
ttr immuno- gn for an animal tha
n is matrial
from a rlat animal. Occasionally, howvr, autoanti
ois, or anti
ois to s
lf- antigns,
xist. This is th xcption rathr than th rul, an this phnomnon is iscus
s in Chaptr
14. Furthrmor, for a su
stanc to licit an immun rspons, it must

su
jct to
antign procssing, which invols nymatic igstion to crat small pptis o
r pics that can

 complx to MHC molculs to prsnt to rsponsiv lymphocyts. If a mac
romolcul cant

 gra an prsnt with MHC molculs, thn it woul
 a poor immunogn
. Th particular
MHC molculs pro- uc also trmin rsponsivnss to iniviual antigns. E
ach iniviual
inhrits th a
ility to prouc a crtain limit rprtoir of MHC molculs, 
iscuss in th
sc- tion on th gns coing for MHC molculs. NATURE OF EPITOPES Although an
immunogn must
hav a molcular wight of at last 10,000, only a small part of th immunogn i
s actu- ally
rcogni in th immun rspons. This ky portion of th immunogn is known as
th trminant
sit or pitop. Epitops ar molcular shaps or configurations that ar rcogn
i
y B or T

clls, an thr is vinc that for protins, pitops rcogni
y B clls m
ay consist of as
fw as 6 to 15 amino acis. 1,2 Larg molculs may hav numrous pitops, an
ach on may

capa
l of trig- gring spcific anti
oy prouction or a T-cll rspons. Epito
ps may

rpating copis, or thy may hav iffr- ing spcificitis. Thy may also

 squntial
or linar (i.., amino acis following on anothr on a singl chain), or thy
may

conformational. A conformational pitop rsults from th foling of on chain o
r multipl
chains,
ringing crtain amino acis from iffrnt sgmnts of a linar squnc
 or squncs
into clos proximity with ach othr so thy can
 rcogni togthr (Fig. 31)
. Epitops
rcogni
y B clls may iffr from thos rc- ogni
y T clls. 1,2 Surfac
anti
oy on B
clls may ract with
oth linar an conformational pitops prsnt on th surf
ac of an
immunogn. Anything that is capa
l of cross- linking surfac immunoglo
ulin mol
culs is a
l to
triggr B-cll activation. Th immunogn os not ncssarily hav to
 gra
 rst. If th
immunogn is a protin, B clls may rcogni th primary, sconary, trtiary,
or vn th
quatrnary structur. For polysaccharis, th
ranch points of
ranch chains
may contri
ut
most to rcognition. 2 T clls, on th othr han, rcogni an pitop only as
a part of a
complx form with MHC protins on th sur- fac of an antign-prsnting cll.
Th
antign-prsnting A Linar pitops B Conformational pitops FIGURE 31. Lina
r vrsus
conformational pitops. (A) Linar pi- tops consist of squntial amino acis
on a singl
polyppti chain. Thr may
 svral iffrnt typs on on chain. (B) Confor
mational pitops
rsult from th foling of a polyppti chain or chains, an nonsquntial amin
o acis ar

rought into clos proximity. 1814_Ch03_039-053.qx 7/10/09 3:46 PM Pag 41 4
2 SECTION 1
Natur of th Immun Systm cll must procss an immunogn first an gra it
into small
pptis for it to
 rcogni
y T clls (s th sc- tion on antign procs
sing). Thus,
T-cll pitops ar linar
ut may
 molculs foun anywhr in th cll, rath
r than strictly
surfac molculs. HAPTENS Som su
stancs ar too small to
 rcogni
y th
m- slvs,
ut if
thy ar complx to largr molculs, thy ar thn a
l to stimulat a rspon
s. Haptns ar
nonim- munognic matrials that, whn com
in with a carrir, crat nw antig
nic trminants.
Onc anti
oy prouc- tion is initiat, th haptn is capa
l of racti
on with anti
oy
vn whn th haptn is not complx to a car- rir molcul; howvr, prci
pitation or
agglutination ractions will not occur,
caus a haptn has a singl tr- min
ant sit an

cannot form th cross-links with mor than on anti
oy molcul that ar ncss
ary for
prcipitation or agglutination (Fig. 32). Haptns may
 complx arti cially with
carrir molculs in a la
oratory stting, or this may occur naturally within a host an s
t off an immun
rspons. An xampl of th lattr is an allrgic raction to poison ivy. Poison
ivy (Rhus
raicans) contains chmical su
stancs call catchols, which ar haptns. Onc
in contact with
th skin, ths can coupl with tissu protins to form th immunogns that giv
ris to contact
rmatitis. Anothr xampl of haptns coupling with normal protins in th
oy
to provok an
immun rspons occurs with crtain rug-protin conju- gats that can rsult in
a
lif-thratning allrgic rspons. Th
st known xampl of this occurs with p
nicillin. Th
most famous stuy of haptns was conuct
y Karl Lanstinr, a Grman scinti
st who was known
for his is- covry of th ABO
loo groups. In his
ook Th Spci city of Srolog
ical Ractions,
pu
lish in 1917, h tail th rsults of an xhaustiv stuy of haptns tha
t has contri
ut
gratly to our knowlg of antignanti
oy ractions. H iscovr that anti
o
is rcogni
not only chmical fa- turs such as polarity, hyropho
icity, an ionic charg,

ut th ovrall
thr-imnsional con guration is also important. 1,2 Th spatial orintation an
th chmical
complmntarity ar rsponsi
l for th lock-an-ky rlationship that allows fo
r tight
ining

twn anti
oy an pitop (Fig. 33). Toay it is known that many thraputic 
rugs an hormons can function as haptns. 2 RELATIONSHIP OF ANTIGENS TO THE HOST Antigns c
an
 plac in

roa catgoris accoring to thir rlationship to th host. Autoantigns ar t
hos antigns
that
long to th host. Ths o not vok an immun rspons unr normal circ
umstancs.
Alloantigns ar from othr mm
rs of th hosts spcis, an ths ar capa-
l
of liciting
an immun rspons. Thy ar important to consir in tissu transplantation an
in
loo
transfusions. Htroantigns ar from othr spcis, such as othr ani- mals, pl
ants, or
microorganisms. Htrophil antigns ar htroantigns that xist in unrlat
plants or
animals
ut ar ithr intical or closly rlat in structur so that anti
o
y to on will
cross-ract with antign of th othr. An xampl of this is th human
loo gro
up A an B
antigns, which ar rlat to
actr- ial polysaccharis. 4 It is
liv tha
t anti-A
anti
oy, which is normally foun in iniviuals with
loo typs othr than A (
.g., typ B an
typ O), is originally form aftr xpo- sur to pnumococci or othr similar

actria.
Naturally occurring anti-B anti
oy is form aftr xposur to a sim- ilar
act
rial cll wall

prouct. Normally in srological ractions, th ial is to us a raction that

is compltly
spci c,
ut th fact that cross- ractivity xists can
 hlpful for crta
in iagnostic
purposs. In, th rst tst for infctious mononuclosis (IM) was
as on a h
trophil
anti
oy raction. During th arly stats of IM, a htrophil anti
oy is form
, stimulat
y
an unknown antign. This anti
oy was foun
+ B A Anti
oy formation Raction wi
th anti
oy
Haptn alon Haptn Com
ination
No a

+ Haptn an carrir Haptn an carrir Ag
glutination
a
X FIGURE 32. Charactristics of haptn. (A) Haptn alon cannot stimulat anti

oy formation.
It can ract with anti
oy,
ut it is monovalnt, so no complxs form. (B) Whn
complx to a
carrir, th haptn can stimulat anti
oy formation. If haptn is complx to
a carrir at
multipl sits, thn agglutination can tak plac. 1814_Ch03_039-053.qx 7/10/0
9 3:46 PM Pag
42 CHAPTER 3 Natur of Antigns an th Major Histocompati
ility Complx 43 to r
act with shp
r
loo clls, an this form th
asis of th Paul-Bunnll scrning tst fo
r mononuclosis
(s Chaptr 22). This procur was a usful scrning tst whn th causativ
agnt of IM ha
not
n intifi. Currnt rapi scrning tsts for IM ar
as on th principl of
tction of htrophil anti
oy. ADJUVANTS Th powr of immunogns to gnrat
an immun
rspons can
 incras through th us of ajuvants. An ajuvant is a su
st
anc
aministr with an immunogn that incrass th immun rspons. It acts

y proucing a
local inflammatory rspons that attracts a larg num
r of immun systm
clls to th
injction sit. 1 Aluminum salts ar th only ons approv for clinical us in
th Unit
Stats, an ths ar us to complx with th immunogn to incras its si an
 to prvnt a
rapi scap from th tissus. 5 It must
 injct into th muscl to work. Th
 hpatitis B
vaccination is an xampl of using this typ of ajuvant. Anothr common ajuvan
t is Fruns
complt aju- vant, which consists of minral oil, mulsifir, an kill myco

actria (0.5
mg/mL). Antign is mix with aju- vant an thn injct. It is rlas
slowly from
th injction sit. Fruns ajuvant proucs granulomas, or larg aras of scar
tissu, an
thus is not us in humans. Ajuvants ar thought to nhanc th immun rspons

y prolonging
th xistnc of immunogn in th ara, incrasing th ffctiv si of th
 immunogn,
an incrasing th num
r of macrophags involv in anti- gn procssing. 1,2 M
AJOR
HISTOCOMPATIBILITY COMPLEX For yars, scintists sarch to intify post
ulat immun
rspons gns that woul account for iffrncs in how iniviuals rspon to
particular

immunogns. Evinc now inicats that th gntic capa
ility to mount a

n immun rspons
is link to a group of mol- culs originally rfrr to as human lukocyt an
tigns (HLA).
Th Frnch scintist Dausst gav thm this nam,
caus thy wr first
fin
y
iscovring an anti
oy rspons to circulating whit
loo clls. 4,6 Ths ant
igns ar also
known as MHC molculs,
caus thy trmin whthr transplant tissu is hi
stocompati
l an
thus accpt or rcogni as forign an rjct. MHC molculs ar actually
foun on all
nuclat clls in th
oy, an thy play a pivotal rol in th vlop- mnt o
f
oth humoral
an cllular immunity. Thir main function is to
ring antign to th cll surfa
c for rcognition
y T clls,
caus T-cll activation will occur only whn antign is co
m
in with MHC
molculs. Clinically, thy ar rlvant,
caus thy may
 involv in trans
fusion ractions,
graft rjction, an autoimmun isass. Gns controlling xprssion of ths
molculs ar
actually a systm of gns known as th major histo- compati
ility complx (MHC)
. Gns Coing
for MHC Molculs (HLA Antigns) Th MHC systm is th most polymorphic systm f
oun in humans.
6,7 It is thought that this polymorphism is ssn- tial to our survival,
caus
MHC molculs
play a pivotal NH 2 Amino
nn (anilin) Antisrum against Ractivity with oAmino
noic aci
Amino
nn + + + 0 0 0 0 + + + 0 0 0 0 + + + + 0 0 0 0 + + +/ + + + + o-Amino

noic aci
m-Amino
noic aci p-Amino
noic aci m-Amino
noic aci p-Amino
noic aci
NH 2 COOH COOH
COOH NH 2 NH 2 FIGURE 33. Lanstinrs stuy of th spci city of haptns. Spatial o
rintation
of small groups is rcogni,
caus anti
ois ma against amino
nn coup
l to a carrir
will not ract with othr similar haptns. Th sam is tru for antisrum to o-a
mino
noic aci,
m-amino
noic aci, an p-amino
noic aci. Anti
oy to a car
oxyl group in on
 location woul
not ract with a haptn, which has th car
oxyl group in a iffrnt location. (
From Lanstinr,
K. Th spci city of srological ractions, rvis ition. Dovr Prss, Nw York
, 1962.)
1814_Ch03_039-053.qx 7/10/09 3:46 PM Pag 43 44 SECTION 1 Natur of th Immu
n Systm rol in
triggring th immun rspons to ivrs immuno- gns. 8 Gns coing for th M
HC molculs in
humans ar foun on th short arm of chromosom 6 an ar ivi into thr cat
goris or
classs. Class I molculs ar co for at thr iffrnt locations or loci, t
rm A, B, an
C. Class II gns ar situat in th D rgion, an thr ar svral iffrnt
loci, known as
DR, DQ, an DP. For th class II molculs, thr is a gn that cos for th a
lpha chain an
on or mor gns that co for th
ta chain. Btwn th class I an class II
rgions on

chromosom 6 is th ara of class III gns, which co for complmnt protins
an cytokins
such as tumor ncrosis factor (Fig. 34). Class III protins ar scrt protins
that hav an
immun function,
ut thy ar not xprss on cll surfacs. Class I an II gn
 proucts ar
involv in antign rcognition an influnc th rprtoir of antigns to whic
h T clls can
rspon. At ach of ths loci, or locations, thr is th possi
il- ity of mult
ipl allls.
Allls ar altrnat forms of a gn that co for slightly iffrnt variti
s of th sam
pro- uct. Th MHC systm is scri
 as polymorphic,
caus thr ar so many
possi
l allls
at ach location. For xam- pl, at last 580 iffrnt allls of HLA-A, 921 al
lls of HLA-B,
an 312 allls of HLA-C hav
n inti  at this tim. 9 Th pro
a
ility that
any two
iniviuals will xprss th sam MHC molculs is vry low. An iniviual inhr
its two copis of
chromosom 6, an thus thr is a possi
ility of two iffrnt allls for ach
gn on th
chromosom, unlss that prson is homoygous (has th sam allls) at a givn l
ocation. Ths
gns ar scri
 as coominant, maning that all allls that an iniviual i
nhrits co for
proucts that ar xprss on clls. Sinc th MHC gns ar closly link, th
y ar inhrit
togthr as a packag call a haplotyp. Thus, ach inhrit chromosomal
rgion consists
of a packag of gns for A, B, C, DR, DP, an DQ. Th full gnotyp woul consi
st of two of ach
gn at a particular locus. Bcaus thr ar numrous all- ls or varint form
s at ach locus,
an iniviuals MHC typ is a
out as uniqu as a ngrprint. Traitionally, HLA nom
nclatur ha

n  n sro- logically through th us of a
attry of anti
ois. Cu
rrntly,
howvr, avancs in DNA analysis hav ma intification of actual gns possi

l. Th
nomnclatur has
com corrsponingly mor complx. For instanc, th notation
HLA DRB1*1301
inicats th actual gn involv in coing for an HLA DR1 antign, wit
h th B staning
for th
ta chain, which is a part of th antign, an th 1301 inicating a sp
ci c alll. Th
uniqunss of th HLA antigns crats a major pro
lm in matching organ onors
to rcipints,

caus ths antigns ar highly immunognic. Howvr, in cass of isput pat
rnity, polymorphisms can
 us as a hlpful inti cation tool. Structur of Class I Molcu
ls Each of th
MHC gns cos for a protin prouct that appars on cll surfacs. All th pro
tins of a
particular class shar structural similaritis an ar foun on th sam typs
of clls. Class
I MHC molculs ar xprss on all nuclat clls, although thy iffr i
n th lvl of
xprssion. Thy ar highst on lymphocyts an low or untct on livr hpat
ocyts, nural

clls, muscl clls, an sprm. 6,10 This may xplain why HLA matching is not o
n in th cas of
livr transplants. Aitionally, HLA-C antigns ar xprss at a lowr lvl t
han HLA-A an
HLA-B antigns, so th lattr two ar th most important to match for transplant
ation. 10 Each
class I antign is a glycoprotin imr, ma up of two noncovalntly link pol
yppti chains.
Th
chain has a molcular wight of 45,000. A lightr chain associat with it,
call a 2
microglo
ulin, has a molcular wight of 12,000 an is nco
y a singl gn o
n chromosom 15
that is not polymorphic. 6 Th
chain is fol into thr omains, 1, 2, an 3, an
it is
insrt into th cll mm
ran via a transmm
ran sgmnt that is hyropho-
i
c. 10 Th thr
xtrnal omains consist of a
out 90 amino acis ach, th transmm
ran om
ain has a
out
25 hyropho
ic amino acis along with a short strtch of a
out 5 hyrophilic ami
no acis, an an
anchor of 30 amino acis (Fig. 35).
2 microglo
ulin os not pntrat th cll m
m
ran,
chain. X-ray crystallograp

ut it is ssntial for propr foling of th
hic stuis
inicat that th 1 an 2 omains ach form an alpha hlix an that ths Class II
DP rgion DQ
rgion DR rgion 2 2 2 2 3 1 1 1 1 C 4 B C 4 A C 2 A C B Class III Clas
Tlomr Cntromr FIGURE 34. Th major histocompati
ility complx. Location of
th class I,
II, an III gns on chromosom 6. Class I consists of loci A, B, an C, whil c
lass II has at
last thr loci, DR, DQ, an DP. 1814_Ch03_039-053.qx 7/10/09 3:46 PM Pag
44 CHAPTER 3
Natur of Antigns an th Major Histocompati
ility Complx 45 srv as th wall
s of a p
groov at th top of th mol- cul that functions as th ppti-
ining sit i
n antign
rcognition. 6,7 This
ining sit is a
l to hol pptis that ar
twn 8 a
n 10 amino acis
long. Most of th poly- morphism rsis in th 1 an 2 rgions, whil th 3 an 2 r
gions
ar similar to th constant rgions foun in immunoglo
ulin molculs. 6,10 Th 3
rgion racts
with CD8 on cytotoxic T clls. Anothr group of molculs call th nonclassica
l class I
antigns ar signat E, F, an G. This group of mol- culs, xcpt for G, ar
 not xprss
on cll surfacs an o not function in antign rcognition
ut may play othr r
ols in th
immun rspons. G antigns ar xprss on tropho
last clls uring th rst tri
mstr of
prgnancy an ar thought to hlp nsur tolranc for th ftus
y pro- tcting
placntal tissu
from th action of NK clls. 6 Structur of Class II Molculs Th occurrnc of
class II MHC
molculs is much mor rstrict than that of class I,
caus thy ar
foun primarily
on antign-prsnting clls, which inclu B lym- phocyts, monocyts, macrophag
s, an nritic
clls. Th major class II molculsDP, DQ, an DRconsist of two noncovalntly
ou

n
polyppti chains that ar
oth nco
y gns in th MHC complx. DR is
xprss at th
highst lvl, as it accounts for a
out on-half of all th class II molculs o
n a particular
cll. 6 Th DR gn is th most highly polymorphic, as 18 iffrnt allls ar
known at this
tim. 6 Both th
chain, with a molcular wight of 33,000, an th
chain, with a
molcular
wight of 27,000, ar anchor to th cll mm
ran. 11 Each has two omains, an
 it is th 1
an th
1 omains that com togthr to form th ppti-
ining sit,
similar to
th on foun on class I molculs 7,10 (s Fig. 35). Howvr,
oth n
s of th
ppti-
ining clft ar opn, an this allows for captur of longr pptis t
han is th cas
for class I molculs. At last thr othr class II gns hav
n scri
DM,
DN, an DO,
th so-call nonclassical class II gns. Proucts of ths gns play a rgula
tory rol in
antign procssing. 7 Th main rol of th class I an class II MHC molculs is
to
in pptis
within clls an transport thm to th plasma mm
ran, whr T clls can rcogn
i thm in th
phnommon known as antign prsntation. T clls can only s an rspon to anti
gns whn
thy ar com
in with MHC molculs. Whil on iniviual can xprss only a sm
all num
r of MHC
molculs, ach molcul can prs- nt a larg num
r of iffrnt antig
nic pptis to
T clls. 12 It is thought that th two main classs of ths molculs hav vol
v to al with
two typs of infctious agnts: thos that attack clls from th outsi (such a
s
ac- tria) an
thos that attack from th insi (viruss an othr intracllular pathogns).Cl
ass I molculs
mainly prsnt pptis that hav
n synthsi within th cll to CD8 (cytot
oxic) T clls,
whil class II molculs prsnt antign to CD4 (hlpr) T clls. Class II molc
uls mainly
in
xognous protinsthos takn into th cll from th out- si an gra. 13
,14 Class I
molculs ar thus th watchogs of viral, tumor, an crtain parasitic antig
ns that ar
synthsi within th cll, whil class II molculs stim- ulat CD4 T clls in
th cas of

actrial infctions or th prsnc of othr matrial that is nocytos
y th
 cll. 13,15 In
ithr cas, for a T-cll rspons to
 triggr, pptis must
 ava
ila
l in
aquat supply for MHC mol- culs to
in, thy must
 a
l to

oun ffc
tivly, 2
-microglo
ulin 1 1 2 2 1 3 2 Cytopl smic t il Tr nsmembr ne segment
Membr ne-proxim l dom ins Membr ne-dist l dom ins Cl ss I molecule Cl ss II mole
cule
Peptide-binding cleft S S S S S S S S S S S S FIGURE 35. Structure of cl ss I nd
II MHC
products. (From Goldsby, RA, Kindt, TJ, nd Osborne, BA. Immunology, ed. 4. WH F
reem n, New York,

2000, p. 178, with permission.) 1814_Ch03_039-053.qxd 7/10/09 3:46 PM P ge 45

46 SECTION 1
N ture of the Immune System nd they must be recognized by T-cell receptor. 16
Some viruses,
such s herpes simplex nd denovirus, h ve m n- ged to block the immune respon
se by interfering
with one or more processes involved in ntigen present tion. 8,17 These viruses
re ble to
m int in lifelong presence in the host (see Ch pter 22 for det ils). The diffe
rence in
functioning of the two molecules is tied to the mech nisms by which processed n
tigen is tr nsported to the surf ce. Both types of molecules, however, must be c p ble of pres
enting n
enormous rr y of differ- ent ntigenic peptides to T cells. The chemistr
y of the MHC
ntigens controls wh t sorts of peptides fit in the binding pockets. These two p
thw ys re
discussed here. Role of Cl ss I Molecules Both cl ss I nd cl ss II molecules r
e synthesized in
the rough endopl smic reticulum, nd for
time they rem in nchored in the endo
pl smic reticulum
membr ne. Cl ss I molecules, however, ctu lly bind peptides while still in the
endopl smic
reticulum. 7 In f ct, binding helps to st bilize the ssoci tion of the
ch
in of cl ss
I with the
2 microglobulin. 16 However, before binding with ntigen occurs, new
ly
synthesized
ch ins freely bind molecule c lled c lnexin. This 88-kd molecule i
s
membr ne-bound in the endopl smic reticulum, nd it keeps the
ch in in
p rti lly
folded st te while it
w its binding to
2 microglobulin. 13,18 When
2 micro
globulin
binds, c lnexin is rele sed, nd three other ch perone molecules c lreticulin, t
p sin, nd
ERp57 re ssoci ted with the complex nd help to st bilize it for peptide bindi
ng 17,18 (Fig.
36). Peptides th t ssoci te with the cl ss I molecules re pproxim tely eight t
o ten mino
cids in length nd re derived from p rti l digestion of proteins synthesized i
n the cytopl sm.
These intr cellul r peptides m y include vir l, Golgi complex Cl ss I MHC Cl ss
II MHC Golgi
complex Cl ss II Cl ss II Note:
Cl ss I = Cl ss II = Inv ri nt = ch in Protei
n Proteosome TAP
Rough endopl smic reticulum Ribosomes Ribosomes Inv ri nt ch in Cytosol Cytosol
Digestion Antigen
Digestion inv ri nt ch in FIGURE 36. Sep r te ntigen processing p thw ys for end
ogenous nd
exogenous ntigens. Cl ss I MHC molecules re m de in the rough endopl smic reti
culum (ER), where
sm ll peptides m de within the cell re tr nsported to the ER. Upon combining wi
th MHC cl ss I,
the peptide-MHC complex is tr nsported through the Golgi complex to the cell sur
f ce. The binding
site of MHC cl ss II molecules re rst occu- pied by n inv ri nt ch in (Ii). Thi
s is degr ded
nd exch nged for short exogenous peptides in n endosom l comp rtment. The exog
enous peptide-MHC

cl ss II complex is then tr nsported to the cell surf ce. 1814_Ch03_039-053.qxd

7/10/09 3:46 PM
P ge 46 CHAPTER 3 N ture of Antigens nd the M jor Histocomp tibility Complex 4
7 tumor, or even
b cteri l ntigens. 12 Such peptides m y be newly m de proteins th t f il to fol
d correctly nd
hence re defective. These re c lled defective ribosom l products (DRiPs). 12 T
wenty to 70
percent of ll proteins synthesized in cell m y f ll into this c tegory. 8,19
Digestion of
these defective or e rly proteins is c rried out by prote ses th t reside in l r
ge cylindric l
cytopl smic complexes c lled pro- te somes. 13 Prote somes re
p cket of enzym
es th t pl y
m jor role in ntigen present tion. 13 Peptides must be unfolded before en
tering the
cylindric l ch mber of the pro- teosome, nd then they re cle ved into the prop
er size for
delivery to cl ss I molecules. Once cle ved, the peptides must then be pumped in
to the lumen of
the endopl smic reticulum by speci lized tr nsporter proteins. 7,15 These two pr
oteins,
tr nsporters ssoci ted with ntigen process- ing (TAP1 nd TAP2), re responsib
le for the
denosine triphosph tedependent tr nsport, from the cytopl sm to the lumen of the
endopl smic
reticulum, of peptides suit- ble for binding to cl ss I molecules. 8,17,18 TAP1
nd TAP2 re
most efficient t tr nsporting peptides th t h ve 12 mino cids or less.
15,17 T p sin
brings the TAP tr nsporters into close proximity to the newly formed MHC molecul
es nd medi tes
inter ction with them so th t peptides c n be lo ded onto the cl ss I molecules.
13,15 Once the
ch in h s bound the peptide, the MHC I-peptide complex is r pidly tr nsported to
the cell surf ce
(see Fig. 36). 6 Of the thous nds of peptides th t m y be processed in this m nne
r, only sm ll
fr ction of them (1 percent or less) ctu lly induce T-cell response. 15 Bindi
ng is b sed on
inter- ction of only two or three mino cid residues with the cl ss I binding
groove. Different
cl ss I molecules will h ve slightly different binding f nities, nd it is these
sm ll differences th t determine to which p rticul r ntigens one individu l will respond
. It is estim ted
th t single cell m y express bout 10 5 copies of e ch cl ss I molecule, so m
ny different
peptides c n be c ptured nd expressed in this m nner. 10 As few s 10 to 100 id
entic l
ntigen-MHC I complexes c n induce
cytotoxic response. 15 In he lthy cells, mo
st of these MHC I
complexes cont in self-peptides th t re ignored by the T cells, while in dise s
ed cells,
peptides re derived from vir l proteins or proteins ssoci ted with c ncerous s
t tes. Displ y of
hundreds of cl ss I molecules complexed to nti- gen llows CD8 T cells to continu
ously check
cell surf ces for the presence of nonself- ntigen. If it recognizes n nti- gen
s being

foreign, the CD8 T cell produces cytokines th t c use lysis of the entire cell (F
ig. 37). Role
of Cl ss II Molecules Unlike cl ss I molecules, cl ss II molecules must be tr ns
- ported from
the endopl smic reticulum (ER) to n endosom l comp rtment before they c n
bind peptides. 7
Dendritic cells re the most potent ctiv tors of T cells, nd they re excellen
t t c pturing
nd digesting exogenous nti- gens such s b cteri . Cl ss II molecules in the e
ndopl smic
reticulum ssoci te with protein c lled the inv ri nt ch in (Ii), which preven
ts inter ction of
the binding site with ny endogenous peptides in the endopl smic reticulum. 7,13
The inv ri nt
ch in is 31-kd protein th t is m de in excess so th t enough is v il ble to b
ind with ll
cl ss II molecules shortly fter they re synthesized. Ii m y be responsible for
helping to bring
nd ch ins together in the ER lumen nd then moving them out through the Golgi c
omplex to the
endocytic vesicles, where digested ntigen is found. 16 Bec use the open structu
re of cl ss II
molecules would per- mit binding of segments of int ct proteins within the ER, I
i serves to
protect the binding site. 20 Once bound to the inv ri nt ch in, the cl ss II mol
ecule is
tr nsported to n endosom l comp rtment, where it encounters peptides deri
ved from
endocytosed, exogenous proteins. Antigen processing m y help to unfold molecules
nd uncover
function l sites th t re buried deep within the n tive protein structure. 3 The
inv ri nt ch in
is degr ded by prote se, le ving just
sm ll fr gment c lled cl ss II inv ri
nt ch in peptide
(CLIP) tt ched to the peptide-binding cleft. 14,21 CLIP is then exch nged for e
xogenous
peptides. Selective binding of peptides is f vored by the low pH of the endosom
l comp rtment. 16
HLA-DM molecules help to medi te the re ction by removing the CLIP fr gment. 6,10,21
Gener lly, peptides of pproxim tely 13 to 18 mino cid residues c n bind,
bec use the
groove is open on both ends, unlike cl ss I molecules, which h ve closed end.
10,14,22,23
Within
centr l core of 13 mino cids, 7 to 10 residues provide the m jor cont
ct points. 10
CD8 MHC I Cytokine rele se Rele se of endonucle ses nd lysosom l enzymes Lysis
T rget
cell
T rget cell T T rget cell C cell T C cell FIGURE 37. The CD8 T cell recogni
es
ntigen in soci tion with MHC cl ss I. If the ntigen is recognized s being fo
reign, cytokines
re rele sed, c using destruction of the t rget cell. 1814_Ch03_039-053.qxd 7/1
0/09 3:46 PM
P ge 47 48 SECTION 1 N ture of the Immune System Hydrogen bonding t kes pl ce l
ong the length of
the c ptured peptide, in contr st to cl ss I molecules, which only bond t th
e mino nd
c rboxy termin l ends. 23,24 There re lso sever l pockets in the cl s
s II proteins

th t e sily ccommod te mino cid side ch ins. This gives cl ss II proteins mor
e exibility in
the types of peptides th t c n be bound. 23,24 Once binding h s occurred, the cl
ss II
protein-peptide complex is st bilized nd is tr nsported to the cell surf ce (se
e Fig. 36). On
the cell surf ce, cl ss II molecules re responsible for forming trimolecul r
com- plex th t
occurs between ntigen, cl ss II molecule, nd n ppropri te T-cell receptor. I
f binding occurs
with T-cell receptor on CD4 T cell, the T helper cell recruits nd triggers
B-cell
response, resulting in ntibody form tion (Fig. 38). Clinic l Signi c nce of MHC Te
sting for MHC
ntigens h s typic lly been done, bec use both cl ss I nd cl ss II molecules c
n induce
response th t le ds to gr ft rejection. Testing methodology h s ch nged from ser
ologic l
principles to molecul r methods, which re much more ccur te. The role of the l
bor tory in
tr ns- pl nt tion is presented in Ch pter 17. MHC ntigens lso ppe r to
pl y role in
development of utoimmune dise ses. The link between MHC ntigens nd utoimmune
dise ses is
discussed more fully in Ch pter 14. However, the evidence th t both cl ss I nd
cl ss II molecules pl y m jor role in ntigen present tion h s more f r-re ching consequen
ces. They
essenti lly determine the types of peptides to which n individu l c n m
ount n immune
response. Although the MHC molecules typic lly h ve bro d binding c p city, sm
ll biochemic l
differences in these proteins re responsible for differences seen in the bilit
y to re ct to
speci c ntigen. 12 It is possible th t non- responders to
p rticul r v ccine su
ch s hep titis
B do not h ve the genetic c p city to respond. On the other h nd, presence of
p rticul r MHC
protein m y confer ddition l protection, s the ex mple of HLA B8
nd i
ncre sed
resist nce to HIV infection shows. 8 Therefore, it will be import nt to know n
individu ls MHC
type for numerous re sons. Much of the recent rese rch h s focused on the types
of peptides th t
c n be bound by p rticul r MHC mole- cules. 2325 Future developments m y inclu
de t iloring
v ccines to cert in groups of such molecules. As more is le rned bout ntigen p
rocessing,
v ccines cont ining cer- t in mino cid sequences th t serve s immunodomin nt
epitopes c n be
specific lly developed. This might void the risk ssoci ted with using live org
nisms.
Addition lly, if n individu l suffers from llergies, knowing persons MHC type
might lso
help predict the types of llergens to which they m y be llergic, bec use rese
rch in this re
is ttempting to group llergens ccording to mino cid structure. 25 It is lik
ely th t
knowledge of the MHC molecules will ffect m ny re s of p tient c re in the fut
ure. SUMMARY To

fully comprehend the specificity of ntigen ntibody combin tion, it is essenti l

to underst nd
the princip l ch r- cteristics of ntigens. Antigens re m cromolecules th t el
icit form tion of
immunoglobulins or sensitized cells in n immunocompetent host. Immunogen is t
erm th t is often
used synonymously with ntigen. The term immuno- gen emph sizes the f ct th t
host response is
triggered, while the term ntigen is sometimes used to denote
sub- st nce th t
does not elicit
host response but re cts with ntibody once it h s been formed. Although immun
ogenicity is
in uenced by f ctors such s ge, he lth, route of inocul tion, nd genetic c p ci
ty, there re
cert in speci c ch r cteristics th t re sh red by most immunogens: molecul r we
ight of t
le st 100,000, molec- ul r complexity, nd foreignness to the host. While
Immunogens
re
f irly l rge molecules, the immune response is keyed to only sm ll portions
of these
molecules, or epitopes, nd very sm ll differences in these epitopes c n be dete
cted by the
immune system. Adjuv nts re subst nces th t c n be mixed with ntigen to enh nc
e the immune
response. Most djuv nts work by CD4 CD 28 B7 Clon l exp nsion Cytokine producti
on
Prolifer tion Memory B cells Pl sm cells
B cell = MHC Cl ss II = Antigen = TCR
PC T
H cell T H cell T H cell FIGURE 38. CD4 T cells recognize exogenous ntigen on ph
go- cytic
ntigen presenting cells long with MHC cl ss II. T cells re stimul ted by cont
ct with ntigen,
nd clon l exp nsion t kes pl ce. These CD4 helper T cells secrete cytokines th t
c use n
ntigen ctiv ted B cell to prolifer te nd produce pl sm cells, which m ke nt
ibody.
1814_Ch03_039-053.qxd 7/10/09 3:46 PM P ge 48 CHAPTER 3 N ture of Antigens n
d the M jor
Histocomp tibility Complex 49 keeping the ntigen in the re nd by incre sing
the num- ber of
cells involved in the immune response. The genetic c p bility to mount n immune
response is
linked to group of molecules known s the MHC ntigens. The two m in cl sses o
f these
molecules, sometimes referred to s HLA ntigens, h ve evolved to de l with infe
ctious gents
th t tt ck cells from the outside (such s b cteri ) nd those th t tt ck from
the inside
(viruses nd other intr - cellul r p thogens). Cl ss I
nd cl ss II molec
ules bind
peptides within cells nd tr nsport them to the pl sm mem- br ne, where they c
n be recognized
by T cells. Cl ss I MHC molecules re found on ll nucle ted cells, nd these mo
lecules ssoci te
with foreign ntigens, such s vir l pro- teins, synthesized within
host cell.
Cl ss I
molecules re st bilized by the binding of ntigen, nd the complex is re d- ily
tr nsported to
the cellul r surf ce. Cl ss II molecules, on the other h nd, h ve more limited
distribution,

nd these ssoci te with foreign ntigens t ken into the cell from the outside.
Binding of
ntigen to the cl ss II molecules occurs in endosom l comp rtments, nd then thi
s complex is
moved to the outside of the cell. The binding sites on both types of molecules m
y limit the size
nd the n ture of the ntigen bound. Thus, these molecules pl y
key role in n
ti- gen
processing nd recognition. 1814_Ch03_039-053.qxd 7/10/09 3:46 PM P ge 49 50
SECTION 1 N ture
of the Immune System A 15-ye r-old boy needs to h ve
kidney tr nspl nt
due to the
effects of severe di betes. His f mily members consist of his f ther, mother, n
d two sisters.
All of them re willing to don te kidney so th t he c n come off di lysis. He
is lso on list
for c d ver kidney. His physi- ci n suggests th t the f mily be tested first f
or the best HLA
m tch. Questions . How m ny lleles would be sh red by mother nd son? F ther
nd son? b. Wh t
re the ch nces th t one of the sisters would be n ex ct m tch? c. Is there p
ossibility th t
c d ver kidney might be better m tch th n ny of the f mily members? CASE STUD
Y
1814_Ch03_039-053.qxd 7/10/09 3:46 PM P ge 50 CHAPTER 3 N ture of Antigens n
d the M jor
Histocomp tibility Complex 51 SPECIFICITY OF ANTIGENANTIBODY REACTIONS Principle
Typing serum is
n ntibody th t will re ct only with the speci c red blood cell ntigen g inst w
hich it is
directed. Agglutin tion indic tes the presence of th t p rticul r ntigen.
Re gents,
M teri ls, nd Equipment Anti-A typing serum Anti-B typing serum Group A, group
B, nd group O
re gent red blood cells Microscope slides Dispos ble stirrers Procedure 1. Divid
e e ch microscope
slide in h lf, using w x m rk- ing pencil. L bel the left side A nd the right s
ide B for
the two ntiser th t will be used. 2. Pl ce one drop of nti-A on the left side
of slide nd one
drop of nti-B on the right side. 3. Add one drop of re gent red blood cell susp
ension to e ch
side. 4. Mix e ch side thoroughly with sep r te dispos ble stirrer. 5. Rock sl
ide gently b ck
nd forth for 2 minutes. 6. Observe for gglutin tion. 7. Repe t this procedur
e for e ch
type of re gent red blood cell. Results 1. Report ny cells th t gglutin te
with nti-A serum
s type A cells nd ny cells th t gglutin te with nti-B serum s type B cells
. 2.
Agglutin tion with both types of ntiser indic tes th t both A nd B ntigens
re present, nd
this is type AB. 3. No gglutin tion indic tes cells h ve neither ntigen, nd t
hese belong to
group O. Interpret tion of Results Red blood cell ntigens consist of lipidsug
r complex th t
is inserted into the cells membr ne. The H ntigen serves s the building block f
or both A nd B
ntigens. As c n be seen in Figure 39, ll three ntigens differ only by the pres
- ence or

bsence of one sug r. If only H ntigen is present, then these cells re typed
s O cells.
Speci c ntibody is ble to detect the one sug r difference in e ch of the ntigen
s, nd n
gglutin tion re ction will occur only with the nti- gen g inst which the nti
body is directed.
EXERCISE FIGURE 39. Structure of H, A, nd B red cell ntigens. (From Cooling, L.
ABO, H, nd
Lewis blood groups nd structur lly rel ted ntigens. In Rob ck, J, Combs, MR, G
rossm n B,
Hillyer, C (eds.): Technic l M nu l, ed. 16. Americ n Associ tion of Blood B nks
, Bethesd , MD,
2008, pp. 36185 with permission.) 1814_Ch03_039-053.qxd 7/10/09 3:46 PM P ge 5
1 52 SECTION 1
N ture of the Immune System 1. All of the following re ch r cteristic of
good
immuno- gen
except . intern l complexity. b. l rge molecul r weight. c. the presence of num
erous epitopes.
d. found on host cells. 2. Which of the following best describes
h pten? . No
t ble to re ct
with ntibody b. Antigenic only when coupled to c rrier c. H s multiple determ
in nt sites d. A
l rge chemic lly complex molecule 3. Which would be the best immunogen? . Prote
in with
molecul r weight of 200,000 b. Nylon c. Polys cch ride with molecul r weight o
f 250,000 d.
Protein with
molecul r weight of 175,000 4. All of the following describe n e
pitope except .
s me s n ntigenic determin nt site. b. re of n immunogen recognized by T c
ells. c. consists
of sequenti l mino cids only. d. key portion of the immunogen. 5. Adjuvents c
t by which of the
following methods? . Complex to ntigen to incre se its size b. Prevent r pid e
sc pe from the
tissues c. Incre se processing of ntigen d. All of the bove 6. A heterophile
ntigen is one
th t . is self- ntigen. b. exists in unrel ted pl nts or nim ls. c. h s been
used previously
to stimul te ntibody response. d. is from the s me species but is different fro
m the host. 7.
Which of the following is true of MHC (HLA) cl ss II ntigens? . They re found
on ll nucle ted
cells. b. They re found on B cells nd m croph ges. c. They ll origin te t on
e locus. d. They
re coded for on chromosome 9. 8. MHC molecules
re ssoci ted with which
of the
following? . Gr ft rejection b. Autoimmune dise ses c. Determining to which nt
igens n
individu l responds d. All of the bove 9. Which of the following best describes
the role of TAP?
. They bind to cl ss II molecules to help block the ntigen-binding site. b. Th
ey bind to cl ss
I proteins in proteosomes. c. They tr nsport peptides into the lumen of the endo
pl smic
reticulum. d. They help cle ve peptides for tr nsport to endosomes. 10. An indiv
idu l is
recovering from b cteri l infection nd tests positive for ntibodies to
pro
tein norm lly
found in the cytopl sm of this b cterium. Which of the following st tements is t
rue of this

situ tion? . Cl ss I molecules h ve presented b cteri l ntigen to CD8 T cells.

b. Cl ss I
molecules h ve presented b cteri l ntigen to CD4 T cells. c. Cl ss II molecules
h ve presented
b cteri l ntigen to CD4 T cells. d. B cells h ve recognized b cteri l ntigen wi
thout help from
T cells. REVIEW QUESTIONS 1814_Ch03_039-053.qxd 7/10/09 3:46 PM P ge 52 CHAPT
ER 3 N ture of
Antigens nd the M jor Histocomp tibility Complex 53 References 1. M k, TW, nd
S unders, ME. The
Immune Response: B sic nd Clinic l Principles. Elsevier, Burlington, MA,
2006, pp.
121146. 2. Kindt, TJ, Goldsby, RA, nd Osborne, BA. Kuby Immunology, ed. 6. WH Fr
eem n nd Co.,
New York, 2007, pp. 76110. 3. Berzofsky, JA, nd Berkower, IJ. Immunogenicity nd
ntigen
structure. In P ul, WE (ed): Fund ment l Immunology, ed. 4. Lippincott Wil
li ms & Wilkins,
Phil delphi , 1999, pp. 651698. 4. Be dling, WV,
nd Cooling, L. Immunohem t
ology. In
Henrys Clinic l Di gnosis nd M n gement by L bor tory Methods, ed. 21. S unders,
Phil delphi ,
2007, pp. 617668. 5. M k, TW, nd S unders, ME. The Immune Response: B sic nd C
linic l
Principles. Elsevier, Burlington, MA, 2006, pp. 695749. 6. M ssey, HD, nd
McPherson,
RA. Hum n Leukocyte Antigen: The M jor Histocomp tibility Complex of M n. In H
enrys Clinic l
Di gnosis nd M n gement by L bor tory Methods, ed. 21. S unders, Phil delphi ,
2007, pp.
876893. 7. M k, TW, nd S unders, ME. The Immune Response: B sic nd Clinic l P
rinciples.
Elsevier, Burlington, MA, 2006, pp. 247277. 8. Groothuis, TAM, Griekspoor, AC,
Neijssen, JJ,
Herberts, CA, et l. MHC cl ss I lleles nd their explor tion of the ntigen- p
rocessing
m chinery. Immunol Rev. 207:6076, 2005. 9. HLA Inform tics Group, The Anthony Nol
n Trust. The
HLA Sequence D t b se. Accessed July 23, 2007,
t
http://www. nthonynol n.org.uk/HIG/lists/cl ss1list.html. 10. Kindt, TJ, Goldsby
, RA, nd
Osborne, BA. Kuby Immunology, ed. 6. WH Freem n nd Co., New York, 2007, pp. 1892
22. 11.
Pieters, J. MHC cl ss II-restricted ntigen processing nd present tion. Adv Imm
unol 75:159208,
2000. 12. Sh stri, N, C rdin ud, S, Schw b, SR, Serwold, T, et l. All the pepti
des th t t: The
beginning, the middle, nd the end of the MHC cl ss I ntigen-processing p thw y
. Immunol Rev
207:3141, 2005. 13. Loureiro, J, nd Ploegh, HL. Antigen present tion nd the
ubiquitin-proteosome system in host-p thogen inter ctions. Adv Immunol 92:226306,
2006. 14. Li,
P, Gregg, JL, W ng, N, Zhou, D, et l. Comp rtment liz tion of cl ss II ntigen
present tion:
Contribution of cytopl smic nd endosom l processing. Immunol Rev 207:206217, 200
5. 15. Koch, J,
nd T mpe, R. The m cromolecul r peptide-lo ding complex in MHC cl ss I-dependen
t ntigen
present tion. Cell Mol Life Sci 63:653662, 2006. 16. Germ in, RN. Antigen process
ing nd

present tion. In P ul, WE (ed): Fund ment l Immunology, ed. 4. Lippincott

Willi ms &
Wilkins, Phil delphi , 1999, pp 287336. 17. Scholz, C, nd T mpe, R. The intr cel
lul r ntigen
tr nsport m chinery TAP in d ptive immunity nd virus esc pe mech- nisms. J Bi
oenerg nd
Biomembr 37:509515, 2005. 18. Cresswell, P, Ackerm n, AL, Giodini, A, Pe per, DR,
et l.
Mech nisms of MHC cl ss-I restricted ntigen processing nd cross-present tion.
Immunol Rev
207:147157, 2005. 19. Yewdell, JW. The seven dirty little secrets of m jor histoc
om- p tibility
complex cl ss I ntigen processing. Immunol Rev 207:818, 2005. 20. M k, TW, nd S
unders, ME.
Antigen processing nd pres- ent tion. In The Immune Response: B sic nd
Clinic l
Principles. Elsevier, Burlington, MA, 2006, pp. 279309. 21. Ghosh, P, Am y , M,
nd Mellins, E,
et l. The structure of n intermedi te in cl ss II MHC m tur tion: CLIP bound t
o HLA-DR3. N ture
378:457462, 1995. 22. Brown, JH, J rdetzky, TS, nd Gorg , JC, et l. Th
reedimension l structure of the hum n cl ss II histocomp tibility ntigen HLA-DR1.
N ture 364:33,
1993. 23. J rdetzky, TS, Brown, JH, nd Gorg , JC, et l. Cryst llogr phic n ly
sis of endogenous
peptides ssoci ted with HLA-DR1 suggests
common, polyproline II-like con
form tion for
bound peptides. Proc N tl Ac d Sci USA 93:734738, 1996. 24. Stern, LJ, Brown, JH,
nd J rdetzky,
TS, et l. Cryst l structure of the hum n cl ss II MHC protein HLA-DR1 complexed
with n in uenz
virus peptide. N ture 368:21521, 1994. 1814_Ch03_039-053.qxd 7/10/09 3:46 PM P
ge 53 4
Antibody Structure nd Function LEARNING OBJECTIVES After nishing this ch pter, t
he re der will
be ble to: 1. Describe the structure of typic l immunoglobulin. 2. Ch r cteri
ze the ve
immunoglobulin types found in hum ns. 3. Differenti te between light nd he vy c
h ins of
immunoglobulins. 4. Describe experiment l evidence for the structure of IgG. 5.
Rel te
immunoglobulin structure to function. 6. Describe how recent knowledge bout imm
unoglobulin genes
supports the clon l selection theory. 7. Discuss the process of monoclon l ntib
ody production.
8. Rel te the in uence of monoclon l ntibodies to current l bor tory testing pr c
tices. KEY
TERMS Allelic exclusion Allotype Bence-Jones proteins Cl ss switching Clon l sel
ection theory
Const nt region F( b) 2 F b fr gment FC fr gment He vy (H) ch in Hinge region Hy
bridom Idiotype
Immunoglobulin Isotype Joining (J) ch in K pp ( ) ch in L mbd ( ) ch in Light (L)
ch in
Monoclon l ntibody Secretory component (SC) V ri ble region 54 1814_Ch04_054-07
1.qxd 7/10/09
3:31 PM P ge 54 CHAPTER 4 Antibody Structure nd Function 55 When B lymphocytes
re stimul ted
by ntigen nd undergo differenti tion, the end product is ntibody or immunoglo
b- ulin.

Immunoglobulins re glycoproteins found in the serum portion of the blood. T

hey re composed
of 82 to 96 percent polypeptide nd 2 to 14 percent c rbohydr te. 1 When subject
ed to
electrophoresis t pH 8.6, immunoglob- ulins ppe r prim rily in the g mm ( ) b n
d (Fig. 41).
E ch of the ve m jor cl sses h s slightly different electrophoretic properties. T
hese cl sses
re design ted IgG, IgM, IgA, IgD, nd IgE (Ig is n bbrevi tion for immunoglobu
lin).
Immunoglobulins re considered to be the humor l br nch of the immune resp
onse. They pl y
n essenti l role in ntigen recognition nd in biologic l ctivities rel ted to
the immune
response such s opsoniz tion nd complement ctiv tion. Although e ch cl ss h s
unique
properties, ll immunoglobulin molecules sh re m ny common fe tures. This ch pte
r presents the
n ture of this gener lized struc- ture nd discusses the ch r cteristics of e ch
immunoglobulin
type. Speci c functions for e ch of the cl sses re ex mined in rel tion to struct
ur l
differences. TETRAPEPTIDE STRUCTURE OF IMMUNOGLOBULINS All immunoglobulin molecu
les re m de up
of b sic four- ch in polypeptide unit th t consists of two l rge ch ins c lled
he vy or H
ch ins nd two sm ller ch ins c lled light or L ch ins. These ch ins re held to
gether by
noncov lent forces nd disul de interch in bridges. The b sic structure of immunog
lobulins w s
elucid ted in the 1950s nd 1960s by the efforts of two men: Ger ld Edelm n, wor
king t the
Rockefeller Institute in the United St tes, nd Rodney Porter t Oxford U
niversity in
Engl nd. For their contri- butions, these men sh red the Nobel Prize in physiolo
gy nd medicine
in 1972. They chose to work with immunoglobulin G. Edelm ns work centered o
n using the
n lytic ultr cen- trifuge to sep r te out immunoglobulins on the b sis of molec
ul r weight. 2 He
found th t int ct IgG molecules h d sediment tion coef cient of 7 S (the Svedber
g unit [S]
indi- c tes the sediment tion r te in n n lytic l ultr centrifuge). CHAPTER OU
TLINE
TETRAPEPTIDE STRUCTURE OF IMMUNOGLOBULINS Cle v ge with P p in Pepsin Digestion
THE NATURE OF
LIGHT CHAINS HEAVY CHAIN SEQUENCING HINGE REGION THREE-DIMENSIONAL STRUCTURE OF
ANTIBODIES IgG
IgM IgA IgD IgE ANTIBODY DIVERSITY Ehrlichs Side-Ch in Theory Clon l Selection GE
NES CODING FOR
IMMUNOGLOBULINS Re rr ngement of He vy Ch in Genes Light Ch in Re rr ngement MON
OCLONAL ANTIBODY
Hybridom Production Selection of Speci c Antibody-Producing Clones Clinic l Appli
c tions SUMMARY
CASE STUDIES EXERCISE: SERUM PROTEIN ELECTROPHORESIS REVIEW QUESTIONS REFERENCES
FIGURE 41.
Serum electrophoresis. (From Widm nn, FK. An Introduction to Clinic l Immunology
. FA D vis,
Phil delphi , 1989, with permission.) 1814_Ch04_054-071.qxd 7/10/09 3:31 PM P
ge 55 56 SECTION

1 N ture of the Immune System L rger molecules will tr vel f rther nd thus h ve
l rger
sediment tion coef cient. On obt ining puri ed prep r - tion of IgG, Edelm n used
7 M ure to
unfold the molecule. Once unfolded, the exposed sulfhydryl bonds could be
cle ved by
reducing gent such s merc ptoeth nol. After such tre tment, the m teri l w s s
ubjected g in to
ultr cen- trifug tion, nd two sep r te fr ctions, one t 3.5 S nd one t 2.2 S
, were obt ined.
The 3.5 S fr ction, with molecul r weight of pproxi- m tely 50,000, w s de
sign ted the H
ch in; the 2.2 S fr ction, with molecul r weight of 22,000, w s n med the L
ch in. 3 These
two pieces occurred in equ l mounts, indi- c ting th t the formul for IgG h d
to be H 2 L 2 .
This is the gener lized formul for ll immunoglobulins. Cle v ge with P p in Po
rters work w s
b sed on the use of the proteolytic enzyme p p in, which w s used to cle
ve IgG into three
pieces of bout equ l size, e ch h ving
sediment tion coef- ficient of 3.5 S
nd representing
molecul r weight of pproxim tely 45,000 to 50,000 d. 3,4 C rboxymethyl cellu- l
ose ion exch nge
chrom togr phy sep r ted this m teri l into two types of fr gments, one of which
spont neously
crys- t llized t 4C. This fr gment, known s the FC fr gment (for fr gment crys
t lliz ble),
h d no ntigen-binding bility nd is now known to represent the c rboxy-term
in l h lves of
two H ch ins th t re held together by SS bonding. 3 The FC fr gment is import
nt in
effector functions of immunoglobulin molecules, which include opsoniz tion nd
complement
x tion. The rem ining two identic l fr gments were found to h ve ntigen-binding
c p city nd
were n med F b fr g- ments (fr gment ntigen-binding). Bec use precipit tion wou
ld not occur if
F b fr gments were llowed to re ct with ntigen, it w s guessed th t e ch fr gm
ent represented
one ntigen-binding site nd th t two such fr gments were pres- ent in n int ct
ntibody
molecule; such
molecule would be ble to form cross-linked complex with nti
gen, nd the
complex would precipit te. E ch F b fr gment thus con- sists of one L ch in
nd one-h lf
of n H ch in, held together by disul de bonding. 4 Pepsin Digestion Alfred Ni
sonoff used
pepsin to obt in ddition l evidence for the structure of immunoglobulins. 3
This
proteolytic enzyme w s found to cle ve IgG t the c rboxy-termin l side of the i
nterch in
disul de bonds, yielding one single fr g- ment with
molecul r weight of 10
0,000 d
nd
ll the ntigen-binding bility, known s F( b) 2 . An ddition l fr g- ment c
lled FC w s
simil r to FC except th t it disintegr ted into sever l sm ller pieces. Thus,
b sic picture of
the four- ch in unit of the immunoglobulin molecule w s obt ined, which indic te
d th t e ch L

ch in w s bonded to n H ch in by me ns of n SS bond, nd the H ch ins were join

ed to e ch
other by one or more SS bonds (Fig. 42). The ex ct number of disulfide bonds diffe
rs mong
ntibody cl sses nd subcl sses. THE NATURE OF LIGHT CHAINS The dif culty in obt i
ning
signi c nt mount of
speci c immunoglobulin for mino cid n lysis w s overcome b
y the
discovery th t Bence-Jones proteins, found in the urine of p tients with mul
tiple myelom ,
were in f ct L ch ins th t were being secreted by the m lign nt pl sm cells.
3 Bence-Jones
proteins h d been discovered in 1845 by Dr. Henry Bence-Jones, who noted the pec
uli r beh vior of
these proteins: When he ted to 60C, they precipit te from urine, but on further h
e ting to 80C,
they redissolve. These ch r cteristics m de it possible to isol te the L ch ins
nd obt in the
mino cid sequence. An lysis of sever l Bence-Jones proteins reve led th t ther
e were two m in
types of L ch ins, design ted k pp ( ) nd l mbd ( ). E ch cont ined between 200
nd 220 mino
cids, nd from position number 111 on (the mino terminus is position number 1)
, it w s
discovered th t e ch type h d essenti lly the s me sequence. This region w s c l
led the const nt
region, nd the mino-termin l end w s c lled the v ri ble region. Thus, ll
L c
h ins h ve n
lmost identic l c rboxy-termin l end, nd the s me is true of
ch ins. The diffe
rence between
the
nd
ch ins lies in the mino cid substitutions t few loc tions long the
ch in. There
re no function l differences between the two types. Both
nd
L ch ins re found
in ll ve
cl sses FIGURE 42. Gener lized structure of n immunoglobulin molecule. The b sic
structure of
n immunoglobulin is tetr peptide, consist- ing of two H nd two L ch ins link
ed by disul de
bonds. Intr ch in disul de bonds cre te ooded regions or dom ins. The mino-termin
l end of e ch
ch in is v ri ble region, while the c rboxy-termin l end is one or more const
nt regions.
Pepsin digestion yields n F( b) 2 fr gment, with ll the ntibody ctivity, nd
n FC 1
fr gment. P p in digestion yields two F( b) fr gments nd n FC portion. 1814_Ch
04_054-071.qxd
7/10/09 3:31 PM P ge 56 CHAPTER 4 Antibody Structure nd Function 57 of immuno
globulins, but
only one type is present in given molecule. HEAVY CHAIN SEQUENCING H ch in seq
uencing
demonstr ted the presence of dom ins simil r to those in the L ch insth t is, v r
i ble nd const nt regions. The first pproxim tely 110 mino cids t the mino-termin l end
constitute the
v ri ble dom in, nd the rem ining mino cids c n typic lly be divided up into
three or more
const nt regions with very simil r sequences, design ted C H 1, C H 2, nd C H 3
. Const nt
regions of the H ch in re unique to e ch cl ss nd give e ch immunoglob- ulin t
ype its n me.

Hence, IgG h s n
H ch in, IgM
ch in, IgA n
ch in, IgD
ch in, nd IgE n
in.
E ch of these represents n isotype,
unique mino cid sequence th t is common
to ll
immunoglobulin molecules of given cl ss in given species. Minor v ri tions o
f these sequences
th t re present in some individu ls but not others re known s llotypes (Fig.
43). Allotypes
occur in the four IgG subcl sses, in one IgA subcl ss, nd in the k pp light ch
in. 3 These
genetic m rkers re found in the con- st nt region nd re inherited in simple M
endeli n f shion.
Some of the best-known ex mples of llotypes re v ri tions of the
ch in known
s G1m3 nd
G1m17. The v ri ble portions of e ch ch in re unique to
specific ntibody mol
ecule, nd they
constitute wh t is known s the idiotype of the molecule. The mino- term
in l ends of
both L nd H ch ins cont in these regions, which re essenti l to the
form tion of
the ntigen-binding site. Together they serve s the ntigen- recognition unit.
HINGE REGION The
segment of H ch in loc ted between the C H 1 nd C H 2 regions is known s the h
inge region. It
h s high content of proline nd hydrophobic residues; the high proline con- te
nt llows for
exibility. 3,5 This bility to bend lets the two ntigen-binding sites oper te in
dependently.
The exibil- ity lso ssists in effector functions such s initi tion of the comp
lement c sc de
(see Ch pter 6 for det ils). G mm , delt , nd lph ch ins ll h ve hinge reg
ion, but mu nd
epsilon ch ins do not. However, the C H 2 dom ins of these l tter two ch ins re
p ired in such
w y s to confer ex- ibility to the F b rms. 1 In ddition to the four polypepti
de ch ins, ll
types of immunoglobulins cont in c rbohydr te portion, which is loc lized betw
een the C H 2
dom ins of the two H ch ins. Functions of the c rbohydr te include (1) incre sin
g the solubility
of immunoglobulin, (2) providing protection g inst degr d tion, nd (3) enh
ncing function l
ctivity of the FC dom ins. This l tter function m y be the most import nt, bec
use recognition
by FC receptors correl tes with the presence of the c rbohydr te moiety. 5 THREE
-DIMENSIONAL
STRUCTURE OF ANTIBODIES The b sic four-ch in structure of ll immunoglobulin mol
- ecules does not
ctu lly exist s str ight
sh pe, but in f ct it is folded into comp ct globul
r subunits,
b sed on the form tion of b lloon-sh ped loops
t e ch of the dom ins. 6
Intr ch in
disulfide bonds st bilize these glob- ul r regions. Within e ch of these regions
or dom ins, the
polypeptide ch in is folded b ck nd forth on itself to form wh t is c lled
-pl
e ted sheet.
The folded dom ins of the H ch ins line up with those of the L ch ins to produce
cylindric l
structure c lled n immunoglobulin fold or b rrel (Fig. 44). 1,3 Antigen is c
ptured within

ch

the b rrel by binding to sm ll number of mino cids t str tegic loc tions on
e ch ch in known
s hyperv ri ble regions. Three sm ll hyperv ri ble regions consisting of pprox
- im tely 30
mino cid residues
re found within the Isotype (s me he vy ch in for e c
h cl ss) Allotype
(v ri tions in const nt regions) Idiotype (v ri tions in v ri ble regions) A B
C H L L H S
S S S S S S S S S S S S S S S FIGURE 43. Antibody v ri tions. (A) Isotypethe H
ch in th t is
unique to e ch immunoglobulin cl ss. (B) Allotypegenetic v ri tions in the const
nt regions. (C)
Idiotypev ri tions in v ri ble regions th t give individu l ntibody molecules sp
eci city.
1814_Ch04_054-071.qxd 7/10/09 3:31 PM P ge 57 58 SECTION 1 N ture of the Immu
ne System
v ri ble regions of both H nd L ch ins. E ch of these regions, c lled
complement rity-determining regions (CDRs), is between 9 nd 12 residues long. 3
They occur s
loops in the folds of the v ri ble regions of both L nd H ch ins, nd the ntig
en-binding site
is ctu lly determined by the pposition of the six hyperv ri ble loops, three f
rom e ch ch in
(see Fig. 44). Antigen binds in the middle of the CDRs, with t le st four of the
CDRs involved
in the bind- ing. 1,3,6,7 Thus, sm ll number of mino cids c n cre te n imme
nse diversity of
ntigen-binding sites. Properties of individu l ntibody cl sses re considered
in the following
sections. IgG IgG is the predomin nt immunoglobulin in hum ns, com- prising ppr
oxim tely 75 to
80 percent of the tot l serum immunoglobulins. As seen in T ble 41, IgG h s the l
ongest
h lf-life of ny immunoglobulin cl ss, pproxim tely 23 to 25 d ys, which m y he
lp to ccount for
its predomin nce in serum. There re four m jor subcl sses, with the following d
istribution:
IgG1, 67 percent; IgG2, 22 percent; IgG3, 7 percent; nd IgG4, 4 percent. 1,3 Th
ese subcl sses
differ m inly in the number nd position of the disul de bridges between the
ch in
s, s seen in
Figure 45. V ri bility in the hinge region ffects the bility to re ch for ntig
en nd the
bility to initi te import nt biologic l functions such s complement ctiv tion
. 8 IgG3 h s the
l rgest hinge region nd the l rgest number of interch in disul de bonds; there- f
ore, it is the
most ef cient t binding complement, followed by IgG1. 1,3 IgG2 nd IgG4 h ve shor
ter hinge
segments, which tend to m ke them poor medi tors of complement ctiv tion. 5 M j
or functions of
IgG include the following: (1) pro- viding immunity for the newborn bec use IgG
c n cross the
pl cent ; (2) fixing complement; (3) co ting ntigen for enh nced ph gocytosis (
opsoniz tion);
(4) neutr lizing toxins nd viruses; nd (5) p rticip ting in gglutin tion nd
precipit tion
re ctions. All subcl sses of IgG ppe r to be ble to cross the pl cent , lthou
gh IgG2 is the
le st efficient. 3 M croph ges, monocytes, nd neutrophils h ve recep- tors on t

heir surf ces

th t re speci c for the FC region of IgG. This enh nces cont ct between ntigen
nd ph go- cytic
cells nd gener lly incre ses the efficiency of ph gocytosis. IgG1 nd Ig
G3 re
p rticul rly good t ini- ti ting ph gocytosis, bec use they bind most strongly
to FC receptors.
1,9 IgG h s high diffusion coef cient th t llows it to enter extr v scul r sp c
es more re dily
th n other immunoglob- ulin types. In f ct, it is distributed lmost equ lly bet
ween the
intr v scul r nd extr v scul r sp ces. 1 Thus, it pl ys
m jor role in neutr l
izing toxins nd
viruses. Agglutin tion nd precipit tion re ctions t ke pl ce in vitro, lthough
it is not known
how signi c nt role these pl y in vivo. IgG is better t precipit tion re ctions
th n t
gglutin tion, bec use precipit tion involves sm ll soluble p rticles, which re
more e sily
brought together by the rel tively sm ll IgG molecule. Agglutin tion is the clum
p- ing together
of l rger p rticles such s red blood cells, nd being
l rger molecule, IgM is
much more
ef cient t this th n IgG. CDR1 CDR3 CDR2 N S S S S C V ri ble dom in Const nt dom
in FIGURE 44.
Three-dimension l structure of n L ch in. In this ribbon di gr m tr cing the po
lypeptide
b ckbone, B str nds re shown s wide ribbons, other regions s n rrow strings.
E ch of the two
globul r dom ins consists of b rrel-sh ped ssembly of seven to nine ntip r l
lel B str nds.
The three hyperv ri ble regions (CDR1, CDR2, nd CDR3) re exible loops th t proj
ect outw rd
from the mino-termin l end of the V L dom in. (From P rslow, TG, et l. Medic l
Immunology, ed.
10. McGr w-Hill/Appleton & L nge, 2001, with permission.) 1814_Ch04_054-071.qxd
7/10/09 3:31 PM
P ge 58 CHAPTER 4 Antibody Structure nd Function 59 IgM IgM is known s m cr
oglobulin,
bec use it h s sedimen- t tion r te of 19 S, which represents molecul r weig
ht of
pproxim tely 970,000. 1 As seen in T ble 41, the h lf-life of IgM is bout 10 d
ysmuch shorter
th n th t of IgG. It ccounts for between 5 nd 10 percent of
ll serum
immunoglobulins.
If IgM is tre ted with merc ptoeth nol, it dissoci tes into ve 7 S units, e ch h
ving
molecul r weight of 190,000 nd four-ch in structure th t resembles IgG. The m
olecul r weight
of the H or
ch in is pproxim tely 70,000. It con- sists of bout 576
mino
cids nd
includes one more const nt dom in th n is found on the
ch in. The pen- t mer f
orm is found in
secretions, while the monomer form occurs on the surf ce of B cells. 5,10 The fi
ve monomeric
units re held together by J or joining ch in, which is
glycoprotein with se
ver l cysteine
residues. These serve s link ge points for disul de bonds between two dj cent mo
nomers. Link ge
occurs t the c r- boxy-termin l end of two of the
ch ins, nd it ppe rs th t t

he J ch in m y
initi te polymeriz tion by st bilizing FC sulfhydryl groups so th t cross-li
nking c n
occur. 3 The molecul r weight of the J ch in is pproxim tely 15,000. One J ch i
n is present per
pent mer. IgM thus con gured ssumes
st rlike sh pe (Fig. 46) with 10 function l
binding
sites; only bout ve of these re used unless the ntigen is extremely sm ll. 1 T
he high v lency
of IgM ntibodies contr venes the f ct th t they tend to h ve low f nity for n
tigen. Bec use
of its l rge size, IgM is found m inly in the intr v scul r pool nd not in othe
r body fluids or
tissues. It c nnot cross the pl cent . IgM is known s the prim ry response nti
body, bec use it
is the first to ppe r fter ntigenic stimul tion, nd it is the first to ppe
r in the m turing
inf nt. It is synthesized only s long s ntigen rem ins present, bec use there
re no memory
cells for IgM. Figure 47 depicts the difference between the pri- m ry response,
which is
predomin ntly IgM, nd the second ry response, which is m inly IgG. The prim
ry response is
ch r cterized by long l g ph se, while the second ry response h s shortened
l g period nd
much more r pid incre se in ntibody titer. T ble 4-1. Properties of Immunoglobu
lins IgG IgM
IgA IgD IgE Molecul r weight 150,000 900,000 160,000 180,000 190,000 Sediment
tion
coef cient 7 S 19 S 7 S 7 S 8 S H ch in H chain su
classs 1, 2, 3
4 None 1, 2 None None H ch in molecul r weight 50,00060,000 70,000 55,00060,000 62
,000
70,00075,000 Const nt dom ins (H ch in) 3 4 3 3 4 Percent of tot l immunoglobulin
7075 10 1015
1 0.002 Serum concentr tion (mg/dL) 8001600 120150 70350 13 0.005 Serum h lf-life (d
ys) 23
6 5 13 23 C rbohydr te content (weight percent) 23 12 711 914 12 Electrophoretic migr
tion
21 112 22 1 1 Complement xation Yes Yes No No No Crosses placenta Yes No
No No IG3 IG1 IG2 IG4 FIGURE 45. IG su
classes. There are four su
classes of
IG: IG1,
IG2, IG3, and IG4. These differ in the num
er and linkaes of the disul de
ond
s. (From
Bryant, NJ. La
oratory Immunoloy and Seroloy, ed. 3. WB Saunders, Philadelphia
, 1992, p. 29,
with permission.) 1814_Ch04_054-071.qxd 7/10/09 3:31 PM Pae 59 60 SECTION 1
Nature of the
Immune System The functions of IM include (1) complement xation, (2) alutinati
on, (3)
opsonization, and (4) toxin neutraliza- tion. IM is the most efficient of all i
mmunolo
ulins at
trierin the classical complement pathway (see Chapter 6),
ecause a sinle mo
lecule can
initiate the reaction as a result of its multiple
indin sites. This pro
a
ly r
epre- sents the
most important function of IM. The larer num
er of
indin sites also ma
kes IM more
efficient at alutination reactions, especially with multivalent anti- ens. T
hus, IM forms
a potent defense aainst many
acterial diseases. Because IM has a J chain,

it can ocasionally acquire a secretory component like IA does, and this allows it to trav
erse epithelial
cells and patrol mucous mem
ranes. 1 IM also serves as a surface receptor for a
ntien. In the
cytoplasm of the pre-B cell,
chains first appear. When they associate with the e
arly surroate
L chains, a sinal is sent to exclude rearranement of the other H chain locus a
nd to
ein
rearranement of the enes controllin L chain synthesis. 5 Later, as L chains a
re synthesized,
IM monomers are formed and
ecome inserted into the plasma mem
rane. The presen
ce of mem
rane
IM classi es lymphocytes as mature B cells. (See Chapter 2 for a complete discuss
ion of B-cell
development.) J chain Bindin site for C1q Antien
indin sites Heavy chain () L
iht chain (
r ) A B S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S
S S S S S S S
S S S S S S FIGURE 46. Structure f immungbuin M. (A) Pentameric structure f
IgM, which is
in ed by a J chain. (B) Spacia cn guratin f IgM. Mnmers can extend in diffe
rent
directins. (Frm Bryant, NJ. Labratry Immungy and Sergy, ed. 3. WB Saun
ders,
Phiadephia, 1992, p. 31, with permissin.) Primary respnse Secndary respn
se IgG IgM IgG
IgM Lag phase First expsure t immungen Subsequent cntact with immungen S e
r u m
a n t i b  d y
Time FIGURE 47. A cmparisn f the primary and secndary r
 e v e  ag
espnse t
immungen. The primary respnse is characterized by a ng ag phase, sw expn
entia increase
in antibdy, and shrt-ived respnse. The secndary r anamnestic respnse has
a shrtened ag
perid, antibdy rise is much mre rapid, and serum eves remain higher fr n
ger perids. This
is caused by the arge number f antigen-speci c memry T and B ces generated du
ring the
primary respnse. 1814_Ch04_054-071.qxd 7/10/09 3:31 PM Page 60 CHAPTER 4 Ant
ibdy Structure
and Functin 61 IgA In the serum, IgA represents 10 t 15 percent f a circua
t- ing
immungbuin, and it appears as a mnmer with a mecuar weight f apprxim
atey 160,000. It
has a sedi- mentatin cef cient f 7 S and migrates between the
and
regins n
eectrphresis. The H chain, caed the
chain, has a mecuar weight betw
een 55,000 and
60,000 and cnsists f abut 472 amin acids. There are tw sub- casses, design
ated IgA1 and
IgA2. They differ in cntent by 22 amin acids, 13 f which are cated in the h
inge regin and
are deeted in IgA2. 11 The ac f this regin appears t ma e IgA2 mre resist
ant t sme
bacteria pr- teinases that are abe t ceave IgA1. 11 Hence IgA2 is the pred
minant frm in
secretins at mucsa surfaces, whie IgA1 is mainy fund in serum. IgA2, is f

und as a dimer
ang the respiratry, urgen- ita, and intestina mucsa, and it as appears
in mi , saiva,
tears, and sweat. 11,12 Since mucsa surfaces are a majr pint f entry fr pa
thgens, IgA2
serves t eep antigens frm penetrating further int the bdy. The dimer cnsis
ts f tw
mnmers hed tgether by a J chain that has a mecuar weight f abut 15,000.
Secretry IgA is
synthesized in pasma ces fund mainy in mucsa-assciated ymphid tissue,
and it is
reeased in dimeric frm. IgA is synthesized at a much greater rate than that f
IgGapprximatey 3 grams per day in the average adutbut because it is mainy in s
ecretry
frm, the serum cncentratin is much wer. 11 A secretry cmpnent (SC), whic
h has a mecuar
weight f abut 70,000, is ater attached t the FC regin arund the hinge prt
in f the
chains. 3,11,13 This prtein, cnsisting f ve immungbuin-i e dmains, is de
rived frm
epitheia ces fund in cse prximity t the pasma ces. 3 As Figure 48 ind
icates, SC
precursr, with a mec- uar weight f 100,000, is actuay fund n the surfac
e f epitheia
ces and serves as a specific receptr fr IgA. Pasma ces that secrete IgA a
ctuay hme t
subepitheia tissue, where IgA can bind as sn as it is reeased frm the pas
ma ces. 3 This
hming f activated ymphcytes depends upn a high eve f certain adhesin m
ecues that
aw binding t epitheia ces. 11 Once binding ta es pace, IgA and SC precu
rsr are ta en
inside the ce and then reeased t the ppsite surface by a prcess nwn as
transcytsis. The
vesice carrying IgA and the SC receptr fuses with the membrane n the ces pp
site side, and
a sma fragment f SC is ceaved t iberate the IgA dimer with the remain- ing
SC. 11 The SC
may thus act t faciitate transprt f IgA t mucsa surfaces. 12 It as ma e
s the dimer mre
resist- ant t enzymatic digestin by mas ing sites that wud be susceptibe t
prtease
ceavage. 3 The main functin f secretry IgA is t patr mucsa surfaces and
act as a rst
ine f defense. It pays an impr- tant re in neutraizing txins prduced by
micrrganisms,
and it heps t prevent bacteria adherence t mucsa sur- faces. 13 Cmpexes
f IgA and
antigen are easiy trapped in mucus and then eiminated by the ciiated epithei
a ces f the
respiratry r intestina tract. This prevents pathgens frm cnizing the muc
sa epitheium.
12 Since IgA is fund in breast mi , breastfeeding heps t maintain the heath
f newbrns. It
appears that IgA is nt capabe f xing cmpement by the cassica pathway, ath
ugh
aggregatin f immune cmpexes may trigger the aternate cmpement pathway. 11
(Refer t
Chapter 6 fr a cmpete discussin f the cmpe- ment pathways.) Lac f cmp
ement activatin

may actuay assist in cearing antigen withut triggering an in amma- try respn
se, thus
minimizing tissue damage. 1113 Additinay, neutrphis, mncytes, and macrpha
ges pssess
speci c receptrs fr IgA. Binding t these sites trig- gers a respiratry burst a
nd
degranuatin. 12 This ccurs fr bth serum and secretry IgA, indicating that
they are capabe f acting as psnins. The success f ra immunizatins such as the Sabin v
accine, which
induces IgA amst excu- sivey, demnstrates the effectiveness f IgAs prtecti
ve re n
mucsa surfaces. IgD IgD was nt discvered unti 1965, when it was fund in a
patient with
mutipe myema. It is extremey scarce in the serum, representing ess than
0.001 percent
f tta immungbuins. It is synthesized at a w eve and has a haf-ife
f ny 2 t 3
days. The mecue has a mecuar weight f apprximatey 180,000, and it migra
tes as a fast
prtein. The
H chain has a mecuar weight f 62,000 and appears t have an ext
ended hinge
regin cnsisting f 58 amin acids. 10 Mst f the IgD present is fund
n the surface
f immuncmpetent but unstimuated B ymphcytes. It is the secnd type f immu
ngbuin t
appear (IgM being the first), and it may pay a re in B-ce activatin. The h
igh eve f
surface expressin and its intrinsic fexibiity ma e it an idea eary respnde
r t antigen. 5
Thse ces bearing ny IgM receptrs appear incapabe f an IgG IgA mnmer Ig
A dimer J chain
Pasma ce Epitheia ce IgA receptr Vacue Ceavage IgA with SC
FIGU
RE 48.
Frmatin f secretry IgA. IgA is secreted as a dimer frm pasma ces and is
captured by
speci c receptrs n epitheia ces. The receptr is actuay an SC, which binds
t IgA and
exits the ce ang with it. 1814_Ch04_054-071.qxd 7/10/09 3:31 PM Page 61 6
2 SECTION 1
Nature f the Immune System respnse, whie thse with bth IgM and IgD receptr
s are capabe f
respnding t T-ce hep and switching t syn- thesis f IgG, IgA, r IgE. 5 Th
us, IgD may pay
a re in reguating B-ce maturatin and differentiatin. 1,10 Because f its
unusuay ng
hinge regin, IgD is mre susceptibe t prteysis than ther immungbuins.
This may be the
main reasn fr its shrt haf-ife. In the secreted frm in the serum, IgD des
nt appear t
serve a prtec- tive functin, because it des nt bind cmpement, it des nt
bind t
neutrphis r macrphages, and it des nt crss the pacenta. 1 IgE IgE is the
east abundant
immungbuin in the serum, accunting fr ny 0.0005 percent f tta
serum
immungbuins. 1 It is an 8 S mecue with a mecuar weight f apprximate
y 190,000. The
r H chain is cm- psed f arund 550 amin acids that are distributed ver ne
variabe and

fur cnstant dmains. A singe disu de bnd jins each

chain t an L chain, and
tw disufide
bnds in the H chains t ne anther. IgE is the mst heat-abie f a immun
gbuins; heating t 56C fr between 30 minutes and 3 hurs resuts in cnfrmatina changes a
nd ss f
abiity t bind t target ces. IgE des nt participate in typica immungbu
in reactins
such as cmpement fixatin, aggutinatin, r psnizatin. Additinay, it is
incapabe f
crssing the pacenta. Instead, shrty after synthesis, it attaches t b
asphis and
tissue mast ces by means f speci c surface prteins, termed high-affinity FC
RI
receptrs,
which are fund excusivey n these ces. 3,5 The mecue binds at the C H 3
dmain n the FC
regin. 9 This eaves the antigen- binding sites free t interact with speci c ant
igen (Fig.
49). Pasma ces that prduce IgE are cated primariy in the ung and in the s
in. 5 Mast
ces are as fund mainy in the s in and in the ining f the respiratry and
aimentary
tracts. One such ce may have severa hundred thusand receptrs, each capa- b
e f binding an
IgE mecue. When tw adjacent IgE mecues n a mast ce bind speci c antigen,
a cascade f
ceuar events is initiated that resuts in degranuatin f the mast ces wit
h reease f
vasactive amines such as his- tamine and heparin. Reease f these mediatrs in
duces what is
nwn as a type I immediate hypersensitivity r aergic reactin (see Chapter 1
3). Typica
reactins incude hay fever, asthma, vmiting and diarrhea, hives, and ife-thre
atening
anaphyactic shc . Whie IgE appears t be a nuisance antibdy, it may serve a
prtective re
by triggering an acute in ammatry reac- tin that recruits neutrphis and esin
phis t the
area t hep destry invading antigens that have penetrated IgA defenses. 1 Esi
nphis,
especiay, pay a majr part in the destructin f arge antigens such as paras
itic wrms that
cannt be easiy phagcytized (see Chapter 20 fr detais). ANTIBODY DIVERSITY A
ttempts t
expain the speci city f antibdy fr a partic- uar antigen began ng befr
e the actua
structure f immungbuins was discvered. The centra issue was whether an a
ntigen seected
ymphcytes with the inherent capabiity f prducing specific antibdy t it r
whether the
presence f antigen added a new speci city t a gener- aized type f antibdy. Eh
richs
Side-Chain Thery One f the rst theries t be frmuated was that f Pau Ehri
ch in the eary
1900s, termed the side-chain thery. Ehrich pstuated that certain ces had s
peci c surface
receptrs fr antigen that were present befre cntact with antigen ccur
red. Once antigen
was intrduced, it wud seect the ce with the prper receptrs, cmbinatin
wud ta e pace,

and then receptrs wud brea ff and enter the circuatin as antibdy mecu
es. New receptrs
wud frm in pace f thse br en ff, and this prcess cud be repe
ated. Athugh
this represented a rather simpistic expanatin fr antibdy synthesis, tw ey
premises
emerged: (1) the c - and- ey cncept f the t f antibdy fr antigen and (2) t
he idea that an
antigen seected ces with the buit-in capacity t respnd t it. Athugh thi
s thery did nt
expain the inetics f the immune respnse r the idea f immungic memry, i
t aid the
fundatin fr further hyptheses. Antigen specific IgE Antigen bridge FcRI rcp
tor Mast cll
with
oun IgE Dgranulation Miators: hparin histamin chmotactic factor
FIG
URE 49. Action
of IgE on mast clls. IgE
ins to spci c rcptors on mast clls. Whn antign

rigs two
nar
y IgE molculs, th mm
ran is is- tur
 an granulation rsults. Ch
mical miators
ar rlas. 1814_Ch04_054-071.qx 7/10/09 3:31 PM Pag 62 CHAPTER 4 Anti
o
y Structur an
Function 63 Clonal Slction In th 1950s, Nils Jrn an Macfarlan Burnt in
pn- ntly
support th ia of a clonal slction procss for anti
oy formation. 1416 Th
ky prmis is
that iniviual lymphocyts ar gntically prprogramm to prouc on typ of
immunoglo
ulin
an that a spci c antign ns or slcts thos particular clls capa
l of rspon
ing to it,
causing thm to prolifrat. Th rcptors Ehrlich origi- nally postulat ar t
h surfac
immunoglo
ulins IgM an IgD, foun on unstimulat B lymphocyts. Rpat contact with
antign woul continually incras a spcific lymphocyt pool. Such a mol
provis an
xplanation for th kintics of th immun rspons. Th main raw
ack to th cl
onal slction
thory was consiration of th gntic
asis for th ivrsity of anti-
oy mo
lculs. If
sparat gns wr prsnt to co for anti
oy to vry possi
l antign, an o
vrwhlming
amount of DNA woul
 n. In 1965, Dryr an Bnntt pro- pos a solution
to this ilmma

y suggsting that th constant an varia
l portions of immunoglo
ulin chains a
r actually co
for
y sparat gns. 17 Thr coul
 a small num
r coing for th constant
rgion an a
largr num
r coing for th varia
l rgion. This woul consi- ra
ly simplify
th task of
coing for such varia
ility. This notion impli that although all lymphocyts s
tart out with
intical gntic grm-lin DNA, ivrsity is crat
y a sris of rcom
ina
tion vnts
that occur as th B cll maturs. Scinti c vinc now inicats that this
is xactly what
happns, as xplain in th following iscussion. GENES CODING FOR IMMUNOGLOBUL
INS Tongawa i
som pionring xprimnts with DNA an iscovr that chromosoms contain
no intact

immunoglo
ulin gns, only
uiling
locks from which gns can
 assm
l. Th
is con rm th
hypothsis of Dryr an Bnntt. 18 Tongawa was awar th No
l Pri in 19
87 for this
monumntal iscovry. Human immunoglo
ulin gns ar foun in thr unlink c
lustrs: H chain
gns ar locat on chromosom 14,
chain gns ar on chromosom 2, an
chain g
ns ar on
chromo- som 22. Within ach of ths clustrs, a slction procss occurs. Th
gns cannot

transcri
 an translat into functional anti
oy molculs until this rarran
gmnt, assist

y spcial rcom
inas nyms, taks plac. Onc this rarrangmnt os occur,
it prmanntly
changs th DNA of th particular lymphocyt. Rarrangmnt of Havy Chain Gns
Th slction
procss
gins with rarrangmnt of th gns for th havy chains. All H chain
s ar riv
from a singl rgion on chromosom 14. Th gns that co for th vari- a
l r
gion ar ivi
into thr groupsV H , D, an J. Thr ar at last 39 V H (varia
l) gns, appr
oximatly 23
functional D (ivrsity) gns, an 6 J (joining) gns. 1921 In aition, thr
is a st of
gns (C) that cos for th con- stant rgion. This inclus on gn for ach
H chain isotyp.
Thy ar locat in th following orr: C , C , C 3, C 1, C 1, C 2, C 4, C , an C 2. Only o
of ths constant rgions is slct at any on tim. For synthsis of th nti
r H chain, a
choic is ma from ach of th sctions so as to inclu on V H gn, on D g
n, on J gn,
an on con- stant rgion. During th procss of B-cll maturation, th pics a
r splic
togthr to commit that B lymphocyt to making anti
oy of a singl spci city. Jo
ining of ths
sgmnts occurs in two stps: First, at th DNA lvl, on D an on J ar rano
mly chosn an
ar join with ltion of th intrvning DNA (Fig. 410). Nxt, a V gn is joi
n to th DJ
complx, rsulting in a rarrang V(D)J gn. Th VJD com
ination cos for th
ntir varia
l
rgion of th havy chain. This rarrang- mnt occurs arly in B-cll vlopm
nt in pro-B
clls. 21,22 (S Chaptr 2 for aitional tails.) Th rcom
inas nyms RAG
-1 an RAG-2,
which ar istinctiv markrs of this stag, ar ssntial for initiating
this procss.
Th rcom
inas nyms rcogni spcific targt squncs call rcom
in
ation signal
squncs that ank all immunoglo
ulin gn sgmnts. 21 Howvr, joining of th V
, J, an D
sgmnts osnt always occur at a fix posi- tion, so ach squnc can var
y
y a small
num
r of nuclotis. This contri
uts aitional ivrsity. 20 If a suc- cs
sful
rarrangmnt of DNA on on chromosom 14 occurs, thn th gns on th s
con chromosom
ar not rarrang; this phnomnon is known as alllic xclusion. If th rst ra
rrangmnt is
nonprouctiv, thn rarrang- mnt of th scon st of gns on th othr chro

mosom 14 occurs.
Th varia
l an constant rgions ar join at th ri
onuclic aci (RN
A) lvl, thus
consrving th DNA of th constant rgions an allowing for a latr phnomnon c
all class
switching, whr
y aughtr plasma clls can prouc anti
oy of anothr typ. D
uring
transcription an synthsis of mssngr ri
onuclic aci (mRNA), a constant rg
ion is splic to
th V(D)J complx. 23 Bcaus C
is th rgion closst to th J rgion, H chains
ar th first
to
 synthsi, an ths ar th markrs of th pr-B lymphocyts. Th C
rg
ion, which lis
closst to th C
rgion, is oftn transcri
 along with C . Th prsnc of DNA
for
oth th
C an C
rgions allows for RNA for IgD an IgM to
 transcri
 at th sam tim
. Thus, a B
cll coul xprss IgD an IgM with th sam varia
l omain on its surfac at t
h sam tim. Th
procss of switching to othr immunoglo
ulin classs occurs latr, rsulti
ng from a looping
out an ltion of othr constant rgions. This allows th sam VJD rgion to

 coupl with a
iffrnt C rgion to prouc anti-
oy of a iffrnt class (i.., IgA, IgG, o
r IgE)
ut with
th intical spcificity for antign. Contact with T clls an with cytokins p
rovis th
signal for switching to tak plac. 23 1814_Ch04_054-071.qx 7/10/09 3:31 PM
Pag 63 64
SECTION 1 Natur of th Immun Systm Light Chain Rarrangmnt Bcaus L chain
rarrangmnt
occurs only aftr chains appar, -chain synthsis rprsnts a pivotal stp in th
 procss. L
chains xhi
it a similar gntic rarrangmnt, xcpt thy lack a D rgion. Rc
om
ination of
sgmnts on chromosom 2, coing for chains, occurs prior to that on chromosom
22, which cos
for
chains. Chromosom 2 contains approximatly 40 functional V
rgions, 5 J rg
ions, an on
C rgion. 21 Th procss of VJ joining is accom- plish
y an xcision of intr
vning DNA.
This rsults in V
an J
sgmnts
coming prmanntly join to on anothr on
th
rarrang chromosom. Transcription
gins at on n of th V
sgmnt an pr
ocs through
th J
an C
sgmnts. Unrarrang J sgmnts ar rmov uring RNA splic
ing, which
occurs in th trans- lation (Fig. 411). A prouctiv rarrangmnt of th
gns w
ith su
squnt protin prouction kps th othr chromosom 2 from rarranging, an
 it shuts own
any rcom
ination of th -chain locus on chromosom 22. 18 Only if a non- functio
ning gn
prouct ariss from
rarrangmnt os chain synthsis occur. Th lam
a locus c
ontains 30V
, 4J
, an four functional C
sgmnts. 20 If functional havy an light chains a
r not
prouc
y ths rarrangmnts, thn th particular B cll is
y apoptosis.
L chains ar thn
join with
chains to form a com- plt IgM anti
oy, which rst appars in immatu

r B clls.
Onc IgM an IgD ar prsnt on th surfac mm
ran, th B lymphocyt is fully
matur an
capa
l of rspon- ing to antign (s Chaptr 2). Th larg varity of V, J, D
, an C
com
inations for ach typ of chain, plus th iffr- nt possi
ilitis for L an
 H chain
com
ination, mak for mor than nough con gurations to allow us to rspon to any
antign in th
nvironmnt. MONOCLONAL ANTIBODY Th knowlg that B clls ar gntically prp
rogramm to
synthsi vry spci c anti
oy has
n us in vl- oping anti
ois for iag
nostic tsting
known as monoclonal anti
ois. Normally, th rspons to an antign is htrognous,
caus
vn a purifi antign has multipl V 1 V 2 V 3 V n D 1 D 2 D 3 D n J 1 J
2 J 3 J 4 C C
3 C 1 C 2 C 4 C C Chromosome 14 DNA re rr ngement-D/J joining V 1 V 2 V 3 V n D
1 D 2
J 1 J 2 C C 3 C 1 C 2 C 4 C C DNA re rr ngement-V/D/J joining V 1 V 2 J 1 J 2 J
3 J
4 C C C 3 C 1 C 2 C C Tr nscription RNA synthesis V 2 J 3 C Tr nsl tion M he
ch in J 3 J 4 C C D 2 D 2
C 4 FIGURE 410. Codin for
immunolo
ulin H chains. Four separate reions on chromosome 14 code for H chain
s. DJ reions are
spliced rst, and then this sement is joined to a varia
le reion. When RNA synth
esis occurs,
one constant reion is attached to the VDJ com
ination.
H chains are made rst,
u
t the cell
retains its capacity to produce immunolo
ulin of another class. V K1 V K2 V K3
V Kn J 1 J 2 J 3
J 4 J 5 C K C K C K V J 3 V/J 3 V/J joinin Transcription RNA splicin Unrearran
ed k locus
Rearraned k ene Primary transcript k mRNA FIGURE 411. Assem
ly and expression o
f the
L chain
locus. A DNA rearranement fuses one V sement to one J sement. The VJ sement
is then
transcri
ed alon with a unique C reion to form mature
mRNA. Unarraned J seme
nts are removed
durin RNA splicin. (From Parslow, TG, et al. Medical Immunoloy, ed. 10. McGra
w-Hill/Appleton &
Lane, 2001, with permission.) 1814_Ch04_054-071.qxd 7/10/09 3:31 PM Pae 64
CHAPTER 4
Anti
ody Structure and Function 65 epitopes that stimulate a variety of B-cell c
lones. In 1975,
Geores Kohler and Cesar Milstein discovered a tech- nique to produce anti
ody a
risin from a
sinle B cell, which has revolutionized seroloical testin. For their pio- neer
in research,
they were awarded the No
el Prize in 1984. Kohler and Milsteins technique fu
ses an
activated B cell with a myeloma cell that can
e rown indefinitely in the la
or
atory. Myeloma
cells are cancerous plasma cells. Normally, plasma cells produce anti
ody, s
o a particular
cell line that is not capa
le of producin anti
ody is chosen. In addition, this
cell line has a
de ciency of the enzyme hypox- anthine uanine phosphori
osyl transferase (HGPRT)
that renders it
incapa
le of synthesizin nucleotides from hypox- anthine and thymidine, whic

h are needed
for DNA synthesis. Hy
ridoma Production A mouse is immunized with a certain ant
ien, and after a
time, spleen cells are harvested. Spleen cells are com
ined with myeloma cells i
n the presence of
polyethylene lycol (PEG), a surfactant. The PEG
rins a
out fusion of plasma c
ells with myeloma
cells, producin a hy
ridoma. Only a small percentae of cells actually fuse, an
d some of these
are like cellsthat is, two myeloma cells or two spleen cells. After fusion, cells
are placed in
culture usin a selective medium containin hypoxanthine, aminopterin, and thymi
- dine (HAT).
Culture in this medium is used to separate the hy
ridoma cells
y allowin t
hem to row
selectively. Myeloma cells are normally a
le to row inde nitely in tis- sue cult
ure,
ut in
this case they cannot,
ecause
oth pathways for the synthesis of nucleotid
es are
locked.
One pathway, which
uilds DNA from deradation of old nucleic acids, is
locked,

ecause the
myeloma cell line employed is deficient in the required enzymes HGPRT and thymidine kinase. 24
The other pathway, which makes DNA from new nucleotides, is
locked
y the prese
nce of
aminopterin. Consequently, the myeloma cells die out. Normal B cells cannot
e m
aintained
continuously in cell culture, so these die out as well. This leaves only the fus
ed hy
ridoma
cells, which have the a
ility (acquired from the myeloma cell) to reproduce inde n
itely in
culture and the a
ility (acquired from the normal B cell) to synthesize nucleoti
des
y the HGPRT
and thymidine kinase pathway (Fi. 412). Selection of Speci c Anti
ody-Producin Cl
ones The
remainin hy
ridoma cells are diluted out and placed in microtiter wells, where
they are allowed
to row. Each well, containin one clone, is then screened for the pres- ence of
the desired
anti
ody
y removin the supernatant. Once identi ed, a hy
ridoma is capa
le of
e
in maintained
in cell culture inde nitely, and it produces a permanent and uniform supply of mon
oclonal
anti
ody that reacts with a sinle epitope. 24 Clinical Applications Monoclonal
anti
odies were
initially used for in vitro dia- nostic testin. A familiar example is prenanc
y testin, which
uses anti
ody speci c for the chain of human chorionic onadotropin, there
y elimi
natin many
false-positive reac- tions. Other examples include detection of tumor antiens a
nd measurement of
hormone levels. Recently, however, there has
een an emphasis on the use of mono
clonal anti
odies
as therapeutic aents. One of the
iest success stories is in the treatment of
two autoim- mune
diseases: rheumatoid arthritis and Crohns disease (a proressive in ammatory coliti
s). Both of
these diseases have
een treated with a monoclonal anti
ody called in ixma
Primar
y and secondary
mouse immunization Mouse spleen cells Antien Mouse myeloma cells Fused in prese

nce of PEG Placed

in HAT restrictive medium Hy
ridoma cells row Spleen cells die Myeloma cells di
e FIGURE 412.
Formation of a hy
ridoma in monoclonal anti
ody production. A mouse is immunized
, and spleen
cells are removed. These cells are fused with nonsecretin myeloma cells and the
n plated in a
restrictive medium. Only the hy
ridoma cells will row in this medium, where the
y synthesize and
secrete a monoclonal immunolo
ulin spe- ci c for a sinle determinant on an anti
en. (From
Barrett, JT. Text
ook of Immunoloy, ed. 5. Mos
y, St. Louis, 1988, with permiss
ion.)
1814_Ch04_054-071.qxd 7/10/09 3:31 PM Pae 65 that
locks the action of tumor
necrosis
factor-alpha. 2529 Another tumor necrosis factor
locker, adalimuma
(Humira
), has also
proven effective in decreasin symptoms of these two diseases. 3032 Monoclonal an
ti
odies have
also
een used to treat var- ious types of cancers. In the case of metastatic
r
east cancer,
trastuzuma
(Herceptin), an anti
ody directed aainst HER-2/neu protein, wh
ich is present in
lare num
ers on tumor cells, has
een helpful in slowin the diseases pr
oress. 33,34
Another example is rituxima
(Rituxan), used to treat non-Hodkin lymphoma. Othe
r monoclonal
anti-
odies approved
y the FDA include cetuxima
(Er
itux) to treat colorectal
cancer and head
and neck cancers and
eva- cizuma
(Avastin) to treat colorectal, non-small lun
, and
reast
cancers. 33 Additionally, some monoclonal anti
od- ies are conjuated with r
adioactive
su
stances that are delivered directly to cancerous cells. Drus in this cate
ory include
i
ritumoma
tiuxetan (Zevalin), for cancerous B lymphocytes, and tositumoma
(Be
xxar) to treat
some non- Hodkin lymphomas that no loner respond to rituxima
. 33 The f
act that
monoclonal anti
odies can now
e humanized
y recom
inant technoloy has cut dow
n on reactions to
the reaents themselves, which used to
e of mouse oriin. This area of research
in pharmacoloy
is rapidly expandin and is likely to continue to row in the future. SUMMARY Th
e
asic
structural unit for all immunolo
ulins is a tetrapeptide composed of two
L and two H
chains joined toether
y disulfide
onds. While
and
L chains are found in all t
ypes of
immunolo
ulins, the H chains differ for each immunolo
ulin class. The five cla
sses are IM,
IG, IA, ID, and IE. IG, ID, and IE exist as monomers, IA has a
dimeric form,
and IM is a pentamer whose su
units are held toether
y a J chain. Each immuno
lo
ulin molecule
has constant and vari- a
le reions. The varia
le reion is at the amino-termina
l 66 SECTION 1
Nature of the Immune System end, called the Fa
frament and this determines the
speci- city of
that molecule for a particular antien. The constant reion, located at the car

oxy-terminal end
of the molecule and named the FC frament, is responsi
le for
indin to effecto
r cells such as
neutrophils,
asophils, eosinophils, and mast cells to amplify the inflammatory
process and speed
up antien removal. Structural differences determine specific functions for each
of the
immunolo
ulin types. For instance, IG is rel- atively small and easily penetra
tes into tissues,
while IM is much larer and excels at complement xation. IA has an SC that prot
ects it from
enzymatic diestion while it patrols mucosal surfaces. An extended hine reion
ives ID an
advantae as a surface receptor for antien. IE
inds to mast cells to initiate
a local
in ammatory reaction. Ehrlichs side-chain theory was the first attempt to ac
count for
anti
ody diversity, and it is
ased on the anti- en selectin the correctly pro
rammed B
lymphocyte. The clonal selection theory took this a step further and postu- late
d that
lymphocytes are enerally pre-endowed to respond to one antien or a roup o
f antiens, with
IM and ID actin as surface receptors that interact with speci c antien to tri
er
proliferation of a clone of identical cells. Genetic preprorammin of lymphocyt
es can
est
e
explained
y the concept of ene recom
ination. More than one ene controls synt
hesis of a
particular immunolo
u- lin, and throuh a random selection process, these indiv
idual sements
are joined to commit that lymphocyte to makin anti
ody of a sinle speci city. A
workin example
of the clonal selection theory is the production of monoclonal anti
odies. A can
cerous cell or
myeloma is fused with an anti
ody-producin cell to form a hy
ridoma. Hy
ridomas
formed
y fusion
of one of each cell type (e.., myeloma and B cell) are identi ed
y usin HAT, a
selective
medium. Then hy
ridomas are diluted out and placed in microtiter wells. The clon
e producin the
desired anti
ody is located
y testin the supernatant in each well. Monoclonal
anti
odies are
used
oth in dianosis and treatment of disease. 1814_Ch04_054-071.qxd 7/10/09
3:31 PM Pae 66
CHAPTER 4 Anti
ody Structure and Function 67 1. A 15-year-old male exhi
ited sym
ptoms of fever,
fatiue, nausea, and sore throat. He went to his primary care physician, and a r
apid strep test
and a test for infectious mononucleosis were performed in the of ce. The rapid str
ep test result
was neative,
ut the test result for infectious mononucleosis was faintly posit
ive. The patient
mentioned that he thouht he had previously had mononucleosis,
ut it was
never officially
dianosed. His serum was sent to a reference la
oratory to test with spec
ific Epstein-Barr
viral antiens. The results indicated the presence of IM only. Question a. Is t
his a reactivated
case of mononucleosis? Explain your answer. 2. A 10-year-old female experienced

one cold after

another in the sprintime. She had missed several days of school, and her mother
was reatly
concerned. The mother took her dauhter to the pediatrician, worried that
she miht
e
immunocompromised
ecause she couldnt seem to fiht off infections. A
lood
sample was
o
tained and sent to a reference la
oratory for a deter- mination of anti
ody
levels,
includin an IE level. The patients IM, IG, and IA levels were all normal
for her ae,

ut the IE level was reatly increased. Question a. What does this indicat
e a
out the
patients state of health? CASE STUDIES 1814_Ch04_054-071.qxd 7/10/09 3:31 PM P
ae 67 68
SECTION 1 Nature of the Immune System SERUM PROTEIN ELECTROPHORESIS Principle El
ectrophoresis is
the miration of chared particles in an electrical eld throuh an electrolyte so
lution toward
the elec- trode of the opposite chare. At a pH of 8.6, all serum proteins are n
eatively
chared, and when placed in an electrical eld, they mirate toward the anode at a
rate that
depends on the net neative chare. Five distinct
ands are o
tained: al
u- min,
alpha 1 , alpha
2 ,
eta, and amma. Immunolo
ulins are located in the amma
and. The relative
proportions of
these fractions is useful in the dianosis of immunode ciency dis- eases and immun
oproliferative
disorders. Sample Preparation O
tain serum from whole
lood
y usin a sterile c
lot tu
e.
Centrifue and remove the supernatant. Discard any sam- ples that appear to
e h
emolyzed. Serum
is sta
le for up to 5 days if the specimen is refrierated. Avoid freezin samples
ecause
this tends to denature protein. Urine and cere
rospinal fluid can also
e
used. They may
e
stored covered at 2 to 8C for up to 72 hours. Reaents, Materials, and Equipment
Aarose el
electrophoresis system Aarose lm Buffer kit Stain Microliter pipette Stir-stain
dishes 5
percent acetic acid Sample cups Other equipment, dependin upon the kit used Pro
cedure Refer to
the procedure for the specific el eletrophoresis system used. Interpretation of
Results All
serum proteins should mirate toward the anode. The rate of miration is influen
ced
y the net
chare on the molecule, the molecules size, the
uffers pH, the
uffers ionic stren
th, and
the el concentration. Al
umin has the reatest neative chare at pH 8.6, and h
ence it will
travel the farthest in a iven time. Because it is present in the reatest conce
ntration, a dark
compact
and should
e seen. Next, a more faint alpha 1
and will
e seen, follo
wed
y an alpha 2

and and the
eta
and. Immunolo
ulins will mirate within the amma reion,
u
t as these are
the least chared, they will remain in a diffuse
and near the oriin.
Protein
ands are
not visi
le until after stainin with amido
lack or acid
lue. Once the film is

dried and then

rinsed in 5 percent acetic acid, the stain will remain local- ized only on prote
in
ands. If
normal controls are run alon with patient serum,
ands can
e visually compared
to deter- mine
any possi
le a
normalities in immunolo
ulin production. Infection may
e in
dicated
y an
overall increase in intensity of color within the amma reion. A sharp
and wit
hin the reion
would indicate monoclonal ammopathies with speci c anti
ody production. Lack of i
mmunocompetence would
e demonstrated
y an overall decrease in color in the amma reion.
If a
densitometer is availa
le, a more quantitative approach is possi
le. The fo
llowin
represents a normal dis- tri
ution for serum proteins: al
umin, 58 to 70 percent
; alpha 1 , 2 to
5 percent; alpha 2 , 6 to 11 percent;
eta, 8 to 14 percent; and amma, 9 to 18
percent. If a
total serum protein determination has
een made, these percentaes can then
e t
urned into actual
m/dL amounts. To determine a
normalities of speci c immunolo
ulin classes, immun
oelectrophoresis must
e performed. EXERCISE 1814_Ch04_054-071.qxd 7/10/09 3:31
PM Pae 68
CHAPTER 4 Anti
ody Structure and Function 69 1. Which is characteristic of v
aria
le domains
of immunolo
ulins? a. They occur on
oth the H and L chains.
. They represent
the complement

indin site. c. They are at the car
oxy-terminal ends of the molecules. d. All
of the a
ove 2.
All of the followin are true of IM except that it a. can cross the placenta.

. xes
complement. c. has a J chain. d. is a primary response anti
ody. 3. How many a
ntien
indin
sites does a typical IM molecule have? a. 2
. 4 c. 6 d. 10 4. Bence-Jones
proteins are
identical to which of the followin? a. H chains
. L chains c. IM molecule
s d. IG
molecules 5. An Fa
frament consists of a. two H chains.
. two L chains. c. on
e L chain and
one-half of an H chain. d. one L chain and an entire H chain. 6. Which of the fo
llowin pairs
represents two different immunolo
ulin allotypes? a. IM and IG
. IM1 and I
M2 c. Antihuman
IM and antihuman IG d. IG1m3 and IG1m17 7. Which of the followin are L
chains of
anti
ody molecules? a.
.
c. d.
8. If the results of serum protein electrophores
is show a
sini cant decrease in the amma
and, which of the followin is a likely possi
il
ity? a. Normal
response to active infection
. Muliple myeloma c. Immunode ciency disorder d. Mon
oclonal
ammopathy 9. The su
classes of IG differ mainly in a. the type of L chain.
.
the arranement
of disul de
onds. c. the a
ility to act as opsonins. d. molecular weiht. 10. Whi
ch
est
descri
es the role of the SC of IA? a. A transport mechanism across endothelial
cells
. A means
of joinin two IA monomers toether c. An aid to trappin antien d. Enhancemen

t of complement
xation
y the classi- cal pathway 11. Which represents the main function of ID?
a. Protection
of the mucous mem
ranes
. Removal of antiens
y complement xation c. Enhancin
proliferation
of B cells d. Destruction of parasitic worms 12. Which anti
ody is
est at alu
tination and
comple- ment xation? a. IA
. IG c. ID d. IM 13. Which of the followin can

e attri
uted to
the clonal selection theory of anti
ody formation? a. Plasma cells make enerali
zed anti
ody.
.
B cells are preprorammed for speci c anti
ody synthesis. c. Proteins can alter th
eir shape to
conform to antien. d. Cell receptors
reak off and
ecome circulatin anti
ody.
14. All of the
followin are true of IE except that it a. fails to x complement.
. is heat sta

le. c.
attaches to tissue mast cells. d. is found in the serum of alleric persons. REV
IEW QUESTIONS
1814_Ch04_054-071.qxd 7/10/09 3:31 PM Pae 69 70 SECTION 1 Nature of the Immu
ne System
References 1. Mak, TW, and Saunders, ME. B cell receptor structure and function.
In Mak, TW,
and Saunders, ME: The Immune Response: Basic and Clinical Principles. Elsevi
er, Burlinton,
MA, 2006, pp. 93120. 2. Edelman, GM. The structure and function of anti
odies. Sc
i Am 223:34,
1970. 3. Kindt, TJ, Golds
y, RA, and Os
orne, BA. Antiens and anti-
odies. In
Kindt, TJ,
Golds
y, RA, and Os
orne, BA: Ku
y Immunoloy, ed. 6. WH Freeman, New York
, 2007, pp.
76110. 4. Porter, RR. The structure of anti
odies. Sci Am 217:81, 1967. 5. Frazer
, JK, and
Capra, JD. Immunolo
ulins: Structure and function. In Paul, WE (ed): Fundamenta
l Immunoloy, ed.
4. Lippincott Williams & Wilkins, Philadelphia, 1999, pp. 3774. 6. Davies, DR, an
d Cohen, GH.
Interactions of protein antiens with anti
odies. Proc Natl Acad Sci USA 93:712,
1996. 7.
Wedemayer, GJ, Patten, PA, and Wan, LH, et al. Structural insihts into the evo
lution of an
anti
ody com
inin site. Science 276:16651669, 1997. 8. Harris, LJ, Larson, SB, a
nd McPherson,
A. Comparison of intact anti
ody structures and the implications for effector fu
nction. Adv
Immunol 72:191208, 1999. 9. Nezlin, R, and Ghetie, V. Interactions of immunolo
u
lins outside
the antien-com
inin site. Adv Immunol 82:155215, 2004. 10. McPherson, RA, and M
assey, HD.
La
oratory evaluation of immunolo
ulin function and humoral immunity. In M
cPherson, RA,
and Pincus, MR (ed): Henrys Clinical Dianosis and Manaement
y La
oratory
Methods, ed.
21. Saunders Elsevier, Philadelphia, 2007, pp. 835848. 11. Brandtzae, P, and Joh
ansen, F.
Mucosal B cells: Phenptypic characteristics, transcriptional reulation, and hom
in prop- erties.
Immunol Rev 206:3263, 2005. 12. Wines, B, and Hoarth, P. IA receptors in health
and disease.
Tissue Antiens 68: 103114, 2006. 13. Woof, JM, and Mestecky, J. Mucosal im

munolo
ulins.
Immunol Rev 206:6482, 2005. 14. MacKay, IR. History of immunoloy in Australia: E
vents and
identities. Int Med J 36:394398, 2006. 15. Jerne, NK. The natural selection theor
y of anti
ody
porduc- tion. Proc Natl Acad Sci USA 41:849857, 1955. 16. Burnet, FM. A modi cation
of Jernes
theory of anti
ody pro- duction usin the concept of clonal selection. Au
st J Sci
20:6769, 1957. 17. Dreyer, WJ, and Bennett, JC. The molecular
asis of anti-
ody
formation: A
paradox. Proc Natl Acad Sci USA 54:864, 1965. 18. Mak, TW, and Saunders, ME.
The
immunolo
ulin enes. In Mak, TW, and Saunders, ME. The Immune Response:
Basic and
Clinical Principles. Elsevier, Burlinton, MA, 2006, pp. 179208. 19. Delves, PJ,
and Roitt, IM.
The immune system. First of two parts. N Enl J Med 343:3749, 2000. 20. Kindt, TJ
, Golds
y, RA,
and Os
orne, BA. Oranization and expression of immunolo
ulin enes. In Kindt,
TJ, Golds
y, RA,
and Os
orne, BA: Ku
y Immunoloy, ed. 6. WH Freeman, New York, 2007, pp.
111144. 21.
Dudley, DD, Chaudhuri, J, Bassin, CH, and Alt, FW. Mechanism and cont
rol of V(d)J
recom
ination versus classswitch recom
ination: Similarities and differences. A
dv Immunol
86:43112, 2005. 22. Co

, RM, Oestreich, KJ, Osipovich, OA, Oltz, EM. Acce
ssi
ility
control of V(D)J recom
ination. Adv Immunol 91:45110, 2006. 23. Chaudhuri, J, Bas
u, U, Zarrin,
A, Yan, C, et al. Evolution of the immunolo
ulin heavy chain class switch recom

ination
mechanism. Adv Immunol 94:157214, 2007. 24. Mak, TW, and Saunders, ME. Explorin
antien-anti
ody interaction. In Mak, TW, and Saunders, ME. The Immune Response:
Basic and
Clinical Principles. Elsevier, Burlinton, MA, 2006, pp. 147176. 25. Maini, R, St
. Clair, EW,
and Breedveld, F, et al. Inflixima
(chimeric anti-tumour necrosis factor-alpha
monoclonal anti
ody) versus place
o in rheumatoid arthritis patients receivin concomitan
t methotrexate: A
randomised phase III trial. ATTRACT Study Group. Lancet 354:19321939, 1999.
26. Nikas, S,
Temekonidis, T, Zikou, A, Aryropoulou, M, et al. Treatment of resistant rheumat
oid arthritis
y
intra-articular inflixima
injections: A pilot study. Ann Rheum Dis 63(1)
:102103, 2004.
27. Kristensen, LE, Saxne, T, Nilsson, J, and Ge
orek, P. Impact of concomitant
DMARD therapy on
adherence to treatment with etanercept and in ixima
in rheumatoid arthritis. Resu
lts from a
six-year o
servational study in southern Sweden. Arthritis Res Ther 8(6):R1
74, 2006. 28. A
e
T, Takeuchi T, Miyasaka N, Hashimoto H, Kondo H, et al. A multicenter, dou
l
e-
lind,
randomized, place
o controlled trial of inflixima
com
ined with low dos
e methotrexate in
Japanese patients with rheumatoid arthritis. J Rheumatol 33(1):3744, 2006. 29. Pr
esent, DH,

Ruteerts, P, and Taran, S, et al. Inflixima
for the treatment of fistulas in

patients with
Crohns disease. N Enl J Med 340:13981405, 1999. 30. Hoch
er, M, Tracy, J, Hawkin
s-Holt, M,
and Flores, R. Comparison of the efficacy of the tumour necrosis factor
lockin
aents
adalimuma
, etanercept, and in ixima
when 15. Which
est descri
es codin for
immunolo
ulin molecules? a. All enes are located on the same chromosome.
. L
chain
rearranement occurs
efore H chain rearranement. c. Four different reions are
involved in
codin of H chains. d.
rearranement occurs
efore
rearranement. 16. What is th
e purpose of
HAT medium in the prepara- tion of monoclonal anti
ody? a. Fusion of the two cel
l types
.
Restrictin the rowth of unfused myeloma cells c. Restrictin the rowth of unf
used spleen cells
d. Restrictin anti
ody production to the IM class 1814_Ch04_054-071.qxd 7/10/
09 3:31 PM Pae
70 CHAPTER 4 Anti
ody Structure and Function 71 added to methotrexate in patient
s with active
rheumatoid arthritis. Ann Rheum Dis 62(Suppl 2): ii13ii16. 2003. 31. FDA approve
s new therapy
for rheumatoid arthritis. www.fda.ov/

s/topics/ANSWERS/2002/ANS01186.html.
Accessed
Septem
er 14, 2007. 32. FDA approves new treatment for Crohns disease. www.fda.o
v/


s/topics/NEWS/2007/NEW01572.html. Accessed Septem
er 14, 2007. 33. Monoclonal
anti
odies.
http://www.cancer.or/docroot/ETO/ content/ETO_1_4X_Monoclonal_Anti
ody_Therapy.
Accessed
Septem
er 10, 2007. 34. Bioloical therapies for cancer: Q and A. http://
cancernet.nci.nih.ov/cancertopics/factsheet/Therapy/
ioloical. Accessed Septe
m
er 1, 2007.
1814_Ch04_054-071.qxd 7/10/09 3:31 PM Pae 71 5 Cytokines Timothy Steall, Ph
D. LEARNING
OBJECTIVES After nishin this chapter, the reader will
e a
le to: 1. De ne cytokin
e. 2. De ne
pleiotropy as it relates to cytokine activities. 3. Distinuish
etween autocrin
e, paracrine, and
endocrine effects of cytokines. 4. Explain the functions of interleukin-1 (IL-1)
in mediatin the
immune response. 5. Explain the effects of tumor necrosis factor (TNF). 6. Compa
re the functions
of type 1 and type 2 interferons (IFN). 7. Descri
e the actions of interleukin-2
(IL-2) on taret
cells. 8. Discuss the
ioloical roles of the hematopoietic rowth factors. 9. D
escri
e the

ioloical role of colony stimulatin factors. 10. Descri
e the current types of
anticytokine
therapies. 11. Descri
e clinical assays for cytokines. KEY TERMS Acute phase res
ponse Adaptive
immune response Adaptive T reulatory 1 cells (TR1) Antaonist Autocrine Chemoki
ne Colony
stimulatin factor (CSF) Cytokines Endocrine Endoenous pyroen Erythropoietin (
EPO)
Granulocyte-CSF (G-CSF) Granulocyte-macrophae- CSF (GM-CSF) Innate immune respo
nse Interins
Interferons (IFN) Interleukins (IL) Macrophae-CSF (M-CSF) Paracrine Pleiotropic

Redundancy
Transformin rowth factor
eta (TGF- ) T reulatory (Tre) cells Tumor necrosis f
actor (TNF) 72
1814_Ch05_072-084.qxd 7/10/09 2:40 PM Pae 72 CHAPTER 5 Cytokines 73 INTRODUC
TION TO CYTOKINES
Cytokines are small solu
le proteins that reulate the immune system, orch
estratin
oth
innate immunity and the adaptive response to infection. These chemical messen- 
ers, produced

y several different types of cells, have activity-modulatin effects on
the
hematopoietic and immune systems throuh activation of cell-
ound receptor prot
eins. 1 Cytokines
are induced in response to speci c stimulisuch as
acterial lipopolysaccha
rides,
flaellin, or other
acterial productsthrouh the liation of cell- adhesion mole
cules or
throuh the reconition of forein antiens
y host lymphocytes. The effects of
cytokines in vivo
include reulation of rowth, differentiation, and ene expression
y many diffe
rent cell types,
includin leukocytes. These effects are achieved throuh
oth autocrine stimu- l
ation (i.e.,
affectin the same cell that secreted it) and paracrine (i.e., affectin
a taret cell in
close proximity) activ- ities. Occasionally, cytokines will also exert sys
temic or
endocrine activities. Individual cytokines do not act alone
ut in conjunction
with many
other cytokines that are induced durin the process of immune activation. The
result- in
network of cytokine expression reulates leukocyte activity and leads to th
e elimination of
the infection. The cytokine cascade produces a spectrum of activities that lead
to the rapid
eneration of innate and adaptive immune responses. In fact, the a
ility or ina

ility to enerate certain cytokine patterns often determines the outcome and the clinical cour
se of infection.
In extreme circum- stances, massive overproduction and dysreulation produces a c
ytokine storm
that leads to shock, multioran failure, or even death, thus contri
utin to pat
hoenesis. 2,3
Initially, cytokines were named
ased on their activities and the types of cells
from which they
were first isolated. The major cytokine families include tumor necrosis factors
(TNF),
interferons (IFN), chemokines, transformin rowth factors (TGF), and colony sti
mulatin factors
(CSF). There are also the interleukins (IL), which currently num
er from IL-1 to
IL-32. 4 The
interleukins are unrelated cytokines that must satisfy three criteria in order t
o
e classified
as interleukins: they must have had their enes cloned, they must
e induci
le i
n leukocytes, and
their
ioloical activ- ities in in ammatory processes must
e cataloued. Cytokin
es were
oriinally thouht to act solely on cells of the immune system,
ut it soon
eca
me apparent that
many also act on cells outside the immune system. The pleiotropic (i.e., havin

many different
effects) nature of cytokine activity relates to the widespread distri
ution of c
ytokine
receptors on many cell types and the a
ility of cytokines to alter expres
sion of numerous
enes. Many different cytokines may share propertiesthat is, they acti- vate some
of the same
pathways and enes. This redundancy may
e explained
y the fact that many cytok
ines share receptor su
units. For instance, IL-6, IL-11, leukemia inhi
itory factor, oncostatin
M, ciliary
neurotrophic factor, and car- diotrophin all utilize the p130 su
unit as
part of their
receptors. 5 The speci city for each cytokine is contained in the other su
units
in the
respective receptors,
ut much of the sinal transmitted throuh the recepto
rs is enerated

y the p130 su
unit. Thus, some cytokines may have overlap- pin effects and ma
y alter the
activity of many of the same enes. However, redundancy of cytokine activities c
annot
e
explained entirely
y shared receptor su
units. TNF- , IL-1, and IL-6 exert
many of the
same
ioloical activities,
ut each utilizes a distinct receptor. Here, the cyt
okines are
activatin similar immune response pathways throuh dif- ferent sinalin pathwa
ys. Fiure 51
illustrates some of the different actions of cytokines. The increasin clinica
l usae of
cytokines, cytokine antaonists, and cytokine receptor antaonists in condition
s such as
rheumatoid arthritis, psoriasis, asthma, Crohns dis- ease, transplantation, and
cancer
treatments will drive demand for cytokine assays in the clinical la
oratory. T
he pattern of
cytokine expression can also determine whether the host will
e a
le to mount an
effective
defense aainst and survive certain infections. In addition, numerous CHAP
TER OUTLINE
INTRODUCTION TO CYTOKINES CYTOKINES IN THE INNATE IMMUNE RESPONSE Interleukin-1
(IL-1) Tumor
Necrosis Factor- (TNF- ) IL-6 Chemokines TGF- IFN- and IFN- CYTOKINES IN THE ADAPTIVE
IMMUNE
RESPONSE Th1 Cytokines Th2 Cytokines Cytokines Associated with T Reulatory Cell
s ERYTHROPOIETIN
AND COLONY STIMULATING FACTORS CYTOKINES AND ANTICYTOKINE THERAPIES CLINICAL ASS
AYS FOR CYTOKINES
SUMMARY CASE STUDY REVIEW QUESTIONS REFERENCES 1814_Ch05_072-084.qxd 7/10/09 2
:40 PM Pae 73
74 SECTION 1 Nature of the Immune System immunode ciency syndromes and leukemias a
re caused
y
defects in cytokines or their receptors/sinal transduction circuits. 6 Genetic
and proteomic
analyses of these defects will occur within the clinical la
oratory in order to
assess treatment
modalities, effectiveness, and potential ene- replacement therapies. CYTOKINES
IN THE INNATE
IMMUNE RESPONSE Cytokines involved in the innate immune response are resp
onsi
le for many
of the physical symptoms attri
uted to in ammation, such as fever, swellin, pain,

and cellular
infiltrates into damaed tissues. The innate immune response is nonspeci c

ut occurs
within hours of rst con- tact with microoranisms (see Chapter 1). It may play a
crucial part in
recovery from infection. The main function of the innate immune response is to r
ecruit effector
cells to the area. Cytokines involved in trierin this response are interleuki
n-1, tumor
necrosis factor-alpha, interleukin-6, chemokines, transformin rowth factor-
et
a, and interferons-alpha and
eta. The function of each is discussed here. Interleukin-1 (IL-1)
The IL-1 family
consists of IL-1 , IL-1 , and IL-1RA (IL-1 receptor antaonist). 7 IL-1 and IL-1 are
proinflamma- tory cytokines produced
y monocytes and macrophaes. IL-1 producti
on may
e induced

y the presence of micro-
ial pathoens,
acterial lipopolysaccharides, or
other cytokines.
IL-1 and IL-1 exhi
it the same activities in many test systems and share a
out 25
percent
sequence homoloy. 7 However, IL-1 remains intracellular within monocytes and mac
rophaes and is
rarely found outside these cells. IL-1 can
e released after cell death and can h
elp attract
inflammatory cells to areas where cells and tissues are
ein killed or damaed.
IL-1 is
responsi
le for most of the systemic activity attri
uted to IL-1, includin feve
r, activation of
phaocytes, and production of acute phase proteins. It is cleaved intra- cellula
rly to an
active form that is then secreted
y monocytes. IL-1 acts as an endoenous
pyroen and
induces fever in the acute phase response throuh its actions on the hypothalamu
s. 8 The
hypothalamus acts as the thermostat for the human
ody, and IL-1 sets the thermo
stat at a hiher
level. Elevated
ody temperatures may serve to inhi
it the rowth of pathoenic

acteria and
viruses and also increases lymphocyte activity. Additionally, IL-1 induces the p
roduc- tion of
vascular cell-adhesion molecules as well as chemokines and IL-6. These chem
okines and
cell-adhesion molecules attract and assist leukocytes to enter the in amed area
y
a process
known as diapedesis (see Chapter 1). IL-1 also induces the production of colony
stimulatin
factors in the
one marrow, there
y increasin the availa
le num
er of phaocyti
c cells that can
respond to the damaed tissues. 9 IL-1RA is also produced
y monocytes and macro
phaes. It acts
as an antaonist to IL-1
y
lockin the IL-1 recep- tor and limitin the availa

ility of the
receptor for IL-1. This helps to reulate the physioloical response to IL-1 and
turn off the
response when no loner needed. Tumor Necrosis Factor- (TNF- ) Tumor necrosis facto
rs (TNF) were
first isolated from tumor cells and were so named
ecause they induced lysis in
these cells.
TNF- is the most prominent mem
er of the TNF superfamily, which consists of at le
ast 19

different peptides that have diverse
ioloical functions. 10 TNF- exists in
o

th
mem
rane-
ound and solu
le forms and causes vasodilation and increased vasop
ermea
ility. The
solu
le form is derived from the mem
rane-
ound form
y Pleiotropy Redundancy Sy
nery Activated T
helper cells Activated T helper cell Activated T helper cells T-cell proliferati
on IL-2 receptors
IL-2 IL-2 IL-4 IL-5 IL-4 IL-5 B-cell proliferation NK cell B-cell proliferation
B cell IE
secretion FIGURE 51. Cytokine characteristics of pleiotropy, redundancy, and anta
onism.
(Adapted from Kindt, TJ, Golds
y, RA, and Os
orne, BA. Ku
y Immunoloy, ed. 6. W
H Freeman, New
York, 2007, with permission.) 1814_Ch05_072-084.qxd 7/10/09 2:40 PM Pae 74 C
HAPTER 5
Cytokines 75 proteolytic cleavae with TNF- -convertin enzyme. However, the s
olu
le form is
unsta
le and has a short half- life. Mem
rane-
ound TNF- can mediate all the cyto
toxic and
in ammatory effects of TNF throuh cell-to-cell con- tact. The main trier for TN
F- production
is the presence of lipopolysaccharide, found in ram-neative
acteria. TNF- secr
eted
y
activated monocytes and macrophaes can activate T cells throuh its a
ility to
induce expression
of MHC class II molecules, vascular adhesion molecules, and chemokines, in a sim
ilar manner to
IL-1. These actions enhance antien presentation and activate T cells to respond
to the
pathoen that triered the initial inflammatory response. However, when se
creted at hiher
levels, TNF can have deleterious systemic effects, leadin to septic shock. This
condition
results from lare amounts of TNF secreted in response to ram-neative
acteria
l infections,
causin a decrease in
lood pressure, reduced tissue perfusion, and dis- seminat
ed intravascular
coaulation. The latter may lead to uncontrolled
leedin. TNF- and IL-1 are
oth
present in
rheumatoid syn- ovial fluids and synovial mem
ranes of patients with rheu
matoid arthritis
(RA). Studies with anti-TNF- and anti-IL-1 demonstrate that TNF- is the central me
diator of
patholoical processes in RA and other inflammatory illnesses such as Crohns dise
ase. 11 TNFR1
(TNF receptor 1) is constitutively expressed on most tissues,
inds solu
le TNF- ,
and is the
primary medi- ator of TNF- sinal transduction in most cell types. TNFR2 i
s usually
expressed in epithelial cells and cells of the immune system and is activated
y
the
mem
rane-
ound form of TNF- . Overall, TNF- activity is at least partially reulate
d
y solu
le
forms of
oth TNF receptors. These act to
ind excess TNF- and, com
ined with the
short
half-life of the solu
le form, serve to limit the cytokines sinalin activity. I
L-6 IL-6 is a
sinle protein produced
y
oth lymphoid and nonlymphoid cell types. It is part
of the cytokine

cascade released in response to lipopolysaccharide and plays an important

role in acute
phase reactions and the adaptive immune response. IL-6 is expressed
y a variety
of normal and
transformed cells, includin T cells, B cells, monocytes and macrophaes, fi
ro

lasts,
hepatocytes, keratinocytes, astrocytes, vascular endothelial cells, and various
tumor cells. IL-1
primarily triers its secretion. This pleiotropic cytokine affects inflammation
, acute phase
reactions, immunolo
ulin synthesis, and the activa- tion states of B cells and
T cells. IL-6
stimulates B cells to proliferate and differentiate into plasma cells and induce
s CD4
T cells
to produce reater quantities of
oth pro- and anti-in ammatory cytokines. 5 Only
one IL-6
receptor has
een identi ed, and it con- sists of IL-6R (the IL-6-speci c receptor) a
nd p130
(the common sinal-transducin receptor su
unit utilized
y several cytokines).
Bindin of IL-6
to the IL-6R induces dimerization of p130 with the
-su
unit (Fi. 52).
Homodimerization followin IL-6
indin causes confor- mational chanes in p130
that expose
tyrosine residues in the intracellular portion of the molecule. Throuh a series
of
phosphorylation reactions, enes for acute phase proteins such as CRP, C3, and
r
inoen are
activated, as is inter- feron reulatory factor-1 (IRF-1). B- and T-cell enes a
re turned on in
the same manner. Chemokines Chemokines are a family of cytokines that enhance mo
tility and
promote miration of many types of white
lood cells toward the source of the ch
emokine
(chemotaxis). 12 Most of the chemotactic activity of leukocytes is reulated
y
the activities of
chemokines, includin the response to infec- tious diseases, autoimmune in ammatio
n, cancer, and
the homin of lymphocytes to all the lymphoid tissues. The chemokines are classi
fied into four
families
ased on the position of N-terminal cysteine residues. The rst roup the
alpha, or
CXC, chemokinescontains a sinle amino acid
etween the first and second cysteine
s. The second
roupthe
eta, or CC, chemokineshas adjacent cysteine residues. The third roupthe
C
chemokineslacks one of the cysteines. CX3C, the last major roup, has three amino
acids
etween
the cysteines. Chemokines play key roles in the initiation and development of in
flammatory
responses in numerous disease processes. Currently, over 40 chemokines and 20 ch
emokine receptors
have
een iden- ti ed (Ta
le 51). Both TNF- and IL-6 are amon the many cytokines th
at induce
chemokine production in the inflammatory response. Com
ined with cell adhesi
on molecules,
the J J  p 1 3 0  p 1 3 0 IL-6 IL- 6R  p 1 3 0  p 1 3 0 IL-6 IL- 6R FIGURE 52
. IL-6
indin
to the IL-6R su
unit recruits two p130 su
units to the receptor complex. Sinal
transduction
takes place throuh tyrosine phosphorylation of the p130 su
units
y one of the

Janus kinases
(J). 1814_Ch05_072-084.qxd 7/10/09 2:40 PM Pae 75 76 SECTION 1 Nature of the
Immune System
chemokines facilitate the extravasation of leukocytes into the tissues. Leukocyt
es rollin on
capillary endothelial cells activate their chemokine receptors in the pres
ence of
chemokines. This, in turn, activates interins, or cell adhe- sion molecules, on
leukocytes and
leads to rm adhesion to the endothelial cells. Shared expression of chemokine rec
ep- tors amon
different types of leukocytes allows for the co-localization of multiple
cell types to the
damaed tissue and helps to
roaden the response to tissue damae. The radient
of chemokine
concentration ena
les the leukocytes to mirate
etween the endothelial cells in
to the tissue in
the direction of increasin chemokine concentration. The spectrum of chemokines
and cytokines
expressed in the in ammatory response determines the types of cells that respond a
nd the enes
that are turned on in response to the stimuli. The types of cell surface recept
ors expressed
y
leukocytes are often developmentally reulatedfor exam- ple, immature T cells pos
sess only the
chemokine receptors related to lymphoid tissue homin. Only mature T cells expre
ss the receptors
that allow them to participate in an onoin immune reaction. The chemokine re
ceptors CXCR4
and CCR5 are utilized
y HIV as co-receptors for infection of CD4 T ly
mphocytes and
macrophaes. 13 Individuals with certain polymorphisms in these chemokine recept
ors are lon-term
nonproressors. They remain asymptomatic, have normal CD4
T-cell counts and norm
al immune
function, and have low or undetecta
le viral loads. The altered protein s
equences of the
receptors
lock or diminish the viruss a
ility to enter the cells and there
y inc
rease the
infected individuals chances of survival. The CCR5- 32 polymor- phism is a 32
p de
letion in the
CCR5 ene and is the most important of the host resistance factors. Homozy
ous individuals
are protected from HIV infection while het- erozyous persons exhi
it loner per
iods from
infection to AIDS development. In addition, certain polymorphisms in SDF1 (the l
iand for CXCR4)
and RANTES (the liand for CCR5) can
lock the viruss a
ility to
ind to and ente
r T cells and
can delay the proression to full-
lown AIDS. TGF- The transformin rowth factor

eta (TGF- )
super- family is composed of three isoforms: TGF- 1, 2, and 3. TGF- was oriinally
characterized as a factor that induced rowth arrest in tumor cells. Later, it w
as identi ed as a
fac- tor that induces antiproliferative activity in a wide variety of cell typ
es. Active
TGF- is primarily a reulator of cell rowth, differentiation, apoptosis, mi
ration, and the
inflammatory response. Thus, it acts as a control to help down-reulate the in amm
atory response

when no loner needed. In the immune response, TGF- functions as
oth an activato
r and an
inhi
itor of proliferation, dependin on the developmental stae of the affected
cells. 14 TGFreu- lates the expression of CD8 in CD4 CD8 thymocytes and acts as an autocrine
inhi
itory
factor for immature thymo- cytes. It inhi
its the activation of macrophaes and
the rowth of
many different somatic cell types and functions as an anti-in ammatory factor for
mature T cells.
TGF-
locks the production of IL-12 and stronly inhi
its the induction of IFN- . I
n addition,
the production of TGF-
y T helper 2 cells is now reconized as an important factor in the
esta
lishment of oral tolerance to
acteria normally found in the mouth. In
activated B
cells, TGF- typically inhi
its proliferation and may function as an autocr
ine reulator
to limit the expansion of activated cells. IFN- and IFN- Interferons were oriinal
ly so named

ecause they inter- fere with viral replication. However, it is the type
I interferons
consistin of IFN- and IFN- that function primarily in this manner. These interfer
ons are
produced
y dendritic cells and induce production of proteins and pathways that
directly
interfere with viral replication and cell division. 15 In most cases, this helps
limit the
infection to one relatively small area of the
ody. Type I IFN acti- vates natur
al killer cells
and enhances the expression of MHC class I proteins, thus increasin the reconi
tion and killin
of virus-infected cells. The type I interferons are also active aainst certain
malinancies and
other inflammatory processes. For instance, IFN- is ef cacious in treatin mult
iple
sclerosis, althouh the exact mechanism of action remains unclear. 16 IFN- has
e
en used to
treat hepatitis C and Kaposis sar- coma, as well as certain leukemias and lymphom
as. 17
CYTOKINES IN THE ADAPTIVE IMMUNE RESPONSE Cytokines involved in the innate immun
e response are
pro- duced
y many different cell types and function mainly to increase acute ph
ase reactants and
to recruit white cells to the area of infection. In contrast, cytokines involved
in the Ta
le
55-2 Comparison of Tests Used CHEMOKINE CHEMOKINE CHEMOKINE GROUP NAMES R
ECEPTORS CC
Chemokines MCP-1, MCAF, JE CCR2 MIP-1 CCR1, CCR5 MIP-1 CCR5 RANTES CCR1, CCR3, CC
R5 Eotaxin
CCR3 CXC Chemokines GRO , MGSA, MIP-2, KC CXCR2 IL-8 CXCR1, CXCR2 IP-10, CRG-2 CXC
R3 SDF-1 CXCR4
Ta
le 5-1. Select Chemokines and Their Receptors 1814_Ch05_072-084.qxd 7/10/09
2:40 PM Pae 76
CHAPTER 5 Cytokines 77 adaptive immune response are mainly secreted
y T cells,
especially T
helper (Th) cells, and affect T- and B-cell func- tion more directly. There are
three main
su
classes of Th cells, Th1, Th2, and Tre (T reulatory cells). Each has a spec
i c function and

produces a different set of cytokines. Once the T-cell receptor (TCR) captures a
ntien, clonal
expansion of those particular CD4
T helper cells occurs. 18 Differentiation into
Th1, Th2, or
Tre cell lineaes is in u- enced
y the spectrum of cytokines expressed in the in
itial response.
19 The Th1 lineae is driven
y the expression of IL-12
y dendritic cells and i
s primarily
responsi
le for cell-mediated immunity, while Th2 cells drive anti
ody- mediated
immunity and are
developmentally reulated
y IL-4. Tre cells are derived from IL-10-responsive
nave T cells and
help to reulate the activities of Th1 and Th2 cells (Fis. 53 and 54). Th1 Cytoki
nes Dendritic
cells in damaed tissues produce IL-12 in response to certain stimuli such as my
co
acteria,
intracellular
acte- ria, and viruses. It is also produced
y macrophaes and B
cells and has
multiple effects on
oth T cells and natural killer (NK) cells. IL-12
inds to i
ts receptor on
nave T cells and causes the expression of a new set of enes, includin those th
at determine
maturation into the Th1 lineae. Activation of Th1 cells induces hih-level
expression of
IFN- . 20 IL-12 also increases the cytolytic a
ility of natu- ral killer (NK) cell
s; therefore,
it serves as an important link
etween the innate and adaptive immune resp
onses
y
enhancin defenses aainst intracellular pathoens. IFN- IFN- is the principal mol
ecule
produced
y Th1 cells, and it affects the RNA expression levels of more
than 200 enes.
21,22 Genes involved in reulation and activation of CD4
Th1 cells, CD8 cytotoxi
c
lymphocytes, NK cells,
actericidal activities, IL-12R and IL-18R are all reula
ted
y IFN- . In
addition, IFN- stimulates antien presenta- tion
y MHC I and MHC II molec
ules. The
increased expression of MHC class I and II molecules on antien- presentin cell
s increases the
likelihood of antien capture and the involvement of additional lymphocytes. In
addition to the
a
ove listed actions, IFN- is a stron stimulator of macrophaes and
oosts their
tumoricidal
activity. Thus, IFN- enhances the immune response in a num
er of key ways. IFN- pr
oduction can

e stimulated in mature Th1 cells
y two means: (1) liation of the T-cell recep
tor (TCR)
y
MHC-peptide antien presentation or (2) cytokine stim- ulation
y IL-12 and
IL-18. IL-12
and IL-18 act syneristically to stimulate IFN- production, even in the a
sence
of TCR
liation. In addition, IL-12 and IL-18 can activate IFN- secretion
y CD8 + lymph
ocytes. IL-2
Th1 cells also secrete IL-2 in addition to IFN- . IL-2 is also known as the T-cell
rowth factor.
. It drives the rowth and differentiation of
oth T and B cells and induces lyt
ic activ- ity in
NK cells. IL-2 and IFN- induce the development of Th1 cells, which, in turn, indu
ces macrophae

activation and delayed type hypersensitivity. Th1 cells stimulate the production
of IG1 and IG3
opsonizin and complement fixin anti
odies
y antien-activated B cells. These
iso- types assist
in the kinds of cell-mediated immune responses that are driven
y Th1 cells. IL2 alone can
activate proliferation of Th2 cells and helps to enerate IG1 and IE producin
cells. The
clonal expansion of activated T helper cells is a necessary part of mountin an
adequate immune
response to any immuno- loic challene. The cytokine network that develops will
continue to
reulate T-cell rowth and differentiation until the challene is one and the r
esponse must
su
side. Transcription of the ene for IL-2 and IL-2R
eins within 1 hour of TC
R liation. The
functional IL-2R con- sists of , , and or
and
su
units. The
and
su
units increase
the
af nity of the receptor for IL-2 and are respon- si
le for most of the sinal
transduction
throuh the receptor. The
chain is also shared
y the receptors for IL-4, IL-7,
IL-9, IL-15,
and IL-21. 23 The importance of the
chain is demonstrated in individuals who hav
e mutations in
this chain. These persons have X-linked severe com
ined immunodeficiency syndrom
e and lack
functional T and B cells. 24 Th2 Cytokines IL-4 As mentioned previously, Th2 cel
ls are primarily
responsi-
le for anti
ody-mediated immunity. IL-4 is one of the key cytokines r
eulatin Th2
immune activities and helps drive anti
ody responses in a variety of diseases. 2
4 The IL-4
receptor is expressed on lymphocytes and on numerous Th1 IFN- Th2 Nave IL-4 Tre
IL-10 IL-10
IL-4 IL-12 FIGURE 53. Development of T helper and T reulatory cells. 1814_Ch05_0
72-084.qxd
7/10/09 2:40 PM Pae 77 78 SECTION 1 Nature of the Immune System nonhematopoie
tic cell types.
IL-4 activity on nave T cells turns on the enes that enerate Th2 cells and turn
s off the enes
that promote Th1 cells, such as IFN- and IFN- R su
units. The chemokine MCP-1 enh
ances IL-4
produc- tion
y nave T cells and could play a role in movin nave T cells toward t
he Th2
pathway. Th2 cells are responsi
le for reulatin many aspects of the immune res
ponse, includin
those related to alleries, autoimmune diseases, and htin off parasites. Amon
the many enes
induced
y IL-4 are MHC-I, IL-5, IL-13, and the co-stimulatory molecules B7.1 an
d B7.2. IL-4
promotes the production of IG2a and IE and, alon with IL-5, drives the differ
entiation and
activation of eosinophils in
oth alleric immune responses and the response to
para- sitic
infections. 24 IL-13 is a cytokine with many of the same properties as IL-4, and

oth cytokines
induce worm expul- sion and favor IE-class switchin. IL-10 IL-10 has anti-in amm
atory and
suppressive effects on Th1 cells. It is produced
y monocytes, macrophaes, CD8 T
cells, and Th2

CD4
T cells. It inhi
its antien presenta- tion
y macrophaes and dendritic cel
ls and
stimulates CD8
T cells. It also induces the production of MHC-II on B cells. How
ever, one of
the major effects of IL-10 is the inhi
ition of IFN- production via the suppressi
on of IL-12
synthesis
y accessory cells and the promotion of a Th2 cytokine pattern. 25 Thu
s, in contrast to
most other cytokines, IL-10 serves as an antaonist to IFN- it is a down-reulator
of the immune
response. Cytokines Associated with T Reulatory Cells The third major su
class
of CD4
T cells
are the T reu- latory (Tre) cells. Tres are CD4
CD25 T cells that are selecte
d in the
thymus. 26 They play a key role in esta
- lishin peripheral tolerance to a wide
variety of
self-antiens, allerens, tumor antiens, transplant antiens, and infec- tious
aents. CD4
CD25
Tres affect T cell activity primarily throuh the actions of TGF- . TGFinduces
expression of Foxp3, a transcription factor that causes Tre cells to suppress t
he activity of
other T cells. 27 Tres may
e found in transplanted tissue and help to esta
lis
h toler- ance to
the raft
y the host immune system throuh the alteration of antien presentati
on. Tres are
also responsi
le for inducin IL-10 and TGF- expression in adaptive T reulatory
1 (Tr1) cells
in the peripheral circulation. Tr1 cells are CD4
T cells that are induced from
antien-activated nave T cells in the pres- ence of IL-10. They exert their suppr
essive
activities on
oth Th1 and Th2 cells
y producin more IL-10, TGF- , or IL-5. T-ce
ll suppression
occurs throuh IL-10 inhi
ition of proin ammatory cytokines and inhi
ition of cost
imula- tory
molecule expression on antien presentin cells (APCs), while TGF- down-reulate
s the
function of APCs Th1 cell IFN- MHC-II ICAM VCAM IL-2 TNF- nd - IFN-R IL-12R IL-18
R
Enhanced activity of: Cytotoxic T cells Natural killer cells Antien presentatio
n Th2 cell IL-4
MHC-I IL-5 IL-6 IL-10 IL-13 IL-4R B7.1 & B7.2 M-CSF Enhanced activity of: Anti
o
dy formation
Alleric response Antiparasite response Antien presentation Tre cell IL-10 TGF
- IL-5 CTLA-4
IL-2R Foxp3 Est blishment of peripher l toler nce Inhibition of: Th1 cells Th2 ce
lls Antigen
presenting cells FIGURE 54. Individu l T cell cl sses produce different prod- uct
s th t
determine the r nge of ctivities for the cl ss. 1814_Ch05_072-084.qxd 7/10/09
2:40 PM P ge 78
CHAPTER 5 Cytokines 79 nd blocks prolifer tion nd cytokine production by CD4
T
cells. All of
these ctivities le d to down-regul tion of the immune response nd the pre
vention of
chronic infl mm tion. These two types of regul tory T cells oper te through
ne
twork of
cytokines to est blish peripher l toler nce to cert in ntigens; therefore, they
pl y key role
in limiting utoimmunity. The rel tive import nce of e ch phenotype depends l rg

ely on the
ntigen involved, the context of nti- gen present tion, nd the biology of the
tissue.
ERYTHROPOIETIN AND COLONY STIMULATING FACTORS The colony stimul ting f ctors (CS
Fs) include IL-3,
ery- thropoietin (EPO) nd gr nulocyte, m croph ge, nd gr nulocyte-m croph ge c
olony stimul ting
f ctors (G-CSF, M-CSF, nd GM-CSF, respectively). 18,2830 In response to infl mm
tory cytokines
such s IL-1, the dif- ferent colony stimul ting f ctors ct on bone m rrow cell
s t different
development l st ges
nd promote specific colony form tion for the v rious c
ell line ges.
IL-3 is mul- tiline ge colony stimul ting f ctor th t induces CD34 bone m rrow
stem cells to
form T nd B cells. In conjunc- tion with IL-3, the CSFs direct imm ture bone m
rrow stem cells
to develop into red blood cells (RBCs), pl telets, nd the v rious types of whit
e blood cells
(Fig. 55). IL-3 cts on bone m rrow stem cells to begin the differenti tion cycle
, nd the
ctivity of IL-3 lone drives the stem cells into the lymphocyte differenti tion
p thw y. GM-CSF
cts to drive differenti tion tow rd other white cell types. If M-CSF is c
tiv ted, the
cells become m croph ges. M-CSF lso incre ses ph gocytosis, chemo- t xis, nd
ddition l
cytokine production in monocytes nd m croph ges. If G-CSF is ctiv ted, the
cells become
neutrophils. G-CSF enh nces the function of m ture neu- trophils nd ffects
the surviv l,
prolifer tion,
nd differenti tion of ll cell types in the neutrophil line ge.
It decre ses
IFN- production nd incre ses IL-4 production in T cells, nd it mobilizes multip
otenti l stem
cells from the bone m rrow. These stem cells re utilized to rep ir d m ged tiss
ues nd cre te
new v scul ture in these re s. These ctivities re necess ry to reconstruct ti
ssues follow- ing
n infection. However, IL-3 in conjunction with GM-CSF drives the develop
ment of b sophils
nd m st cells, while the ddition of IL-5 to IL-3 nd GM-CSF drives the cells t
o develop into
eosinophils. The net effect is n incre se in white blood cells to respond to th
e ongoing
in mm tory processes. EPO regul tes RBC production in the bone m rrow but is prim
rily produced
in the kidneys. The protein in its phys- iologic l form is 34 kD monomer with
high
c rbohydr te content. Glycosyl tion of the protein is required for ctiv- ity
nd ccounts for
the structur l differences between recombin nt EPOnd EPO- . Both proteins h
ve the s me
mino cid sequence nd simil r ctivities s endoge- nous EPO, but EPO- is the f
orm licensed
for clinic l use by the FDA. EPO- is often prescribed to improve the red cells co
unts for
individu ls with nemi nd for those with c ncer who h ve undergone r di
tion nd
chemother py. RBC prolifer tion induced by EPO improves oxygen - tion of the

tissues
nd
eventu lly switches off EPO production. The norm l serum EPO v lues r nge fro
m 5 to 28 U/L but
must be interpreted in rel tion to the hem - tocrit, s levels c n incre se by u
p to
thous ndfold during nemi . CYTOKINES AND ANTICYTOKINE THERAPIES Cytokine-inhibi
ting biologics
disrupt the inter ction between cytokines nd their cogn te receptors. 31 Init
i l stud- ies
used murine monoclon l ntibodies th t were ble to function only for short
periods of time
before the host mounted n immune response g inst them. Recombin nt DNA tech
niques h ve
llowed for the production of hum n- ized monoclon l ntibodies th t re much le
ss immunogenic
nd th t function s cytokine nt gonists. An ex mple is inflixim b (Remic de),
chimeric
ntibody cont ining hum n const nt regions nd murine ntigen-speci c rms th t b
ind hum n
TNF- . Remic de blocks the ctivity of TNF- in rheum toid rthritis (RA) nd Crohns
dise se. A
single dose llevi tes symptoms nd reduces swollen joints BM Stem T & B Cells I
L-3 IL-3 GM-CSF
GM-CSF M-CSF G-CSF IL-3, GM- CSF, IL-5 IL-3, GM- CSF B sos Eos PMN M FIGURE 55. Ma
turation o

white blood cell lineages under the in uence o

colony stimulating
actors. 1814_C
h05_072-084.qxd
7/10/09 2:40 PM Page 79 80 SECTION 1 Nature o
the Immune System in RA patien
ts
or up to 4
weeks. Multiple in
usions achieve 60 to 70 percent improvement in symptoms
in these
individuals. Monoclonal antibodies can be
urther humanized so that the
complementarity-determining regions are the only murine components remaini
ng (e.g.,
Zenapax, which is directed at human IL-2R ). These antibodies generally have low
er a
nity and
extended hal
-li
e, but reduced immuno- genicity. As an alternative, transgenic
mice that have
human immunoglobulin genes are able to produce
ully human monoclonal antibodies

rom mouse
hybridoma cells
ollow- ing immunization. These antibodies have high a
nities and
long
hal
-lives and are not immunogenic. Another approach to anti-cytokine therapy is
the development o
hybrid proteins containing cytokine receptor binding sites attached to
immunoglobulin
constant regions. The only member o
this class that has been approved
or human
use is
etanercept (Enbrel). Enbrel consists o
the extracellular domains o
the type 2
TNF receptor

used to the heavy chain constant region o

IgG1. This
usion pro- tein can bind
TNF- , block its
activity, and has a 4.8-day hal
-li
e in serum. However, about 16 percent o
tre
ated individuals
develop antibodies to the TNFR2 moiety. Some o
these antibodies may be abl
e to bind to
native receptors and activate signaling pathways, thus mimicking TNF- activity an
d negating the
proteins therapeutic e

ects. 32 CLINICAL ASSAYS FOR CYTOKINES Activation o

the
immune system

is a signi cant component o

autoimmune diseases such as rheumatoid arthritis, 11
pso- riasis, 33
and in ammatory bowel disease. 34,35 In addition, the role o
cytokines in develop
ing an
e

ective immune response to certain in

ectious diseases has been well studied.
2 For example,
tuberculosis is controlled by cell-mediated immu- nity, not humoral immunity, an
d immunity is
there
ore driven by Th1 cytokines. Clinical evaluation o
the cytokine pro le in t
he patient
could be o
prognostic and diagnostic value to the physician. 36,37 Although the
re are several
cytokine assay
ormats avail- able in Europe, none have been approved
or clinic
al use by the FDA
at this time. The most basic test is the ELISpot assay, which employs the enzyme
-linked
immunosorbent assay (ELISA; see Fig. 56) technique on in vitro activated peripher
al white blood
cells. In this process, either a mon- oclonal or polyclonal antibody speci
i
c
or the
chosen cytokine is precoated onto a microplate. Antigen-stimulated, mitogen-stim
ulated (positive
control), or saline-stimulated (negative control) white blood cells are pipetted
into the wells,
and the microplate is placed into a humidi
ied CO 2 incubator at 37C
or a speci ed
period o

time. During the incubation period, the immobilized antibody, in the imme- diate
vicinity o

the secreting cells, binds the secreted cytokine. A

ter any cells and unbou
nd substances are
washed away, a biotinylated polyclonal antibody speci
ic
or the chosen cytokine
is added to the
wells. Following a wash to remove any unbound biotinylated antibody, alkaline- p
hosphatase
conjugated to streptavidin is added. Unbound enzyme is subsequently washed away,
and a substrate
solu- tion is added. A colored precipitate
orms and appears as spots at the sit
es o
cytokine
localization, with each spot representing an individual cytokine-secreting cell.
The spots can be
counted with a stereomicroscope or with an auto- mated ELISpot reader. Multiplex
ed ELISAs utilize
several detector antibodies bound to individual microwells or antibody microarra
ys and allow
or
simultaneous detection o
several cytokines
rom serum. 38 Current
ormulations
allow
or the
detection o
12 to 25 pro- and anti-in ammatory cytokines in one reaction. In the
microarray

ormat, each well on the slide contains a microarray o

spotted antibodies, with

our spots
or
each o
the 12 cytokines plus additional spots
or positive and negative control
s. The replicate
spots allow
or acquisition o
reliable quantitative data
rom a single sample.
New microbead
assays allow
or the simultaneous detec- tion o
multiple cytokines in a single
tube. Each bead
type has its own uorescent wavelength, which, when combined with the uorescent sec
ondary
antibody bound to a speci c cytokine, allows
or the detection o
up to 100 di

er
ent ana- lytes

in one tube. The use o

a multiplexed bead array enabled simultaneous m
easurement o

representative proin- ammatory cytokines such as IL-1 , IL-1RA, IL-6, IL-8, and TN
F- ; Thl/Th2
distinguishing cytokines IFN- , 1. Capture antibody attached to well. 2. Cell c
ulture
supernatant containing cytokines is added. Wash o

unbound material. 3. Enzymelabeled secondary

antibody is added. Wash o

unbound antibody. 4. Substrate changes color in the

presence o
the
enzyme-labeled antibody bound to the cytokine. FIGURE 56. A standard ELISA assay.
1814_Ch05_072-084.qxd 7/10/09 2:40 PM Page 80 IL-2, IL-4, IL-5, and IL-10; no
nspeci
ic acting
cytokines IFN- , IL-13, IL-15, and IL-17; and chemokines IP-10, MCP-1, MIP-1 , and R
ANTES. One
drawback o
protein-based technologies is the short hal
-li
e o
certain cytokin
es. This can be
overcome by look- ing at RNA expression in cells using reverse transcription pol
ymerase chain
reaction (PCR). The PCR product is made using a uorescent-labeled primer and can
be hybridized
to either solid-phase or liquid microarrays. Solid-phase arrays have up to 40,00
0 spots
containing speci c ssDNA oligonu- cleotides representing individual genes. Clinica
lly use
ul
arrays generally have substantially
ewer genes represented. Fluorescence o
a s
pot indicates
that the gene was expressed in the cell and that the cell was producing the cyto
kine. The liquid
arrays utilize the same beads as the antibody microbead arrays discussed earlier
. However,
instead o
anti- bodies, these beads have oligonucleotides on their sur
aces and
allow up to 100
di

erent cDNAs to be identi ed. The combination o

the bead uorescence and the uore
scence o

the labeled cDNA produces an emission spectrum that identi es the cytokine gene th
at was
expressed in the cells. SUMMARY Cytokines are the
unctional regulatory proteins

or the immune
system and are secreted by white blood cells and a variety o
other cells. They
exert
activity-modulating e

ects on cells o
the hematopoietic and immune systems thr
ough the
activation o
cell-bound receptor proteins. Cytokines are induced in response to
speci c stimuli
such as bacterial lipopolysaccharides,
lagellin, or other bacterial products th
rough the
ligation o
cell-adhesion molecules or through the recognition o

oreign antige
ns by host
lymphocytes. The e

ects o
cytokines in vivo include regulation o
growth, di

erentiation, and
gene expression by many di

erent cell types, including leukocytes, through both

autocrine and
paracrine activities. Occasionally, cytokines will also exert systemic or endocr
ine activities.
Individual cytokines do not act alone but in conjunction with many other cyto
kines that are
induced during the process o
immune activation. The resulting network o
cyto
kine expression
regulates the activities o
leukocytes by modulating cytokine activities; this l

eads to the
elimination o
the in
ection. The combined cytokines produce a spec- trum o
act
ivities that lead
to the rapid generation o
innate and adaptive immune responses. In
act, the ab
ility or inability to generate certain cytokine patterns o
ten determines the outcome and clini
cal course o

in
ection. In certain cir- cumstances, massive overproduction produces a cytokine
CHAPTER 5
Cytokines 81 storm that leads to shock, multiorgan
ailure, or even death, thus c
ontributing to
pathogenesis. The cytokines were originally thought to act solely on cells o
th
e immune system,
but we now know that many cytokines also act on cells outside the immune
system. Vascular
endothelial cells, astrocytes, liver cells, muscle cells, and many other cell
types respond
to cytokines. The pleiotropic nature o
cytokine activity relates to the widespread
distribution o
cytokine receptors on many cell types and the ability o
cytokin
es to alter
expression o
many genes. Several di

erent cytokines may share properties (i.e.

, they activate
some o
the same pathways and genes). This redundancy may be explained by the sh
aring o
cytokine
receptor subunits by many cytokines. However, redundancy o
cytokine activities
cannot be
explained entirely by shared receptor subunits. TNF- , IL-1, and IL-6 exert many o

the same
biological activities, but each utilizes a distinct receptor. Here, the cytokine
s are activating
similar immune response pathways through di

erent signaling pathways. The major

cytokines
involved in the initial stages o
the in ammatory response are IL-1, IL-6, TN
F- , and the
chemokines. These cytokines are responsible
or many o
the physical symptoms at
tributed to
in ammation such as
ever, swelling, pain, and cellular in ltrates into damaged tiss
ues.
Engagement o
the TCR by the appropriate peptide- MHC complex triggers clonal ex
pansion o
CD4
T helper cells. Nave T cells can di

erentiate into Th1, Th2, or Treg cell linea

ges, with the
spectrum o
cytokines expressed in the initial response determining the line
age. The Th1
lineage is driven by the expression o
IL-12 by dendritic cells and is primarily
responsible
or
cell-mediated immu- nity, while Th2 cells drive antibody-mediated immunity and a
re
developmentally regulated by IL-4. Treg cells are derived
rom IL-10-responsive
nave T cells and
help to regulate the activities o
Th1 and Th2 cells through the activities o
I
L-10 and TGF- .
The colony stimulating
actors (CSFs) include IL-3; ery- thropoietin (EPO); an
d granulocyte,
macrophage, and granulocyte-macrophage colony stimulating
actors (G-CSF, M-CSF
, and GM-CSF,
respectively). The CSFs are respon- sible
or inducing di

erentiation and growt

h o
all white
cell classes and help to drive the immune response by ensur- ing that the num

bers and types

o
cells available are adequate. Anticytokine therapies continue to be develo
ped
or many
pathological conditions such as Crohns disease and rheuma- toid arthritis. Thes
e therapies
will drive the need
or laboratory assays
or cytokines and other markers o

immune
activation. 1814_Ch05_072-084.qxd 7/10/09 2:40 PM Page 81 82 SECTION 1 Nature
o
the Immune
System A 55-year-old woman being treated
or acute lymphocytic leukemia (ALL) wa
s
ound to be
severely neutropenic (477 neutrophils/mL). Her physicians
elt that increasing t
he dosage o
her
chemotherapy drugs was necessary to eliminate the cancer, but they could n
ot risk
urther
lowering o
the neutrophil count due to the increased risk o
in
ection. Since t
imely
ull-dose
chemotherapy greatly improves survival, it was considered necessary to continue
with treatment.
However, in order to continue with chemotherapy, treatment
or the neutrope
nia was also
necessary. While undergoing treatment, the patient was also enrolled in a
research study
designed to look at cytokine expression in ALL patients with neutropenia. The st
udy utilized a
liquid bead array that included the colony stimulating
actors and the cyt
okines typically
seen in the innate immune response and in Th1 and Th2 responses. Questions a. Wh
at colony
stimulating
actor should the physi- cians prescribe to overcome the neutropenia
? b. What are
some o
the cytokines that might be detected in a Th1 type response? c. Wh
at are some o

the cytokines that might be detected in a Th2 type response? CASE STUDY
1814_Ch05_072-084.qxd 7/10/09 2:40 PM Page 82 CHAPTER 5 Cytokines 83 1. The a
bility o
a
single cytokine to alter the expression o
several genes is called a. redundancy
. b. pleiotropy.
c. autocrine stimulation. d. endocrine e

ect. 2. Which o
the
ollowing can be
attributed to
IL-1? a. Mediator o
the innate immune response b. Di

erentiation o
stem cells
c. Halts growth
o
virally in
ected cells d. Stimulation o
mast cells 3. Which o
the
ollowing
are target cells

or IL-3? a. Myeloid precursors b. Lymphoid precursors c. Erythroid precursors d

. All o
the
above 4. A lack o
IL-4 might result in which o
the
ollowing? a. Inability to g
ht o

viral
in
ections b. Increased risk o
tumors c. Lack o
IgE d. Decreased eosinophil co
unt 5. Which o

the
ollowing is also known as the T-cell growth
actor? a. IFN- b. IL-12
c. IL-2 d.
IL-10 6. Which chemokine receptor does HIV need
or cell- speci c binding? a. CCR1
b. CCR3 c.
CCR4 d. CCR5 7. IFN- and IFN- di

er in which way
rom IFN- ? a. IFN- and IFN- are cal
led
immune inter
erons, and IFN- is not. b. IFN- and IFN- primarily activate macrophage
s, while
IFN- halts viral activity. c. They are made primarily by activated T cells, while
IFN- is made

by broblasts. d. IFN- and IFN- inhibit cell proli

eration, while IFN- stimulates ant
igen
presentation by class II MHC molecules. 8. A patient in septic shock caused by a
gram-negative
bacterial in
ection exhibits the
ollowing symptoms: high
ever, very low blood
pressure, and
disseminated intravascular coagulation. Which cytokine is the most likely contri
butor to these
symptoms? a. IL-2 b. TNF c. IL-12 d. IL-7 9. IL-10 acts as an antagonist to what
cytokine? a.
IL-4 b. TNF- c. IFN- d. TGF- 10. Which would be the best assay to measure a speci
i
c cytokine?
a. Blast
ormation b. T-cell proli
eration c. Measurement o
leukocyte chemotaxi
s d. ELISA
testing REVIEW QUESTIONS 1814_Ch05_072-084.qxd 7/10/09 2:41 PM Page 83 84 SEC
TION 1 Nature o

the Immune System Re

erences 1. Thomson, A. The Cytokine Handbook. Academic
Press, London,
1991. 2. Lucey, DR, Clerici, M, and Shearer, GM. Type 1 and type 2 cytokine dysr
egulation in
human in
ectious, neoplastic, and in ammatory diseases. Clin Microbiol Rev 9:532562
, 1996. 3.
Glickstein, LJ, and Huber, BT. Karoushideath by overwork in the immune system. J
Immunol
155:522524, 1995. 4. http://www.genenames.org/gene
amily/il.php. Accessed March 1
1, 2008. 5.
Heinrich, PC, Behrmann, I, Muller-Newen, G, Schaper, F, and Graeve, L. Interleuk
in-6-type
cytokine signaling through the gp130/Jak/STAT pathway. Biochem J 334:297314, 1998
. 6. Lim, MS,
and Elenitoba-Johnson, KSJ. The molecular pathol- ogy o
primary immunode ciencies
. J Mol Diag
6:5983, 2004. 7. Dinarello, CA. Interleukin-1 and interleukin-1 antagonism. Blood
77:16271652,
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the hypothalamic- pituitary
-adrenal axis by
cytokines: Actions and mechanisms o
action. Physiol Rev 79:171, 1999. 9. Metcal

, D. The
molecular control o
cell division, di

eren- tiation commitment, and maturation

in hematopoietic
cells. Nature 339:2730, 1989. 10. Lockesley, RM, Killeen, N, and Lenardo, MJ. The
TNF and TNF
receptor super
amilies: Integrating mammalian biology. Cell 104:487501, 2001. 11.
Feldmann, M,
Brennan, FM, and Maini, RN. Role o
cytokines in rheumatoid arthritis.
Ann Rev
Immunol 14:397440, 1996. 12. Kim, CH. Chemokine-chemokine receptor network i
n immune cell
tra

icking. Curr Drug TargetsImmune, Endocrine, Metab Disord 4:343361, 2004. 1

3. Reiche,
EMV, Bonametti, AM, Voltarelli, JC, Morimoto, HK, and Watanabe, MAE. Genetic
polymorphisms
in the chemokine and chemokine receptors: Impact on clinical course and therapy
o
the Human
Immunode
iciency Virus type 1 in
ection (HIV-1). Curr Medicin Chem 14:13251334, 2
007. 14.
Rahimi, RA, and Leo
, EB. TGF- signaling: A tale o
two responses. J Cell Biochem
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eron-producing cells and plas
macytoid

dendritic cell precursors. Ann Rev Immunol 23:275306, 2005. 16. Van Weyenbergh, J
, Weitzerbin,
J, Rouillard, D, Barral-Netto, M, and Liblau, R. Treatment o
multiple sclerosis
patients with
inter
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eron al
acon-1: A review
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its
pharmacology and therapeutic e
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chronic hepatitis C. Drug
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2001. 18. Currier, JR. T-lymphocyte activation and cell signalling. In Detrick,
B, Hamilton, RG,
and Folds, JD (eds): Manual o
Molecular and Clinical Laboratory Immunology, ed.
7. ASM Press,
Washington, DC, 2006. 19. Tsugi, NM. Antigen-speci
ic CD4 + T cells in the intes
tine. In amm
Aller Drug Targets 5:191201, 2006. 20. Szabo, SJ., Costa, GL, Zhang, X, and Glimc
her, LH. A
novel transcription
actor, T-bet, directs Th1 lineage commitment. Cell 100:65566
9, 2000. 21.
Boehm, U, Klamp, T, Groot, M, and Howard, JC. Cellular responses to inter
eron
gamma. Ann
Rev Immunol 15:749795, 1997. 22. Murphy, KM, and Reiner, SL. The lineage decisio
ns o
helper T
cells. Nature Rev Immunol 2:933944, 2002. 23. Sugamura, K, Asao, H, Kondo, M, Tan
aka, N, Isii,
N, Ohbo, K, Nakamura, M, and Takeshita, T. The interleukin-2 recep- tor
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ts role in
multiple cytokine receptor complexes and T cell development in XSCID. Annu
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14:179205, 1996. 24. Abbas, AK, Murphy, KM, and Sher, A. Functional diversity o

helper T
lymphocytes. Nature 383:787793, 1996. 25. Assadullah, K, Sterry, W, and Volk
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Interleukin-10 therapyreview o
a new approach. Pharmacol Rev 55:241269, 2003. 26.
Wan, YY, and
Flavell, RA. The roles
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regu
latory T cells.
Immunolog Rev 212:114130, 2006. 27. Fonenot, JD, and Rudensky, AY. A well adapted
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orkhead
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Kremastinos, DT, and Uhal, BD. Hematopoietic colony stimulating
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Sloand, EM, Kim, S, Maciejewski, JP, Van Rhee, F, Chauduri, A, Barrett, J, and Y
oung, NS.
Pharmacologic doses o
granu- locyte colony stimulating
actor a

ect cytokine p
roduction by
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Griswold, DE, and Shealy, D. Coming o
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2002. 32. Engelmann, H, Holtmann, H, Nophar, Y, Hadas, E, Leitner, O, and Wallac

h, D. Antibodies
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orm o
tumor necrosis
actor (TNF) receptor have TNF-like activity
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265:1449714504, 1990. 33. Jacob, SE, Nassiri, M, Kerdel, FA, and Vincek,
V. Simultaneous
measurement o
multiple Th1 and Th2 serum cytokines in psoriasis and correlation
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Judmaier, G. Interleukin-1 receptor antagonist in di

erential diagnosis o
in
l
ammatory bowel
diseases. Eur J Gastroenterol Hepatol 7:10311036, 1995. 35. Desai, D, Faubion, WA
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inter
eron gamma assay in the diagnosis o
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Dosanjh, DPS, Hinks, TSC, Innes, JA, Deeks, JJ, Pasvol, G, Hack
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/10/09 2:41 PM
Page 84 6 Complement System 85 LEARNING OBJECTIVES A
ter nishing this chapter, th
e reader will
be able to: 1. Describe the nature o
the complement components. 2. Di

erentiat
e between the
classical and the alternative pathways, including proteins and activators involv
ed in each. 3.
Discuss
ormation o
the three principal units o
the classical pathway: recogni
tion, activation,
and membrane attack units. 4. Describe how initiation o
the mannose-binding lec
tin (MBL) pathway
occurs. 5. Explain how C3 plays a key role in all pathways. 6. Describe regulato
rs o
the
complement system. 7. Discuss the role o
the
ollowing cell membrane receptors:
CR1, DAF, MCP,
CR2, CR3, CR4, CD59, and MCP. 8. Relate biological mani
estations o
complement
activation to
generation o
speci c complement products. 9. Describe the complement de ciency asso
ciated with
the
ollowing condi- tions: hereditary angioedema and paroxysmal nocturnal hemog
lobinuria. 10.
Di

erentiate tests
or
unctional activity o
complement
rom measure- ment o

individual
complement components. 11. Analyze laboratory ndings and indicate disease implica
tions in
relation to complement abnormalities. KEY TERMS Activation unit Alternative path
way Anaphylatoxin
Bystander lysis C1 inhibitor (C1INH) C4-binding protein (C4BP) Classical pathway
Complement
receptor type 1 (CR1) Decay-accelerating
actor (DAF) Factor H Factor I Hemolyti
c titration (CH

50 ) assay Hereditary angioedema Immune adherence Lectin pathway Mannose-binding

lectin (MBL)
Membrane attack complex Membrane co
actor protein (MCP) Paroxysmal nocturnal hem
oglobinuria (PNH)
Properdin Recognition unit S protein 1814_Ch06_085-106.qxd 7/10/09 2:47 PM Pa
ge 85 86 SECTION
1 Nature o
the Immune System As described in Chapter 1, complement is a complex
series o
more
than 30 soluble and cell-bound proteins that interact in a very speci c way to enh
ance host
de
ense mechanisms against
oreign cells. Originally recognized in the 1890s as
a heat-labile
substance present in normal nonimmune serum, complement was so coined by Paul Eh
rlich because it
complements the action o
antibody in destroying microorganisms. 1 Jules B
ordet was awarded
the Nobel Prize in 1919
or his role in elucidating the nature o
complement. Wh
ile complement
promotes opsonization and lysis o

oreign cells and immune complexes, chronic a
ctivation can
lead to in ammation and tissue damage. It is the latter that occurs in autoimmune
diseases;
there
ore, complement acti- vation needs to be care
ully regulated in order to m
inimize tissue
damage. In addition to the major proteins involved in activation, there are nume
rous proteins
that act as con- trols or regulators o
the system. The three pathways
or activ
ation o

complement will be discussed along with major system controls. Most plasma compl
ement proteins
are synthesized in the liver, with the exception o
C1 components, which
are mainly
produced by intestinal epithelial cells, and
actor D, which is made in adipose
tissue. 1,2 Other
cells, such as mono- cytes and macrophages, are additional sources o
ear
ly complement
components, including C1, C2, C3, and C4. 3,4 Most o
these proteins are inactiv
e precursors, or
zymogens, which are converted to active enzymes in a very precise order. Table 61
lists the
characteristics o
the main com- plement proteins. CHAPTER OUTLINE THE CLASSICAL
PATHWAY The
Recognition Unit The Activation Unit The Membrane Attack Complex THE ALTERNATIVE
PATHWAY THE
LECTIN PATHWAY SYSTEM CONTROLS Regulation o
the Classical and Lectin Pathways R
egulation o
the
Alternative Pathway Regulation o
Terminal Components COMPLEMENT RECEPTORS AND T
HEIR BIOLOGICAL
ROLES BIOLOGICAL MANIFESTATIONS OF COMPLEMENT ACTIVATION COMPLEMENT AND DISEASE
STATES COMPLEMENT
DEFICIENCIES Major Pathway Components Regulatory Factor Components LABORATORY DE
TECTION OF
COMPLEMENT ABNORMALITIES Immunologic Assays o
Individual Components Assays
or
the Classical
Pathway Alternative Pathway Assays Interpretation o
Laboratory Findings Complem
ent Fixation
Testing SUMMARY CASE STUDIES EXERCISE: EZ COMPLEMENT CH 50 ASSAY REVIEW QUESTION
S REFERENCES
Table 6-1. Proteins o
the Complement System SERUM PROTEIN MOLECULAR WEIGHT, k
D CONCENTRATION

(g/mL) FUNCTION Classical Pathway C1q 410 150 Binds to FC region o

IgM and IgG
C1r 85 50
Activates C1s C1s 85 50 Cleaves C4 and C2 C4 205 300600 Part o
C3 convertase (C4
b) C2 102 25
Binds to C4b
orms C3 convertase C3 190 1200 Key intermediate in all pathways C5 1
90 80 Initiates
membrane attack complex C6 110 45 Binds to C5b in MAC C7 100 90 Binds to C5bC6 i
n MAC C8 150 55
Starts pore
ormation on membrane C9 70 60 Polymerizes to cause cell lysis 1814_
Ch06_085-106.qxd
7/10/09 2:47 PM Page 86 CHAPTER 6 Complement System 87 The complement system c
an be activated
in three di
-
erent ways. The rst is the classical pathway, which involves nine
proteins that
are triggered by antigenantibody com- bination. Pillemer and colleagues later
discovered
an antibody-independent pathway in the 1950s, and this plays a major role as a n
atural de
ense
system. 5 The second path- way, the alternative pathway, was originally called t
he properdin
system, because the the protein properdin was thought to initiate this path
way. Now,
however, it is known that properdins major
unction is to stabilize a key enzyme
complex
ormed
along the pathway. The third pathway, the lectin pathway, is another antibody-in
dependent means
o
activating complement proteins. Its major constituent, mannose- (or mannan-)
binding lectin
(MBL), adheres to mannose
ound mainly in the cell walls or outer coating o
bac
teria, viruses,
yeast, and protozoa. While each o
these pathways will be considered separately,
activation
seldom involves only one pathway. The complement system plays a major part in th
e in am- matory
response directed against
oreign antigens. Although the end product o
compleme
nt activation is
lysis o
the invading cell, many other important events take place along the way
. Complement

ragments acting as opsonins,

or which speci
ic receptors are present on phagoc
ytic cells,
enhance metabolism and clear immune complexes. In
act, uptake o
immune complex
es in the spleen
appears to be complement-dependent. 1 Complement components are also able to inc
rease vascular
permeability, recruit monocytes and neutrophils to the area o
antigen concentra
tion, and trigger
secretion o
immunoregulatory molecules that ampli
y the immune response. 4
Any de ciencies
in the com- plement system can result in an increased susceptibility to in
ectio
n or in the
accumulation o
immune complexes with possible autoimmune mani
estations. Thus,
these proteins
play a signi cant role as a host de
ense mechanism. THE CLASSICAL PATHWAY The cla
ssical pathway
or cascade is the main antibody- directed mechanism
or triggering complem
ent activation.
However, not all immunoglobulins are able to activate this pathway. Those classe
s that can
include IgM, IgG1, IgG2, and IgG3. IgM is the most e
cient, because it has multip
le binding

sites; thus, it takes only one molecule attached to two adjacent antigenic deter
minants to
initiate the cascade. Two IgG molecules must attach to antigen within 30 to 40 n
m o
each other
be
ore complement can bind, and it may take at least 1000 IgG molecules to ensur
e that two are
close enough to initiate such binding. 1,6 Some epitopes, notably the Rh group,
are too
ar apart
on the cell
or this to occur, so they are unable to x complement. Within the IgG
group, IgG3 is
the most e

ective,
ollowed by IgG1 and then IgG2. 6 In addition to antibody, t
here are a
ew
substances that can bind complement directly to initiate the classical cas- cade
. These include
C-reactive protein, several viruses, mycoplasmas, some protozoa, and certain gra
m-negative
bacteria such as Escherichia coli. 6 However, most in
ectious agents can directl
y activate only
the alternative pathway. Complement activation can be divided into three main st
ages, each o

which is dependent on the grouping o

cer- tain reactants as a unit. The rst stag
e involves C1,
which is known as the recognition unit. Once C1 is xed, the next components activ
ated are C4,
C2, and C3, known collec- tively as the activation unit. C5 through C9 comprise
the membrane
attack complex, and it is this last unit that com- pletes lysis o
the
oreign p
article. Each o

these is discussed in detail in the

ollowing sections. Figure 61 depicts a simpli ed scheme
o
the entire pathway. The Recognition Unit The rst complement component to bind
is C1, a
molecu- lar complex o
740,000 d. It consists o
three subunits: C1q, C1r, and C
1s, which are
stabilized by calcium. 1,6 The com- plex is made up o
one Clq subunit and two e
ach o
the C1r
and C1s subunits. Although the C1q unit is the part that binds to antibody molec
ules, the C1r and
C1s subunits gen- erate enzyme activity to begin the cascade. The (C1r, C1s) 2 c
omplex is an
S-shaped structure with several domains o
Table 6-1. Proteins o
the Complement
SystemContd
SERUM PROTEIN MOLECULAR WEIGHT, kD CONCENTRATION (g/mL) FUNCTION Alternativ
e Pathway
Factor B 93 200 Binds to C3b to
orm C3 convertase Factor D 24 2 Cleaves
actor
B Properdin 55
1525 Stabilizes C3bBbC3 convertase MBL Pathway MBL 200600 0.000210 Binds to mannose
MASP-1 93
1.512 Unknown MASP-2 76 Unknown Cleaves C4 and C2 FC
Fragment crystallizable; Ig
immunoglobulin; MAC
membrane attack complex; MBL mannose-binding lectin; MASP
MBL-associated serine protease. 1814_Ch06_085-106.qxd 7/10/09 2:47 PM Page 87
88 SECTION 1
Nature o
the Immune System unequal size. The sequence o
binding is C1s-C1r-C1r
-C1s. 1 It is
hypothesized that this structure assumes the shape o
a distorted gure eight, and
it wraps
itsel
around the arms o
C1q (Fig. 62). C1q has a molecular weight o
410,000 an
d is composed
o
six strands that
orm six globular heads with a collagen- like tail portion
. This

structure has been likened to a bouquet with six blossoms extending outward
(see Fig. 62).
Each o
the six stalks is composed o
three homologous polypeptide chainsA, B, an
d Cthat
orm a
triple helix. 1 Alanine residues, which interrupt the triple helix, make a semi ex
ible joint that
allows the stems to bend. As long as calcium is present in the serum, C1r and C1s
remain associated with C1q. C1q recognizes the
ragment crystallizable (FC) region o
two adj
acent
antibody molecules, and at least two o
the globular heads o
Clq must be bound
to initiate the
classi- cal pathway. This binding occurs at the C H 2 region
or IgG and at the
C H 3 region
or
IgM. C1r and C1s are both serine protease proenzymes or zymogens. As binding o

C1q occurs,
both are converted into active enzymes. Autoactivation o
C1r results
rom
a con
ormational
change that takes place as C1q is bound. Mechanical stress trans- mitted
rom th
e stems as
binding occurs opens up the active site on C1r. 7 Once activated, C1r cleaves a
thioester bond on
C1s, which activates it. Activated C1r is extremely speci c, because its o
nly known
substrate is C1s. Likewise, C1s has a limited speci
icity, with its only
substrates being
C4 and C2. Once C1s is activated, the recognition stage ends. The Activation Uni
t Phase two, the

ormation o
the activation unit, results in the production o
an enzyme known a
s C5 convertase.
C4 is the second-most-abundant complement protein, with a serum concentrat
ion o

approximately 600 g/mL. 6 It consists o

three polypeptide chains and has a mol
ecular weight
o
approximately 198,000. C1s cleaves C4 to split o

a 77-amino acid
ragment c
alled C4a. In the
process, it opens a thioester-containing active site on the remaining part, C4b.
C4b must bind to
protein or carbohydrate within a
ew seconds, or it will react with water molecu
les to
orm iC4b,
which is rapidly degraded. Thus, C4b binds mainly to antigen in clusters that ar
e within a 40 nm
radius o
C1. This represents the rst ampli cation step in the cascade, because
or
every one C1
attached, approximately 30 mol- ecules o
C4 are split and attached. 2 C2 is the
next component
to be activated. Complement proteins were named as they were isolated, be

ore the
sequence o
activation was knownhence the irregularity in the numbering system. C
2 is a
single-chain glycoprotein with a molecular weight o
102,000. 2 The C2 gene is c
losely associated
with the gene
or
actor B (alternative pathway) on chromosome 6 in the major hi
stocompatability
complex, and each serves a similar purpose in its particular pathway. 8 When com
bined with C4b in
the presence o
magne- sium ions, C2 is cleaved by C1s to
orm C2a (which has a
Recognition unit
Activation unit Membrane attack complex C1qrs C4 C2 C4a C2b C4b2a C3 C3a C5 C5a
Anaphylatoxin

kinin-like activity Anaphylatoxin chemotaxin C4b2a3b C5b C5b67 C5b678 C5b6789 Ly

sis (C3
convertase) (C5 convertase) Anaphylatoxin FIGURE 61. The classical complement cas
cade. C1qrs is
the recog- nition unit that binds to the FC portion o
two antibody molecules. C
1s is activated
and cleaves C4 and C2 to
orm C4b2a, which is known as C3 convertase. C3 convert
ase cleaves C3 to

orm C4b2a3b, known as C5 convertase. The combination o

C4b2a3b is the activation unit. C5
convertase cleaves C5. C5b attracts C6, C7, C8, and C9, which bind together,
or
ming the membrane
attack complex. C9 polymerizes to cause lysis o
the target cell. Activation o

C1qrs C1q C1s C1r

FIGURE 62. Structure o
C1qrs. When two or more globular heads o
C1q attach to b
ound
immunoglobulin molecules, the collagen- like stalks change their con guration. The
resulting
shape change causes C1r to become a serine protease. This cleaves a small
ragment o

C1s,
uncovering the C1s protease, whose only targets are C4 and C2. 1814_Ch06_085-106
.qxd 7/10/09
2:47 PM Page 88 CHAPTER 6 Complement System 89 molecular weight o
70,000) and
C2b (which has a
molec- ular weight o
34,000) (Fig. 63A). This is the only case
or the designati
on a to be
given to the cleavage piece with enzyme activity. C2 must be within a 60 nm radi
us o
bound Cls

or cleavage to occur. Binding o

C2 to C4b can occur in the
luid phase, but C4
b attached to
antigen is much more e
cient in accepting C2. This serves to keep the reac- tion
localized. The
combination o
C4b and C2a is known as C3 conver- tase (Fig. 63B). This is writte
n as C4b2a to
indicate that the complex is an active enzyme. This complex is not very stable.
The hal
-li
e is
estimated to be between 15 seconds and 3 minutes, so C3 must be bound quickly. I

binding does
occur, C3 is cleaved into two parts, C3a and C3b. C3 is the major constituent o

the complement
system and is present in the plasma at a concentration o
1200 g/mL. 6 It serves
as the pivotal
point
or all three pathways, and cleavage o
C3 to C3b represents the most sign
i cant step in
the entire process o
complement activation. 9 The molecule has a molecular weig
ht o
190,000,
and it consists o
two polypeptide chains, alpha and beta. The alpha chain conta
ins a highly
reactive thioester group, and when C3a is removed by cleavage o
a single bond i
n the chain, the
thioester is exposed; the remaining piece, C3b, is then capa- ble o
binding to
hydroxyl groups
on carbohydrates and proteins in the immediate vicinity. 6,7,9 Only a small perc
ent- age o

cleaved C3 molecules bind to antigen; most are hydrolyzed by water mol

ecules and
decay in the
luid phase. 6,10 C3b is estimated to have a hal
-li
e o
60 mic
rosec- onds i

not bound to antigen. The cleavage o

C3 represents a second and major ampli- cat
ion process,

because about 200 molecules are split

or every molecule o
C4b2a. 1 In addition
to being
required
or the
ormation o
the membrane attack complex, C3b also serves as a
power
ul opsonin.
Macrophages have speci
ic receptors
or it (discussed later in the chapter), and
this contributes greatly to the process o
phagocytosis. However, a large number o
mole
cules are needed

or this to occur; hence the need

or ampli cation. I
C3b is bound within 40 nm o

the C4b2a,
then this creates a new enzyme known as C5 convertase. Figure 63C depicts this la
st step in the

ormation o
the activation unit. The cleaving o
C5 with deposition o
C5b at a
nother site on
the cell membrane constitutes the beginning o
the mem- brane attack complex (MA
C). The Membrane
Attack Complex C5 consists o
two polypeptide chains,
and , which are linked by d
isul de bonds
to
orm a molecule with a molec- ular weight o
about 190,000. C5 convertase, co
nsisting o

C4b2b3b, splits o

a 74-amino-acid piece known as C5a, and C5b attaches to the

cell membrane,

orming the begin- ning o

the MAC. The splitting o
C5 and the cleavage o
C3 r
epresents the
most signi cant biological consequences o
the complement system, as explained in
the Biological
Mani
estations o
Complement Activation section. However, C5b is extremely labile
, and it is
rapidly inactivated unless binding to C6 occurs. 1 Subsequent binding involves C
6, C7, C8, and
C9. None o
these proteins have enzymatic activity, and they are all present in
much smaller
amounts in serum than the preced- ing components. C6 and C7 each have molecular
weights o

approximately 110,000, and both have similar physical and chemical properties. C
8 is made up o

three dissimilar C4b C4a C2 C4 C3 C3b C4b C4b C2a C3b C3b C3b C3b C2a C3a C2b
C2a Cell sur
ace
Recognition unit Anaphylatoxin Anaphylatoxin Kinin-like activity + + + C1qrs B C
A C3 convertase
C5 convertase FIGURE 63. Formation o
the activation unit. (A) C1qrs is able to c
leave C4 and
C2. (B) The larger pieces, C4b and C2a, bind to the target cell sur
ace and
orm
the enzyme C3
convertase. This cleaves C3 into C3a and C3b. (C) C3b is a power
ul opsonin, and
it binds to the
target in many places. I
some remains associated with C4bC2a, the enzyme C5 con
vertase is

ormed. The association o

C4bC2aC3b is known as the activation unit. 1814_Ch06_
085-106.qxd
7/10/09 2:47 PM Page 89 90 SECTION 1 Nature o
the Immune System chains joined
by disul de
bonds and has a total molecular weight o
about 150,000. 2 C9 is a single polype
ptide chain with
a molecular weight o
70,000. The carboxy-terminal end is hydrophobic, while
the
amino-terminal end is hydrophilic. The hydrophobic part serves to anchor the M
AC within the
target membrane. Formation o
the mem- brane attack unit is pictured in Figure 64
. Membrane

damage is caused by at least two di

erent mechanisms: channel

ormation and the
binding o
phospholipids. The latter causes a reordering and reorientation o
molecules that re
sults in leaky
patches. 6 When comple- ment proteins are bound, membrane phospholipids rea
rrange
themselves into domains surrounding the C5b6789 complex, and the integrity o

the membrane is
destroyed. Ions then are able to pass
reely out o
the cell. The membrane attac
k unit begins
when C6 binds to C5b, thereby stabilizing it. Then this complex attaches to the
cell sur
ace. C7
binds next,
orming a trimolecular complex that has a high a

inity
or lipid co
nstituents o
the
cell mem- brane. This allows
or insertion o
the C7 part o
the C5b67 complex i
nto the membrane
o
the target cell. 3 Next, C8 binds to C7 and exposes a hydrophobic region that
interacts with
the cell membrane to
orm a small hole in the membrane as C8 is inserted into th
e lipid bilayer.
3 Lysis can be observed at this stage, but the addition o
C9 accelerates the pr
ocess. Binding o

C8 causes a loss o
potas- sium
rom the cell, which is
ollowed by leakage o
a
mino acids and
ribonucleotides. The pore
ormed by the binding o
C5b678 is small, capable o
l
ysing
erythrocytes but not nucleated cells. 1 For lysis o
nucleated cells to be achie
ved, C9 must bind
to the complex. When C9 binds to the C5C8 complex, it un
olds, becomes in
serted into the
lipid bilayer, and polymerizes. Anywhere
rom 1 to 18 molecules o
C9 can bind t
o one C8
molecule. 3 C9 polymerizes only when bound, and it is believed that the
C5C8 complex
acts as a catalyst to enhance the rate o
reaction. 2 Polymerized C9
orms a
hol- low,
thin-walled cylinder, which can be seen on electron microscopy and constitutes t
he transmembrane
channel. The completed MAC unit has a
unctional pore size o
70 to 100. 1,3 One
such unit can
lyse erythrocytes, but lysis o
nucleated cells is a multihit phenomenon; it was
originally
believed that lysis o
all cells was a single-hit phenomenon. Destruction o
tar
get cells
actually occurs through an in ux o
water and a corresponding loss o
electrolytes
. THE
ALTERNATIVE PATHWAY Pathogens can be destroyed in the absence o
antibody by mea
ns o
the
alternative pathway, which acts as part o
innate or natural immunity. This path
way is important
as an early de
ense against pathogens. Phylogenetically, this represents the old
est o
the C3
activating pathways. First described by Pillemer and his associates in the early
1950s, the pathway was originally named
or the protein properdin, a constituent o
norm
al serum with a
concentration o
approx- imately 5 to 15 g/mL. 11 Now it is known that properdin
does not
initiate this pathway but rather stabilizes the C3 convertase
ormed
rom activa
tion o
other


actors. In addi- tion to properdin, the serum proteins that are unique to this
pathway include

actor B and
actor D. C1, C2, and C4,
ound in the classical pathway, are not u
sed at all in
this sys- tem. C3, however, is a key component o
both pathways. The alternative
pathway is
summarized in Figure 65. Triggering substances
or the alternative pathway includ
e bacterial
cell walls, especially those containing lipopoly- saccharide;
ungal cell walls;
yeast; viruses;
virally in
ected cells; tumor cell lines; and some parasites, especially try- pa
nosomes. 1 All o

these can serve as sites

or binding the complex C3bBb, one o
the end products
o
this pathway.
The conversion o
C3 is the rst step in this pathway. In plasma, native C3 is
not stable.
Water is able to hydrolyze a thioester bond and thus spontaneously activates
a small number o

these molecules. 12 Once activated, it can C6 C7 C5b Formation o

membrane attac
k complex C6 C7
C5b C8 penetrates the cell membrane C6 C7 C5b Transmembrane channel Lysis Poly C
9 C8 C8 FIGURE
64. Formation o
the membrane attack unit. C5 conver- tase cleaves C5 into C5a an
d C5b. C5b
binds to the target cell, and C6 and C7 become attached to it. C8 binds to these
associated molecules and begins to penetrate the cell membrane. C9 binds to the complex and po
lymerizes to
orm
the transmembrane channel, which causes lysis o
the cell. 1814_Ch06_085-106.qxd
7/10/09 2:47
PM Page 90 CHAPTER 6 Complement System 91 bind to
actor B, which has a molecul
ar weight o

93,000 and is
airly abundant in the serum, at a level o
200 g/ mL. 6,8 Binding
o
C3b
(sometimes called iC3) to B causes a con
ormational change that makes B more sus
ceptible to
cleavage by serine proteases. Thus, only bound
actor B can be cleaved by
actor
D. The role o

actor B is thus analo- gous to that o

C2 in the classical pathway, because it

orms an integral
part o
a C3 convertase. Factor D is a plasma protein that goes through a con
or
- mational change
when it binds to
actor B. 7,8 It is a serine protease with a molecular weight o

24,000, and its

only sub- strate is bound
actor B. When bound to
actor B, the cataly
tic site on

actor D opens, making it an active enzyme. The concentration o

actor D in the
plasma is the
lowest o
all the complement proteins, approximately 2 g/mL. 6 It cleaves
actor
B into two
pieces: Ba (with a molecular weight o
33,000) and Bb (with a molecular weight o

approximately
60,000). Bb remains attached to C3b,
orming the initial C3 convertase o
the al
ternative
pathway. Bb is rapidly inacti- vated unless it becomes bound to a site o
n one o
the
triggering cellular antigens. As the alternative pathway convertase, C3bBb is th
en capable o

cleaving additional C3 into C3a and C3b. Some C3b attaches to cellular sur
aces
and acts as a

binding site
or more
actor B. This results in an ampli cation loop that
eeds C3
b into the
classical and alternative pathways. All C3 present in plasma would be rapidly co
nverted by this
method were it not
or the
act that the enzyme C3bBb is extremely unstable unle
ss properdin
binds to the complex. Binding o
properdin increases the hal
-li
e o
C3bBb
rom
90 seconds to
several minutes. 6,8 In this manner, optimal rates o
alter- native pathway acti
vation are
achieved. 11 C3bBb can also cleave C5, but it is much more e
cient at cleaving C3
. 13 I
,
however, some o
the C3b produced remains bound to the C3 convertase, the enzyme
is altered to

orm C3bBb3bP, which has a high a

nity
or C5 and exhibits C5 convertase activity
. 12,13 C5 is
cleaved to produce C5b, the rst part o
the membrane attack unit. From this point
on, both the
alternative and classical pathways are identical. THE LECTIN PATHWAY The lectin
pathway
represents another means o
activating complement without antibody being present
. Lectins are
proteins that bind to carbohydrates. This pathway provides an additional link be
tween the innate
and acquired immune response, because it involves nonspeci c recognition o
car- b
ohydrates that
are common constituents o
microbial cell walls and that are distinct
rom those

ound on human
cell sur
aces. 14,15 One key lectin, called mannose-binding, or mannan- binding,
lectin (MBL),
binds to mannose or related sugars in a calcium-dependent manner to initiate
this pathway.
16 These sugars are
ound in glycoproteins or carbohydrates o
a wide variety o

microorganisms
such as bacteria, yeasts, viruses, and some parasites. MBL is considered an acut
e phase protein,
because it is produced in the liver and is normally present in the serum bu
t increases
during an initial in ammatory response. 15 It plays an important role as a de
e
nse mechanism
in in
ancy, during the interval between the loss o
maternal antibody and th
e acquisition o

a
ull- edged antibody response to pathogens. 17 In
act, de ciencies o
MBL have be
en associated
with serious in
ec- tions such as neonatal pneumonia and sepsis. 15,18 The struc
ture o
MBL is
similar to that o
C1q, and it is associated with three MBL-serine proteases
(MASPs):
MASP-1, MASP-2, and MASP-3. Once MBL binds to a cellular sur
ace, MASP-2, which
is homologous to
C1s, autoactivates. 19 MASP-2 thus takes the active role in cleav- ing C4 and C2
, while the

unctions o
MASP-1 and MASP-3 are unclear at this time. 16,19 Once C4 and C2 ar
e cleaved, the
rest o
the pathway is identical to the classical pathway. Figure 66 shows the co
nvergence o

all three pathways. C3 C3 C3b C3b B Factor B B Spontaneously hydrolyzes + + Mic

robial cell
Factor D Mg ++ C3b Bb Ba C3 convertase C3b Bb P C3 convertase stabilized by pr
operdin C3b C3a

C3b Bb P C5 convertase Formation o

MAC C5 Anaphylatoxin FIGURE 65. The alternati
ve pathway. C3
is hydrolyzed by water to produce a C3b sometimes called iC3. This molecule can
bind
actor B.
When
actor B is bound to C3b, B is cleaved to
orm C3Bb, an enzyme with C3 conv
ertase activity.
More C3 is cleaved to
orm more C3bBb. This enzyme is stabilized by properdin, a
nd it continues
to cleave additional C3. I
a molecule o
C3 remains attached to the C3bBbP enzy
me, the
convertase now has the capability to cleave C5. The C5 convertase thus consists
o
C3bBb3bP.
A
ter C5 is cleaved, the path- way is exactly the same as the classical pathway.
1814_Ch06_085-106.qxd 7/10/09 2:47 PM Page 91 92 SECTION 1 Nature o
the Immu
ne System SYSTEM
CONTROLS Activation o
complement could cause tissue damage and have devastating
systemic e

ects
i
it was allowed to pro- ceed uncontrolled. To ensure that in
ectious agents an
d not
sel
-antigens are destroyed and that the reaction remains localized, sever
al plasma
proteins act as system regulators. In addition, there are speci
ic receptors on
certain cells
that also exert a controlling in
luence on the activation process. In
act, appr
oximately
one-hal
o
the complement components serve as controls
or critical steps in th
e activation
process. Because activation o
C3 is the pivotal step in all pathways, the major
ity o
the
control pro- teins are aimed at halting accumulation o
C3b. However, there are
controls at
all crucial steps along the way. Regulators will be discussed according to
their order o

C5b C4 C2 C3 C4b C3b C3b C3b C3b C3b C2a C4b Factor D C2a C3 convertase P Clqrs
Classical
Pathway Lectin Pathway Alternative Pathway MBL MASP-1 MASP-2 MASP-3 + Mannose C
3 convertase
Properdin stabilizes C5 convertase C5 convertase Membrane attack complex B Bb B
b C5 C5a C6 C7
C8 FIGURE 66. Convergence o
the classical, alternative, and lectin pathways. The
binding o

C1qrs to two anti- body molecules activates the classical pathway, while the alt
ernative pathway
is started by hydrolysis o
C3. The lectin pathway is triggered by binding o
MB
P to mannose on
bacterial cell walls. MASP-1, MASP-2, and MASP- 3 bind to
orm an activated C1-l
ike complex.
MASP-2 cleaves C2 and C4 and proceeds like the classical pathway. Factor B and

actor D operate
in the alternative pathway. While C3 convertase is
ormed di

erently in each pa
th- way, C3 is a
key component in each one. The C5 convertase in the alternative pathway consists
o
C3bBb3bP. In
the classical pathway, C5 convertase is made up o
C4b2a3b. A
ter C5 is cleaved,
the pathway is
common to all. 1814_Ch06_085-106.qxd 7/10/09 2:47 PM Page 92 CHAPTER 6 Comple
ment System 93
appearance in each o
the three pathways. A brie
summary o
these is
ound in T
able 62.
Regulation o
the Classical and Lectin Pathways C1 inhibitor, or C1INH, is a gly

coprotein with a
molecu- lar weight o
105,000 that inhibits activation at the
irst stages o
bo
th the classical
and lectin pathways. Like most o
the other complement proteins, it is mainly sy
nthesized in the
liver, but monocytes also may be involved to some extent in its manu
acture. Its
main role is to
inactivate C1 by binding to the active sites o
C1r and C1s. Clr and Cls become
instantly and
irreversibly dissociated
rom C1q. 2,6 Clq remains bound to antibody, but all en
zymatic activity
ceases. C1INH also inactivates MASP-2 binding to the MBL-MASP complex, thus halt
ing the lectin
pathway. 12,19 Further
ormation o
C3 convertase in the classical and lectin pa
thways is
inhibited by
our main regulators: soluble C4b-binding protein (C4BP) and three
cell-bound receptors, complement receptor type 1 (CR1), membrane co
actor protein (MCP), a
nd decay
accelerating
actor (DAF). 1,7 All o
these act in concert with
actor I, a seri
ne pro- tease
that inactivates C3b and C4b when bound to one o
these regulators. C4BP is abun
dant in the
plasma and has a molec- ular weight o
about 520,000. It is capable o
combining
with either
uid-phase or bound C4b, so C4b cannot bind C2 and is made available
or degradati
on by
actor I.
1 I
C4BP attaches to cell-bound C4b, it can dissociate it
rom C4b2a complexes,
thus causing the
cessation o
the classical pathway. CR1, also known as CD35, is a large polymorp
hic glycoprotein with a molecular weight between 165,000 and 280,000. 3 It is
ound
mainly on
peripheral blood cells, including neutrophils, monocytes, macrophages, erythro
- cytes,
eosinophils, B lymphocytes, some T lymphocytes, and
ollicular dendritic cells.
2 It binds C3b
and C4b but has the greatest a
nity
or C3b. 2,20 Once bound to CR1, both C4b and
C3b can then
be degraded by
actor I Perhaps one o
the main
unctions o
CR1, however, is to
act as a
receptor on platelets and red blood cells and to mediate transport o
C3b-coated
immune complexes
to the liver and spleen. 3,20 It is there that xed tissue macrophages strip the
immune
complexes
rom the red blood cells, process the complexes, and return the r
ed blood cells
intact to circulation. The ability o
cells to bind complement- coated particles
is re
erred to
as immune adherence. Membrane co
actor protein (MCP), or CD46, has a mole
cular weight
between 50,000 and 70,000 and is
ound on virtually all epithelial and endotheli
al cells except
ery- throcytes. 3 MCP is the most e

icient co
actor
or
actor Imediated cleavag
e o
C3b. It
also serves as a co
actor
or cleavage o
C4b, but it is not as e

ective as C4B
P. MCP also helps
to control the alternative pathway, since binding o

actor B to C3b is inhibite
d.
Decay-accelerating
actor (DAF), or CD55, a 70,000 d membrane glycoprotein, is t

he third main
receptor, and it has a wide tissue distribution. It is
ound on peripheral blood
cells, on
endothelial cells and broblasts, and on numerous types o
epithelial cells. 3,6,2
1,22 DAF is
capable o
dissociat- ing both classical and alternative pathway C3 convertases.
It can bind to
both C3b and C4b in a similar manner to CR1. 3 It does not prevent initial bindi
ng o
either C2
or
ac- tor B to the cell but can rapidly dissociate both
rom their binding sit
es, thus
preventing the assembly o
an active C3 convertase. The carboxy-terminal porti
on o
DAF is
covalently attached to a glycophospholipid anchor that is inserted into the oute
r layer o
the
membrane lipid bilayer. This arrange- ment allows DAF mobility within the membra
ne so it can
reach C3 convertase sites that are not immediately adjacent to it (Fig. 67). 21 T
he presence o

DAF on host cells pro- tects them

rom bystander lysis and is one o
the main me
chanisms used in
discrimination o
sel

rom nonsel
, because
oreign cells do not possess this s
ubstance.
However, it does not permanently modi
y C3b or C4b; thus, they are capable o
re
-
orming
elsewhere as active convertases. Regulation o
the Alternative Pathway The princ
ipal soluble
regulator o
the alternative pathway is
actor H, which has a molecular weight o

160,000. 7,12
It acts by binding to C3b, thus preventing the binding o

ac- tor B. C3b in the

luid phase has

a hundred
old greater a
nity
or
actor H than
or
actor B, but on cell sur
aces
, C3b
pre
erentially binds
actor B. However,
actor H also accelerates the dissocia
tion o
the
C3bBb complex on cell sur
aces. When
actor H binds to C3bBb, Bb becomes Table
6-2. Plasma
Complement Regulators SERUM PROTEIN MOLECULAR WEIGHT (KD) CONCENTRATION (mg/
mL) FUNCTION C1
inhibitor (C1INH) 105 240 Dissociates C1r and C1s
rom C1q Factor I 88 35 Cleave
s C3b and C4b
Factor H 150 300450 Co
actor with I to inactivate C3b; prevents binding o
B to C
3b C4-binding
protein 520 250 Acts as a co
actor with I to inactivate C4b (C4bp) S protein (vi
tronectin) 84 500
Prevents attachment o
the C5b67 complex to cell membranes 1814_Ch06_085-106.qxd
7/10/09 2:47
PM Page 93 94 SECTION 1 Nature o
the Immune System displaced. In this manner,
C3 convertase
activity is curtailed in plasma and on cell sur
aces. Additionally,
actor H act
s as a co
actor
that allows
actor I to break down C3b. It appears that only those molecules wit
h tightly bound

actor H acquire high-a

inity binding sites

or
actor I. 23 When
actor I bind
s, a
con
ormational change takes place and allows it to cleave C3b. 23 On cellu- lar
sur
aces, C3b is
cleaved to C3
, a small piece that is released into the plasma, and to iC3
b, which
remains attached but is no longer an active enzyme. iC3b is
urther broken down

to C3c and C3dg

by
actor I in conjunction with another co
actor, the CR1 receptor (Fig. 68). 1 R
egulation o

Terminal Components S protein is a soluble control protein that acts at a deeper

level o

complement activation. Also known as vitronectin, S protein interacts with the C

5b67 complex as
it
orms in the
luid phase and prevents it
rom binding to cell membr
anes. 6 Binding
o
C8 and C9 still proceeds, but polymerization o
C9 does not occur, and the co
mplex is unable
to insert itsel
into the cell membrane or to produce lysis. 3 A recepto
r known by
various terms, including mem- brane inhibitor o
reactive lysis (MIRL), or CD59,
also acts to
block
ormation o
the membrane attack complex. MIRL is widely distribu
ted on the
cell membranes o
all circulating blood cells, including red blood cells, and
on endothelial,
epithelial, and many other types o
cells. 2,3 Its main
unction is to bind to C
8 and prevent
insertion o
C9 into host cell membranes. 6,11 Table 63 lists the receptors and i
ndicates the
type o
cell on which they are
ound. COMPLEMENT RECEPTORS AND THEIR BIOLOGICAL
ROLES Some
complement receptors
ound on host cells ampli
y and enhance the immune response
by augmenting
phago- cytosis and stimulating accessory cells, rather than acting as regulators
. Table 63
summarizes the main characteristics o
each and describes the cell types on whic
h they are
ound.
CR1 has been discussed in the previous section. A second receptor, CR2 or CD21,
is
ound mainly
on B lymphocytes and
ollicular dendritic cells. 2,4 Ligands
or CR2 include deg
radation products
o
C3b, such as C3dg, C3d, and iC3b. In addition, the Epstein-Barr virus gains e
ntry to B cells
by binding to this receptor. CR2 is present only on mature B cells, and it is lo
st when
conversion to plasma cells occurs. A B C4b C4b C2a DAF Classical Pathway DAF B
b DAF DAF C3b C3b
Alternative Pathway FIGURE 67. Inhibitory e

ects o
DAF. DAF acts in both the cl
assical and
alternative pathways to dissociate C3b. When C3b binds to cell sur
aces that hav
e DAF present,
DAF helps dissociate Bb
rom binding to C3b in the alternative pathway. In the c
lassical pathway,
DAF dissociates C2a
rom C4b. Nonactivating sur
ace (sel
-antigen) Factor I H C3
b CRI C3
Factor
B compete Factor H Factor I C3c CRI is co
actor iC3b C3dg CRI CRI FIGURE 68. Comp
lement
controls. CR1 receptor acts as a co
actor in the inactivation o
C3b. Factor I c
leaves C3b to

orm C3dg and C3c. C3dg is not an e

ective opsonin, and it is not capable o

u
rther
participation in the complement cascade. 1814_Ch06_085-106.qxd 7/10/09 2:47 PM
Page 94 CHAPTER
6 Complement System 95 CR2 plays an important role as part o
the B-cell corecep
- tor
or
antigen. Acting in concert with CD19, it binds complement-coated antigen

and cross-links
it to membrane immunoglobulin to activate B cells. In this manner, immune comple
xes are more
e

ective at enhancing B-cell di

eren- tiation and production o

memory cells t
han is antigen by
itsel
. 3,4,21 Another receptor, CR3 (CD11b/CD18),
ound on mono- cytes, macroph
ages,
neutrophils, and natural killer cells, speci
ically binds particles opsonized
with iC3b, a
C3b degradation product. 3 It does this in a calcium-dependent manner. The CR3 r
eceptor plays a
key role in mediating phagocytosis o
particles coated with these complement
ra
g- ments (Fig.
69). It consists o
an and a
chain, with molecular weights o
165,000 and 95,000,
respectively. 24 These proteins trigger sur
ace adhesion and increased activ- it
y o
phagocytic
cells. 6 Patients whose white blood cells lack these receptors
ail to exhibit

unctions such as
chemotaxis, sur
ace adherence, and aggregation. De ciencies in phago- cytosis are
also noted.
These individuals have an impaired capacity to bind iC3b-coated particles and ar
e subject to
recurrent in
ections. The CR4 (CD11c/CD18) receptor is very similar to CR3 in th
at it also binds
iC3b
ragments in a calcium-dependent
ashion. CR4 proteins are
ound on neutrop
hils, monocytes,
tissue macrophages, activated T cells, dendritic cells, NK cells, and activated
B cells. 3
Neutrophils and monocytes, Table 6-3. Receptors on Cell Membranes
or Complement
Components
RECEPTOR LIGAND CELL TYPE FUNCTION CR1 (CD35) C3b, iC3b, C4b RBCs, neutrop
hils, monocytes,
Co
actor
or
actor I; mediates transport o
macrophages, eosinophils, B and im
mune complexes T
cells,
ollicular dendritic cells CR2 (CD21) C3dg, C3d, iC3b B cells,
ollicular
dendritic cells,
B-cell coreceptor
or antigen with CD19 epithelial cells CR3 (CD11b/CD18) iC3b,
C3d, C3b
Monocytes, macrophages, Adhesion and increased activity o
neutrophils, NK cell
s phagocytic
cells CR4 (CD11c/CD18) iC3b, C3b Monocytes, macrophages, Adhesion and increased
activity o

phagocytic neutrophils, NK cells, activated cells T and B cells, dendritic cell

s DAF (CD55) C3b,
C4b RBCs, neutrophils, platelets, Dissociates C2b or Bb
rom binding sites, mon
ocytes,
endothelial cells, thus preventing
ormation o
C3 convertase broblasts, T cell
s, B cells,
epithelial cells MIRL (CD59) C8 RBCs, neutrophils, platelets, Prevents insertio
n o
C9 into cell
membrane monocytes, endothelial cells, epithelial cells MCP (CD46) C3b, C4b Neut
rophils,
monocytes, Co
actor
or
actor I cleavage o
C3b and C4b macrophages, platelets
, T cells, B
cells, endothelial cells RBC red blood cell; NK
natural killer Antibody-coated b
acterial cell
Complement activation Phagocytic cell Phagocytosis Fc receptor CR3 iC3b FIGURE 69
. Role o
C3b
in opsonization. C3b-coated antigens are destroyed more easily due to interactio
n with the CR1

receptors
ound on phagocytic cells. 1814_Ch06_085-106.qxd 7/10/09 2:47 PM Pa
ge 95 96 SECTION
1 Nature o
the Immune System however, possess smaller amounts o
CR4 than o
CR
3. Their
unction
appears to be similar to that o
CR3, and they may assist neutrophil adhesion to
the endothelium
dur- ing in ammation. Receptors speci
ic
or Clq are
ound on neutrophils, monocyt
es,
macrophages, B cells, platelets, and endothe- lial cells. 3,23 These receptors,
known as
collectin receptors, bind the collagen portion o
Clq and generally enhance the
binding o
Clq
to FC receptors. Interacting only with bound Clq, the receptors appear to in
crease phagocytic cells uptake o
immune complexes opsonized with Clq. Additionally, on neutr
ophils, they
may act to enhance the respiratory burst triggered by IgG binding to FC
receptors.
BIOLOGICAL MANIFESTATIONS OF COMPLEMENT ACTIVATION Activation o
complement i
s a very
e

ective means o
ampli
ying the in
lammatory response to destroy and clear

oreign antigens.
The cycle does not always have to pro- ceed to lysis
or this to be accomplished
; hence, some o

the initiating proteins are much more plenti

ul than pro- teins that
orm the me
mbrane attack
complex. Complement proteins also serve as a means o
linking innate and natu- r
al immunity. They
act as opsonins to
acilitate destruction by phagocytic cells, and they play a m
ajor role in
uptake and presentation o
antigens so a speci
ic immune response can occur. The
y also
acilitate
B-cell activation and may, in
act, be necessary
or maintaining immunological m
em- ory. E

ector
molecules generated earlier in the cascade play a major role in all these areas.
Such molecules
can be classi
ied into three main categories: anaphylatoxins, chemotaxins,
and opsonins. An
anaphylatoxin is a small peptide that causes increased vascular permeability, co
ntraction o

smooth muscle, and release o

histamine
rom basophils and mast cells. Proteins
that play such a
part are C3a, C4a, and C5a. All o
these have a molecular weight between 9,000 a
nd 11,000, and
they are
ormed as cleavage products
rom larger complement components. O
these
molecules, C5a
is the most potent, at least 200 times more power
ul than C3a, while C4a is the
least e

ective.
6 C3a, C4a, and C5a attach to speci c receptors on neu- trophils, basophils, mast
cells,
eosinophils, smooth muscle cells, and vascular endothelium. 4 C3a and C4a attach
to the C3a
receptor (C3aR), and C5a attaches to the C5a recep- tor (C5aR). When binding occ
urs on basophils
and mast cells, histamine is released, thereby increasing vascular per- meabilit
y and causing
contraction o
smooth muscles. C5a causes neutrophils to release hydrolytic enzy
mes, oxygen
radicals, and prostaglandins, aiding in destruction o

oreign antigens. 4
,6 C5a also

serves as a chemotaxin
or neutrophils, basophils, eosinophils, mast cells,
monocytes, and
dendritic cells. In this manner, these cells are directed to the source o
antig
en concentration.
Because o
increased vascular permeability, neutrophils migrate
rom blood vesse
ls to the tissues
and tend to aggregate. Binding o
C5a to monocytes causes them to undergo an oxi
dative burst
that includes increased production o
hydrolytic enzymes, neutrophil chemota
ctic
actor,
platelet activating
actors, interleukin-1, and toxic oxygen metabo- lites. 13 I
nterleukin-1 is
a protein that enhances T-cell activation. This may produce
ever and an inc
rease in acute
phase reactants, both o
which are characteristic o
an in ammatory respon
se. As a means
o
localizing and controlling the e

ects o
C3a, C4a, and C5a, these substances
are rapidly
inactivated by an enzyme in the plasma called carboxypeptidase N. C3a and C4a ar
e cleaved in
seconds, while conversion o
C5a occurs more slowly. The last major e

ect o
co
mplement-derived
peptides is opsonization. C4b, C3b, and iC3b, which accumulate on cell membranes
as complement
activation proceeds, bind to speci c receptors on erythrocytes, neutrophils, monoc
ytes, and
macrophages, as discussed earlier. This
acilitates phago- cytosis and clearance
o

oreign
substances, and it is one o
the key
unctions o
the complement system. In addi
tion, attachment
o
C3 products to an antigen has been
ound to enhance the B-cell response. COMP
LEMENT AND
DISEASE STATES Although complement acts as a power
ul weapon to com- bat in
ecti
on by ampli
ying
phagocytosis, in some cases it can actually contribute to tissue damage o
r death.
Complement can be harm
ul i
(1) activated systemically on a large scale as
in
gram-negative septicemia, (2) it is activated by tissue necrosis such a
s myocardial
in
arction, or (3) lysis o
red cells occurs. In the case o
septicemia due to a
gram-negative
organism, large quan- tities o
C3a and C5a are generated, leading to neutrophil
aggregation and
clotting. Damage to the tiny pulmonary capillaries and interstitial pulmonary
edema may
result. 6 Tissue injury
ollowing obstruction o
the blood supply, such as occur
s in myocardial
in
arction, or heart attack, can cause complement activation and deposition o
m
embrane attack
complexes on cell sur
aces. Receptors
or C3a and C5a have been
ound in coronar
y plaques,
indicating that com- plement components may increase the damage to heart
tissue. 26,27
Lysis may be another end result o
complement activa- tion. Hemolytic diseases s
uch as cold
autoimmune hemolytic anemia are characterized by the presence o
an autoanti- bo
dy that binds at
low temperatures. When these cells warm up, complement xation results in lysis (s
ee Chapter 14


or a more complete discussion o
complement-mediated autoimmune diseases).
1814_Ch06_085-106.qxd 7/10/09 2:47 PM Page 96 CHAPTER 6 Complement System 97
COMPLEMENT
DEFICIENCIES Major Pathway Components Although excess activation o
the compleme
nt system can
result in disease states, lack o
individual components also has a deleterious e

ect. Hereditary
de ciency o
any com- plement protein, with the exception o
C9, usually mani
ests
itsel
in
increased susceptibility to in
ection and delayed clear- ance o
immune complexe
s. Most o
these
conditions are inherited on an autosomal recessive gene, and they are quite rare
, occurring in
0.03 percent o
the general population. 26 The de ciency that occurs most o
ten is
that o
C2,
which is
ound in 1 in 20,000 individuals. 2830 Recent evidence indi- cates that
atherosclerosis
may be related to a C2 de ciency. 28 Additionally, C2-de cient individuals may be mo
re prone to
recurrent streptococcal and staphylococcal in
ections. 28 The
actor B locus is
nearby, and o
ten
C2-de cient persons are reported to have decreases in
actor B. Other types o
com
- plement
de ciencies are less common. A second de ciency that occurs with some
requency is t
hat o

mannose-binding lectin,
ound in 3 to 5 percent o
the population. MBL is im
portant during
the interval between the loss o
passively acquired maternal antibodies and the
ability o

in
ants to produce their own antibody. 10 Lack o
MBL has been associated with p
neumonia, sepsis,
and meningococcal disease in in
ants. 15,17,18 The most serious de
iciency,
however, is
that o
C3, because it is the key mediator in all pathways. Individuals with a
C3 de ciency are
prone to developing severe, recur- rent li
e-threatening in
ections with encapsu
lated bacteria
such as Streptococcus pneumoniae and may also be subject to immune complex disea
ses. 17 Such
complexes can be lodged in the kidney and result in glomerulonephritis. 26,31 It
appears that a
de ciency o
any o
the terminal com- ponents o
the complement cascade (C5C8)
causes
increased susceptibility to systemic Neisseria in
ections, including meningo
coccal meningitis
and disseminated gon- orrheal disease. 10,28 Table 64 lists the complement co
mponents and
the disease states associated with the absence o
each individual
actor. R
egulatory Factor
Components A prime example o
a disease due to a missing or de
ective regulatory
component is
paroxysmal nocturnal hemoglo- binuria (PNH). Individuals with this disease have
red blood cells
that are de cient in DAF; hence, the cells are subject to lysis by means o
the by
stander e

ect
once the complement system has been triggered. These individuals appear to have
a de
iciency in
the glycophospholipid anchor o
the DAF molecule that prevents its insertion
into the cell
mem- brane. 10,21 When C3b is deposited on erythrocytes because o
activation o


either pathway,
the result is complement- mediated intravascular and extravascular hemolysis res
ulting in a
chronic hemolytic anemia. Some studies indicate that a DAF de
iciency is associated with a lack
o
CD59 (MIRL), and both are implicated in PNH. 17,32 CD59 has the same glycopho
spholipid anchor

ound in DAF, so the gene de

iciency a

ects both mole- cules. As mentioned prev

iously, CD59
prevents insertion o
C9 into the cell membrane by binding to the C5b678 complex
, thus
inhibiting
ormation o
transmembrane channels. 26 There
ore, the presence
o
both DAF
and CD59 is important in protecting red blood cells against bystander lysis. Rec
urrent attacks o

angioedema that a

ect the extrem- ities, the skin, the gastrointestinal tract,

and other mucosal
sur
aces are characteristic o
hereditary angioedema. This disease is caused by
a de ciency or
lack o
C1INH, which occurs with a population
requency o
2 in 10,000. 17 Lack
o
C1INH results
in excess cleavage o
C4 and C2, keeping the classical pathway going and creatin
g kinin-related
pro- teins that increase vascular permeability. Thus, the response to a localize
d antigen is
continued swelling or edema. The edema can be subcutaneous or within the bowel o
r upperrespiratory tract. Normally, this spontaneously subsides in 48 to72 hours, b
ut i
this
occurs in the area o
the oropharynx, li
e-threatening upper-airway obstruc
tion may develop.
26 The condition may be either hereditary or acquired. It is inherited as an aut
osomal dominant
gene that codes
or either a dys
unctional or an inactive protein. One normal Ta
ble 55-2
Comparison o
Tests Used DEFICIENT ASSOCIATED COMPONENT DISEASE C1 (q, r, or
s) Lupuslike
syndrome; recurrent in
ections C2 Lupuslike syndrome; recurrent in
ections; athe
rosclerosis C3
Severe recurrent in
ections; glomer- lonephritis C4 Lupuslike syndrome C5-C8 N
eisseria
in
ections C9 No known disease association C1INH Hereditary angioedema DAF Parox
ysmal nocturnal
hemoglobinuria MIRL Paroxysmal nocturnal hemoglobinuria Factor H or
actor I Rec
urrent pyogenic
in
ections MBL Pneumococcal diseases, sepsis, Neisseria in
ections Properdin N
eisseria
in
ections MASP-2 Pneumococcal diseases C1INH C1 inhibitor; DAF
decay accelerati
ng
actor;
MIRL membrane inhibitor o
reactive lysis; MBL
mannose-binding lectin; MASP-2 ma
nnoseasociated serine protease. Table 6-4. De ciencies o
Complement Components 1814_Ch
06_085-106.qxd
7/10/09 2:47 PM Page 97 98 SECTION 1 Nature o
the Immune System gene does not
produce enough
inhibitor to keep up with the demand, and the condition is apparent in the heter
ozygous state. I

the condition is hereditary, laboratory results indi- cate normal levels o

C1q
and C3 but
reduced levels o
C4 and C2. 30 In contrast, individuals with an acquired C1INH

de
i- ciency
show reduced levels o
C1q because o
excess activation o
the classical
cascade by
antibody. This may result
rom a B-cell malignancy; presence o
an autoimmune di
sease; or, in
some rare instances, appearance o
an autoan- tibody that binds to the active si
te o
the C1
inhibitor. 14 In each instance, C1INH is present but quickly becomes depl
eted. Other
inhibitors
or which there are genetic de ciencies include
actors H and I. Lack o

either
component produces a constant turnover o
C3 to the point o
depletion.
Recurrent
bacterial in
ections may ensue. LABORATORY DETECTION OF COMPLEMENT ABNORMALITIES
Determining the
levels o
complement components can be a use
ul adjunct in diagnosing disease. H
ereditary
de cien- cies can be identi
ied, and much can be learned about in ammatory
or autoimmune
states by
ollowing the con- sumption o
complement proteins. Techniques to dete
rmine complement
abnormalities generally
all into two categories: (1) measurement o
components
as antigens in
serum and (2) measurement o

unctional activity. 6 Many assays are not availabl
e in routine
clinical laboratories and are restricted to research laboratories. Some o
the m
ore common assays
will be discussed here. Immunologic Assays o
Individual Components The methods
most

requently used to assay individual components include radial immunodi

usio
n (RID) and nephelometry. 17,26 Components that are usually measured and
or which there are sta
ndardized
reagents include C1q, C4, C3, C5,
actor B,
actor H,
actor I, and C1 inhibitor
. Kits are
available
or C3a, C4a, and C5a. Radial immunodi

usion uses agarose gel into wh

ich speci c
antibody is incorporated. Serum serves as the antigen and is placed in wells tha
t are cut in the
gel. Di

usion o
the antigen
rom the well occurs in a circular pattern. The ra
dius o
the
resulting circle can be related to antigen concentration (see Chapter 8
or
urther details on
RID). This is a sensitive technique when per
ormed correctly, but it takes at le
ast 24 hours
be
ore test results are available. Nephelometry measures concentration according
to the amount o

light scattered by a solution containing a reagent antibody and a measured patie

nt sample (re
er
to Chapter 8
or more details on nephelometry). Generally, the more antig
enantibody
complexes that are present, the more a beam o
light will scatter as it passes t
hrough the
solution. Such sys- tems have a high degree o
accuracy, results are available q
uickly, and
processing is easy because o
the use o
automa- tion. However, it is necessary
to purchase
expensive equipment. Nephelometry and RID are both sensitive tests. 33 None o
t
he assays
or
individual components are able to distinguish whether the molecules are
unction

ally active.
Thus, although the preceding techniques give quantitative results and are relati
vely easy to
per
orm, test results must be interpreted care
ully. Assays
or the Classical Pa
thway Assays that
measure lysis, the end point o
complement acti- vation, are
unctional tests
that are

requently run in conjunction with testing o

individual components. The hemol
ytic titration
(CH 50 ) assay is most commonly used
or this purpose. 17,26 This measures the a
mount o
patient
serum required to lyse 50 percent o
a standardized concen- tration o
antibodysensitized sheep
erythrocytes. Because all proteins
rom C1 to C9 are necessary
or this to occur
, absence o
any
one component will result in an abnormal CH 50 , essentially reducing this numbe
r to zero. The
titer is expressed in CH 50 units, which is the recip- rocal o
the dilution tha
t is able to lyse
50 percent o
the sensitized cells. 6,17 The 50 percent point is used because th
is is when the
change in lytic activity per unit change in com- plement is a maximum. Most labs
need to
establish their own normal values. An additional CH 50 test has also been develo
ped based on
lysis o
liposomes that release an enzyme when lysed. This can be read on an ana
lyzer and is more
accurate than traditional CH 50 testing. 26 However, lytic assays in general are
complicated to
per
orm and lack sensitivity. Individual labs must establish their own norm
al values. When
the results are abnormal, the reasons cannot be determined. Such procedures are
use
ul in
establishing
unctional activ- ity or lack thereo
. Additional testing
or
individual
components should be per
ormed to
ollow up on any abnormality. Lytic ac
tivity can also
be measured by radial hemolysis in agarose plates. Rabbit red blood cells that h
ave been sensitized with antibody are implanted in agarose, and patient serum is added to we
lls punched in
the gel. Lysis appears as a clear zone around each well, and i
complement stand
ards are run, the
size o
the zone can be related to complement concentration. ELISAs have been de
signed as another
means o
meas- uring activation o
the classical pathway. 17,26,34 Solid-phase I
gM attached to
the walls o
microtiter plates is used to ini- tiate complement activation. Anti
human antibody to
C9 conjugated to alkaline phosphatase is the indicator o
com- plement activatio
n. When a
substrate is added, i
any C9 is present and the antibody conjugate has attached
, a color change
will be evident. (Re
er to Chapter 10
or a complete discussion o
the princi
ple o
ELISA
techniques.) This type o
testing, which is very sensitive, is probably the bes
t screen
or
complement abnormalities. 26 Additionally, split products that result
rom compl
ement activation
can also 1814_Ch06_085-106.qxd 7/10/09 2:47 PM Page 98 CHAPTER 6 Complement S

ystem 99 be
detected by this same method. These products include C4a, C4d, C3a, and C5a, whi
ch are generated
only i
com- plement activation has occurred. Alternative Pathway Assays Alterna
tive pathway
activation can be measured by several di

erent means. An AH 50 can be per

ormed
in the same
manner as the CH 50 , except magnesium chloride and eth- ylene glycol tetraaceti
c acid are added
to the bu

er, and calcium is le

t out. 33 This bu

er chelates calcium, which b

locks classical
pathway activation. Rabbit red cells are used as the indicator, because these pr
ovide an ideal
sur
ace
or alternative pathway activation. An additional means o
testing
or a
lternative
pathway
unction is via ELISA. One such test can detect C3bBbP or C3bP complexes
in very small
quantities. Microtiter wells are typically coated with bacterial polysaccharide
to trigger
activation o
the alternative pathway. One test system has been developed that c
an determine the
activity o
all three pathways: the classical pathway, the alternative pathway,
and the
mannose-binding lectin path- way. 17,34 In this test system, strips used

or the
classical pathway are coated with IgM, strips
or the alternative path- way are
coated with
lipopolysaccharide, and strips
or the mannose-binding lectin pathway are coated
with mannose.
Such testing is easy to per
orm and is not dependent upon the use o
animal eryt
hrocytes, which
may be hard to obtain. De ciencies can be detected using the combined test results
.
Interpretation o
Laboratory Findings Decreased levels o
complement components
or activity may
be due to any o
the
ollowing: (1) decreased production, (2) consumption, or (3
) in vitro
consumption. The third con- dition must be ruled out be
ore either o
the other
two is
considered. Specimen handling is extremely important. Blood should be colle
cted in a clot
tube with no serum sep- arator. 34 The tube should be spun down, and the serum s
hould be
rozen
or placed on dry ice i
it is not tested within 1 to 2 hours. I
a specimen has
been inadequately
re
riger- ated, been subjected to multiple
reezethaws, or been in prolonged stor
age, the
results may be invalid, and the test needs to be repeated with a
resh specimen.
Control serum
should also be included with each batch o
test sera. A typical screening test

or complement
abnormalities usually includes determination o
the
ollowing: C3, C4, and
acto
r B levels, as
well as hemolytic content. 17 Testing
or products o
complement activation such
as C3a, C4a,
C5a, and Ba as well as breakdown products, including C3dg, iC3b, and C4d, can al
so be per
ormed
as a means o
moni- toring in ammatory processes such as rheumatoid arthritis and
systemic lupus
erythematosus. Table 65 presents some o
the possible screening results
rom

ELISA
testing and correlates these with de ciencies o
individual
actors. An understan
ding o
these
patterns may be help
ul in di

erentiating hereditary de ciencies

rom activa
tional states
that consume available complement components. Additional testing would be necess
ary, however, to
actually pinpoint hereditary de ciencies. Complement Fixation Testing Complement i
tsel
can
actually be used as a reagent in the test known as complement xation. Because com
plement xation occurs a
ter the binding o
antigen and antibody, uptake o
comple
ment can be used
as an indicator o
the presence o
either speci c antigen or antibody. This technique has been
used in the detection o
viral,
ungal, and rickettsial antibodies. The test inv
olves a two-stage
process: (1) a test system with antigen and antibody, one o
which is unknown, a
nd (2) an
indicator system consisting o
sheep red blood cells coated with hemolysin, whic
h will lyse in
the presence o
complement. To destroy any complement present, patient serum mus
t be heated to
56C
or 30 minutes prior to testing. Then dilutions o
the serum are combined wit
h known antigen
and a measured amount o
guinea pig complement. Guinea pig complement can be use
d
or testing,
because comple- ment is not species-speci c. I
patient antibody is present, it wi
ll combine with
the reagent antigen, and complement will be bound. The test can also be per
orme
d with reagent
antibody to detect the presence o
antigen in the sample. The second step involv
es adding sheep
red blood cells that are coated with hemolysin. A
ter an additional incuba- tion
, the tubes are
centri
uged and then read
or hemolysis. I
hemolysis is present, this means tha
t no patient
antibody was present, and the test is negative. Lack o
hemolysis is a positive
test (Fig. 610).
Results are expressed as the high- est dilution showing no hemolysis. Table 55-2
Comparison o

Tests Used IMPAIRED FUNCTION/ CLASSICAL LECTIN ALTERNATIVE DEFICIENCY PATH

WAY PATHWAY
PATHWAY C1q, C1r, C1s Low Normal Normal C4, C2 Low Low Normal MBL, MASP2 Normal
Low Normal B, D,
P Normal Normal Low C3, C5, C6, C7, Low Low Low C8, C9 C1INH Low Low Low Factor
H and I Low Low
Low Improperly Low Low Low handled sera Adapted
rom Seelen, MA, et al. An enz
yme-linked
immunosorbent assay-based method
or
unctional analysis o
the three pathways o

the complement
system. In Detrick, B, Hamilton, RG, and Folds, JD (eds): Manual o
Molecular an
d Clinical
Laboratory Immunology, ed. 7. ASM Press, Washington, D.C., 2006, p. 124. Table 6
-5. Diagnosis o

Complement Abnormalities 1814_Ch06_085-106.qxd 7/10/09 2:47 PM Page 99 100 SE

CTION 1 Nature o

the Immune System The use o

controls is extremely important to ensure the accur
acy o
test
results. These include running known posi- tive and negative sera, an antigen co

ntrol, a patient
serum control, a cell control, and a complement control. The anti- gen and pat
ient serum
controls test
or the ability o
complement to bind in the absence o
antib
ody. These controls should show lysis with the sheep red blood cells. The cell control is a ch
eck on
spontaneous lysis. Lysis should be absent or extremely low. The complement
control tube
should show lysis to indicate that the complement is work- ing correctly. Due to
the amount o

manipulation required and the need

or standardization and numerous controls, th
e use o

complement xation in the clinical laboratory is limited. SUMMARY The complement s

ystem is a
series o
more than 30 proteins normally
ound in serum that play a major role i
n phagocy- tosis
and clearance o

oreign antigens
rom the body. While the end product o
comple
ment activation
is lysis, other important events, such as opsonization, increase in vascu- lar
permeability,
and chemotaxis o
monocytes and neutrophils take place along the way to enha
nce host de
ense
mechanisms. Most o
the proteins o
the complement system are inactive enzyme pr
ecursors, or
zymogens, that are con- verted to active enzymes in a very precise order. One su
ch means o

activation is the classical pathway, which is trig- gered by antigenantibody comb

ination and
involves nine o
the proteins. The alternative pathway acts as a natural means o

de
ense against
in
ection; it is triggered not by antigenantibody combination but by the presence
o

microorganisms. The lectin pathway also involves nonspeci

ic recognition o

microorganisms
by means o
sugars such as mannose that are common constituents o
microbial cel
l walls but that
are distinct
rom those
ound on human cell sur
aces. Although initial activatio
n o
these
pathways involves proteins that are unique to each, C3 serves a major role in li
nking all three
pathways. Cleavage o
C3 to C3a and C3b represents one o
the most signi cant step
s in all
pathways, because C3b is a power
ul opsonin that greatly enhances phagocytosis.
C3a is an
anaphylatoxin that causes release o
reactants
rom mast cells that ampli
y the
in ammatory
response. The end result is lysis o
cells due to production o
a membrane attac
k com- plex that
drills holes in the cell membrane. I
activation o
the complement system was to
proceed
uncontrolled, tissue damage and li
e-threatening systemic e

ects might result.

There
ore,
several proteins act as reg- ulators to keep the reaction localized and e
nsure that
in
ectious agents and not sel
-antigens are destroyed. Some o
these are
ree-
l
oating in the
plasma, while others are
ound on cell sur
aces as speci c receptors. One class o

membrane
receptors on host cells acts in conjunction with complement components to ampli

y the immune
response. These receptors bind antigenantibody complexes and
acilitate phagocyto
sis. Other

unctions may include enhancing activity o

phagocytes and production o
increas
ed memory B
cells. Complement plays a major role in several disease states. Decreased comple
ment levels or a
decline in lytic activity can indicate either hereditary de ciencies or extreme ac
ti- vation o

the system. Paroxysmal nocturnal hemoglobinuria and hereditary angioedema are tw

o conditions that
result
rom missing or very low production o
certain regulators. De ciencies o
s
ome components,
especially that o
C3, may cause an increased risk o
li
e-threatening in

ection or
glomerulonephritis due to a lack o
clearance o
immune complexes. Several labor
atory assays have
been devised to detect abnormal complement levels. One o
these, the CH 5
0 hemolytic
assay, uses 50 percent lysis o
a standard concen- tration o
antibody-sensitize
d sheep
erythrocytes as an end point. This measures the presence o
all proteins involve
d in the
classical pathway. A similar assay, the AH 50 assay, meas- ures the
unctioning
o
the
alternative pathway. Other assays using RID or ELISA techniques measure levels o

individ- ual
components. Proper handling o
specimens is essential to ensure correct interpre
tation o

laboratory testing. Complement xation tests use complement as a reagent to detect

the presence
o
antigen or antibody. Antigen and antibody are allowed to combine with a measu
red amount o

complement. Then indicator sheep red blood cells are added. These are coa
ted with
hemolysin (antisheep cell anti- body), and i
complement has not been tied up in
the rst
reaction, then lysis o
the cells occurs. Thus, lysis is a neg- ative test, indi
cating that
either antigen or antibody was not present. As in all complement testing, the us
e o
standards
and control is necessary
or accurate results. ag+ab +C Incubate SRBC +
ag-ab-c
+ ag+ab +
Lysis No lysis = positive test Lysis = negative test
SRBC SRBC C C
Incubate FIGU
RE 610.
Patient serum and antigen are incubated, and then a measured amount o
complemen
t is added. A
ter
a second incubation, sheep red blood cells, which are coated with antibody, are
added to the
reaction tube. I
patient antibody to the speci c antigen is present, no hemolysis
occurs. Lack
o
patient antibody allows the complement to combine with coated sheep red blood
cells and lysis
occurs. 1814_Ch06_085-106.qxd 7/10/09 2:47 PM Page 100 CHAPTER 6 Complement S
ystem 101 1. A
3-year-old child has a history o
serious in
ections and is currently hospitaliz
ed with
meningitis. The doctor suspects that he may have a complement de ciency and orders
testing. The

ollowing results are obtained: decreased CH 50 , normal C4 and C3 lev

els, and
decreased radial hemolysis in agarose plates using a bu

er that chelates calciu

m. Questions a.
What do the results indicate about possible path- way(s) a

ected? b. Which comp

onent(s) are
likely to be lacking? c. What sort o
additional
ollow-up would be recom- mende
d? 2. A
25-year-old
emale appeared at the emergency room with symptoms o
abdominal pai
n with severe
vomiting and swelling o
the legs and hands. She stated that she has had these s
ymptoms on
several previous occasions. A
ter appendicitis was ruled out, a battery o

tests,
including some
or abnormalities o
complement com- ponents, was per
ormed. The

ollowing results
were obtained: red and white blood cell count normal, total serum protein normal
, CH 50
decreased, alternative path- way
unction normal, C3 level normal, and C4 and C2
levels
decreased. Questions a. What symptoms led physicians to consider a possi- ble co
mplement
abnormality? b. What are possible reasons
or a decrease in both C4 and C2? c. W
hat other testing
would con rm your suspicions? CASE STUDIES 1814_Ch06_085-106.qxd 7/10/09 2:47 PM
Page 101 102
SECTION 1 Nature o
the Immune System Principle Total hemolytic complement activ
ity is measured
by calcu- lating the dilution o
patient serum that is needed to lyse 50 percent
o
a standard
concentration o
optimally sensi- tized sheep red blood cells. Sheep erythrocyte
s have been
previously sensitized with speci
ic antibody. The value obtained is compar
ed with those
reported
or a normal con- trol. The degree o
hemolysis is directly proportiona
l to the total
complement activity. Sample Preparation Collect at least 2 mL o
blood by venipu
ncture using
sterile technique. Do not use any anticoagulants. Allow the blood to clot at roo
m temperature.
Separate the serum
rom the clot. I
not used within a
ew hours, the serum can
be aliquoted in
0.5 mL portions and quick-
rozen at 70C. Do not use plasma. Reagents, Materials, a
nd Equipment
Sheep erythrocytes sensitized with antisheep antibody and preserved with sodium
azide (Diamedix
Corp., Miami, FL). These should be stored upright at 2C to 8C. Complement re
erenc
e serum
(Diamedix or in-house controls) High and low controls (Diamedix) Pipette and dis
posable tips to
dispense 5 L Centri
uge Photometer that will read absorbance at 415 nm CAUTION Th
e sensitized
cells contain sodium azide. Azides are reported to react with lead and copper in
plumbing to
orm
compounds that may detonate on percussion. When disposing o
solutions containin
g sodium azide,

lush with large volumes o

water to minimize the buildup o
metal azide c
ompounds.
Procedure* 1. Reconstitute the standard with cold (2C to 8C) distilled water
or i
mmediate use.
Discard any unused portion. 2. Reconstitute high and low controls with cold dist

illed water
or
immediate use. Discard the unused portion. 3. Place test tubes with sensitized c
ells in a rack
and allow them to warm to room temperature. Set up one tube
or each specimen, o
ne
or the
re
erence serum, one each
or the high and low controls, and one
or the spontaneous lysis
control. It is recommended that no more than 12 tubes be run at once. EZ COMPLEM
ENT CH 50 ASSAY
4. Vortex tubes or shake vigorously
or 10 seconds to resuspend cells. 5
. Remove the
stopper and trans
er 5 L o
sample or standard into a tube. Replace the sto
pper and mix
immediately by inverting the tube three to
our times. The spontaneous lysis
tube receives
no sample but should also be mixed. 6. Allow tubes to stand at room temperatur
e
or 60 minutes.
7. Mix contents o
all tubes again by inverting three to
our times or by vortex
ing. 8.
Centri
uge the tubes at approximately 1000 rpm
or at least 5 minutes. 9. Read t
he absorbance o

the supernatants at 415 nm within 15 minutes. I

possible, read directly through
the original
tube, or use a photometer with a sipping device that can withdraw the supernatan
t without disturbing the sedimented cells. a. To read the absorbance, zero the photometer wit
h a water blank.
b. Read and record the absorbance o
the spontaneous lysis control. I
the absor
bance value is
greater than 0.1, the results o
the assay will not be valid, and the test must
be repeated with
a new lot o
sensitized cells. c. Zero the photometer again with the spontaneous
lysis control.
This will correct
or any spontaneous lysis in test specimens. d. Read and recor
d the absorbance
value o
the re
er- ence serum. e. Read and record the absorbance value o
each
test sample.
Note: The volume o
individual tubes is at the lowest limit o
detection on Spec
trometer 20
instruments used in many clinical laboratory science programs. An EZ Reader phot
ometer designed
speci cally to read these tubes is available
rom Diamedix. 10. Calculate test res
ults as

ollows: Divide the absorbance value o

the supernate by the absorbance value o

the re
erence
serum and multiply by 100 to give the per- cent o
re
erence. * From the package
insert
or the
EZ Complement CH 50 Assay by Diamedix Corporation, Miami, FL. EXERCISE 1814_Ch06
_085-106.qxd
7/10/09 2:47 PM Page 102 CHAPTER 6 Complement System 103 Results Percent o
Re

erence Standard
Complement Level 050 Absent or low 51150 Normal 150 High Interpretation o
Results
This test
will provide a quantitative value
or the
unctional activity o
total complemen
t. It cannot
identi
y the individ- ual component or components that are abnormal. Abnormal sp
ecimens should be
assayed
or levels o
each o
the individ- ual components o
complement (C1C9). D
epressed
complement levels may be due to any o
the
ollowing: genetic de ciencies; decreas

ed complement
syn- thesis accompanying liver disease; or xation o
complement due to chronic
glomerulonephritis, rheumatoid arthritis, hemolytic anemias, gra
t rejection, SL
E, or other
autoim- mune phenomena. Elevated levels are
ound in acute in ammatory cond
itions,
leukemia, Hodgkins disease, sar- coma, and Behets disease. 1814_Ch06_085-106.qxd 7
/10/09
2:47 PM Page 103 104 SECTION 1 Nature o
the Immune System 1. The classical com
plement pathway
is activated by a. most viruses. b. antigenantibody complexes. c.
ungal cell wal
ls. d. All o

the above 2. All o

the
ollowing are characteristic o
complement components ex
cept a. normally
present in serum. b. mainly synthesized in the liver. c. present as active enzym
es. d.
heat-labile. 3. All o
the
ollowing are true o
the recognition unit except a.
it consists o

C1q, C1r, and C1s. b. the subunits require calcium

or binding together. c. bind
ing occurs at the
FC region o
antibody molecules. d. Clq becomes an active esterase. 4. Which is
re
erred to as C3
convertase? a. C4b2a b. C3bBb c. iC3Bb d. All o
the above 5. Mannose-binding pr
otein in the
lectin pathway is most similar to which classical pathway component? a. C3 b. C1
rs c. C1q d. C4
6. Which o
the
ollowing describes the role o
properdin in the alternative pat
hway? a.
Stabilization o
C3/C5 convertase b. Conversion o
B to Bb c. Inhibition o
C3 c
onvertase

ormation d. Binding to the initiating antigen 7. Which best characterizes the m

embrane attack
complex? a. Each pathway utilizes di

erent
actors to
orm it. b. C5 through C9
are not added in
any particular order. c. One MAC unit is su
cient to lyse any type o
cell. d. C9
polymerizes to

orm the transmembrane channel. 8. All o

the
ollowing represent
unctions o
t
he comple- ment
system except a. decreased clearance o
antigenantibody complexes. b. lysis o

o
reign cells. c.
increase in vascular permeability. d. migration o
neutrophils to the tissues. 9
. Which o
the

ollowing is true o
the ampli cation loop in complement activation? a. It is
oun
d in the
alternative pathway. b. iC3 binds
actor B to generate ampli cation. c. C3b is the
product that
is increased. d. All o
the above 10. Factor H acts by competing with which o
t
he
ollow- ing

or the same binding site? a. Factor B b. Factor D c. C3B d. Factor I 11. A lack
o
CR1 receptors
on red blood cells would result in which o
the
ollowing? a. Decreased binding
o
C3b to red
blood cells b. Lack o
clearance o
immune complexes by the spleen c. Increased
breakdown o
C3b
to C3d and C3dg d. All o
the above 12. A lack o
CR2 on cell membranes would re
sult in which o

the
ollowing? a. Decrease in hemolysis b. Increased susceptibility to in
ection
c. Decreased
antibody production d. Increase in antibody o
the IgG class 13. Why is compleme
nt not activated

with anti-Rh (D) antibodies? a. Rh antigens stimulate little antibody production

. b. Rh antigens
are too
ar apart on red blood cells. c. Rh antibodies are not capable o
xing co
mplement. d.
DAF protects red blood cells
rom buildup o
anti- body. 14. The CH 50 test meas
ures which o
the

ollowing? a. The dilution o

patient serum required to lyse 50 percent o
a sta
ndard
concentration o
sensi- tized sheep red blood cells b. Functioning o
both the c
lassical and
alternative pathways c. Genetic de ciencies o
any o
the complement components d.
All o
the
above 15. Which o
the
ollowing would be most e

ective in pre- venting bystand

er lysis o
red
blood cells? a. C1INH b. Factor B c. DAF d. Factor H REVIEW QUESTIONS 1814_Ch06_
085-106.qxd
7/10/09 2:47 PM Page 104 CHAPTER 6 Complement System 105 16. Decreased CH 50 l
evels may be
caused by a. inadequate re
rigeration o
specimen. b. coagulation-associated com
plement
consumption. c. autoimmune disease process. d. All o
the above Re
erences 1. Ki
ndt, TJ, Goldsby,
RA, and Osborne, BA. Kuby Immunology, ed. 6. WH Freeman and Co., New York, 2007,
pp. 168188. 2.
Prodinger, WM, et al. Complement. In Paul, WE (ed): Fundamental Immunol
ogy, ed. 4.
Lippincott-Raven, Philadelphia, 1999, pp. 967995. 3. Mak, T, and Saunders, M. The
Immune
Response: Basic and Clinical Principles. Elsevier, Burlington, MA, 2004, pp. 5535
81. 4. Carroll,
MC. The complement system in regulation o
adap- tive immunity. Nat Immunol 5:98
1986, 2004. 5.
Pillemer, L, et al. The properdin system and immunity: Demonstration and
isolation o
a
new serum protein, properdin, and its role in immune phenomena. Science 120:279,
1954. 6. Massey,
HD, and Mcpherson, RA. Mediators o
in
lamma- tion: Complement, cytokines, and a
dhesion
molecules. In McPherson, RA, and Pincus, MR (ed): Henrys Clinical Diagnosi
s and
Management by Laboratory Methods, ed. 21. Saunders Elsevier, Philadelphia, 2007,
pp. 850875. 7.
Arlaud, GJ, Barlow, PN, Gaboriaud, C, Gross, P, et al. Deceiphering com
plement
mechanisms: The contributions o
structural biology. Mol Immunol 44:38093822, 200
7. 8. Xu, Y,
Narayana, SVL, and Volanakis, JE. Structural biology o
the alternative pathw
ay convertase.
Immunol Rev 180:123135, 2001. 9. Janssen, BJC, Christodoulidou, A, McCarthy, A,
Lambris, JD, et
al. Structure o
C3b reveals con
ormational changes that underlie complement act
ivity. Nature
444:213216, 2006. 10. Walport, MJ. Complement. Part I. New Engl J Med 344
:10581066,
2001. 11. Mollnes, TE, Song, WC, and Lambris, JD. Complement in in
lammatory tis
sue damage and
disease. Trends Immunol 23:6164, 2002. 12. Schwaeble, WJ, and Reid, KBM. Does pro
perdin
crosslink the cellular and the humoral response? Immunol Today 20:1721, 19
99. 13. Rawal,

N, and Pangburn, MK. Structure/

unction o
C5 con- vertases o
complement. Int I
mmunopharmacol
1:415422, 2001. 14. Medzhitov, R, and Janeway, C. Innate immunity. New Engl J Med
343:33843,
2000. 15. Eisen, DP, and Minchinton, RM. Impact o
mannose-binding lection on
susceptibility
to in
ectious diseases. CID 37:14961505, 2003. 16. Arnold, JN, Dwek, RA, Rudd,
PM, and Sim,
RB. Mannan binding lectin and its interaction with immunoglobulins in health and
in disease.
Immunol Lett 106:103110, 2006. 17. Mollnes, TE, Jokiranta, TS, Truedsson, L, Nils
son, B, et al.
Complement analysis in the 21st century. Mol Immunol 44:38383849, 2007. 18
. Frakking,
FNJ, Brouwer, N, van Eijkelenburg, NKA, Merkus, MP, et al. Low mannose-bindi
ng lectin (MBL)
levels in neonates with pneumonia and sepsis. Clin Exp Immunol 150:25526
2, 2007. 19.
Kerr, FK, Thomas, AR, Wijeyewickrema, LC, Whisstock, JC, et al. Elucidation o
t
he substrate
speci city o
the MASP-2 protease o
the lectin complement pathway and identi cation
o
the
enzyme as a major physiological target o
the serpin, C1-inhibitor. Mol Immunol
45:670677, 2008.
20. Smith, BO, Mallin, RL, Krych-HGoldberg, M, Wang, X, et al. Structure o
the
C3b binding site
o
CR1 (CD35), the immune adherence receptor. Cell 108:769780, 2002. 21. Carroll,
MC, and
Fischer, MB. Complement and the immune response. Curr Opin Immunol 9:6469, 1997.
22. Lubin, DM,
and Atkinson, J. Decay-accelerating
actor: Biochemistry, molecular biology
, and
unction.
Adv Immunol 7:35, 1989. 23. DiScipio, RG. Ultrastructures and interactions o
co
mplement
actors
H and I. J Immunol 149:2592, 1993. 24. Sun, X, et al. Role o
decay-accelerating

actor in
regulating complement activation on the erythrocyte sur
ace as revealed by gene
targeting. Proc
Natl Acad Sci, USA 96:628633, 1999. 25. Kozono, Y, et al. Cross-linking CD21
/CD35 or CD19
increases both B71 and B72 expression on murine splenic B cells. J Immunol 160:156
51572, 1998.
26. Wen, L, Atkinson, JP, and Giclas, PC. Clinical and laboratory evaluation o

complement
de ciency. J Allergy Clin Immunol 113:585593, 2004. 27. Oksjoki, R, Laine, P, Helsk
e, S,
Vehmaan-Kreula, P, et al. Receptors
or the anaphylatoxins C3a and C5a are expre
ssed in human
atherosclerotic coronary plaques. Atherosclerosis 195:9099, 2007. 28. Sjoholm, AG
, Jonsson, G,
Braconier, JH, Stur
elt, G, et al. Complement de ciency and disease: An update. Mo
l Immunol
43:7885, 2006. 29. Pickering, MC, Botto, M, Taylor, PR, Lachmann, et al.
Systemic lupus
erythematosus, complement de
iciency, and apoptosis. Adv Immunol 76:227324, 2000.
30. Lewis, MJ,
and Botto, M. Complement de ciencies in humans and animals: Links to autoimmun
ity.
Autoimmunity 39:367378, 2006. 31. Hughes, J, et al. C5-C9 membrane attack complex
mediates

endothelial cell apoptosis in experimental glomerulonephri- tis. Am J Physiol Re

nal Physiol
278:F74757, 2000. 32. Song, WC. Complement regulatory proteins and autoimmu- nity
. Autoimmunity
39:403410, 2006. 33. Giclas, PC. Analysis o
complement in the clinical laborator
y. In Detrick,
B, Hamilton, RG, and Folds, JD (eds): Manual o
Molecular and Clinical Laborator
y Immunology, ed.
7. ASM Press, Washington, D.C., 2006, pp. 115117. 34. Seelen, MA, Roos, A, Wi
eslander, J,
and Daha, MR. An enzyme-linked immunosorbent assay-based method
or
unc- tio
nal analysis o

the three pathways o

the complement system. In Detrick, B, Hamilton, RG,
and Folds, JD
(eds): Manual o
Molecular and Clinical Laboratory Immunology, ed. 7. ASM Press,
Washington,
D.C., 2006, pp. 124127. 1814_Ch06_085-106.qxd 7/10/09 2:47 PM Page 105 1814_Ch
06_085-106.qxd
7/10/09 2:47 PM Page 106 II SECTION Basic Immunological Procedures 107 1814_Ch
07_107-122.qxd
7/10/09 2:49 PM Page 107 7 Sa
ety and Specimen Preparation Susan K. Strasinger
, DA, MT (ASCP)
and Christine Stevens LEARNING OBJECTIVES A
ter nishing this chapter, the reader
will be able
to: 1. List the components o
the chain o
in
ection and the sa
ety precautions
that will break
the chain. 2. Correctly per
orm routine handwashing. 3. Describe the types o
pe
rsonal protective
equipment used by laboratory personnel. 4. Di

erentiate between universal preca

utions, body
substance isolation, and standard precautions. 5. Describe the acceptable method
s
or disposal o

biological waste in the laboratory. 6. Following

ederal regulations and guideli
nes, prepare
patient specimens
or transport. 7. Sa
ely dispose o
sharp objects. 8. Describe
the components
o
the Occupational Exposure to Bloodborne Pathogens Standard. 9. Describe sa
et
y precautions
utilized when handling chemicals. 10. Discuss the components o
chemical hygiene
plans. 11.
Identi
y the symbol
or radiation. 12. Describe the
ollowing: serial dilution,
solute, diluent,
compound dilution. 13. Explain how an antibody titer is determined. 14. Calculat
e the amount o

diluent needed to prepare a speci c dilution o

a serum specimen. KEY TERMS Biohaz
ardous Body
substance isolation Chain o
in
ection Chemical Hygiene Plan Diluent Material sa

ety data sheets

Occupational Sa
ety and Health Administration (OSHA) Personal protective equipme
nt Postexposure
prophylaxis Serial dilution Solute Standard precautions Titer Universal precauti
ons 108
1814_Ch07_107-122.qxd 7/10/09 2:50 PM Page 108 CHAPTER 7 Sa
ety and Specimen
Preparation 109
The clinical laboratory contains a wide variety o
sa
ety hazards, many capable
o
producing
serious injury or li
e- threatening disease. To work sa
ely in this environment,
clinical
laboratorians must learn what hazards exist and the basic sa
ety precautions ass
ociated with

them, and they must apply the basic rules o

common sense required
or every- da
y sa
ety. Some
hazards are unique to the health-care environment, and others are encounter
ed routinely
through- out li
e (Table 71). BIOLOGICAL HAZARDS In the immunology laboratory,
the most
signi
icant hazard exists in obtaining and testing patient specimens. An unders
tanding o
the
transmissionthe chain o
in
ectiono
microorganisms is necessary to prevent in
e
ction. The
chain o
in
ection requires a continuous link between three elements: a source,
a method o

trans- mission, and a susceptible host. The most likely source o

in
ection in s
erological
testing is through contact with patient specimens, and the main concern is expos
ure to viruses
such as the hepatitis viruses and human immun- ode
iciency virus (HIV). There
or
e, sa
ety
precautions are designed to protect health-care workers
rom exposure to potenti
ally harm
ul
in
ectious agents. The ultimate goal o
biological sa
ety is to prevent completi
on o
the chain
by preventing transmission. Figure 71 contains the uni- versal symbol
or biohaza
rdous material
and illustrates the chain o
in
ection and how it can be broken by
ollow- ing s
a
ety practices.
Preventing the transmission o
microorganisms
rom in
ected sources to susceptib
le hosts is
critical in control- ling the spread o
in
ection. Procedures used to prevent mi
croorganism
transmission include handwashing, wearing personal protective equipment (PPE), i
solating highly
in
ective or highly susceptible patients, and properly dis- posing contaminated
materials.
Strict adherence to guidelines published by the Centers
or Disease Control an
d Prevention
(CDC) and the Occupational Sa
ety and Health Administration (OSHA) is essential.
CHAPTER OUTLINE
BIOLOGICAL HAZARDS Handwashing Personal Protective Equipment Procedure Precautio
ns Biological
Waste Disposal Transporting Patient Specimens SHARPS HAZARDS Governmental Regula
tions
Occupational Exposure to Bloodborne Pathogens CHEMICAL HAZARDS General Precautio
ns Material
Sa
ety Data Sheets Chemical Hygiene Plan Chemical Waste Disposal RADIOACTIVE HAZ
ARDS General
Precautions Radioactive Waste Disposal SEROLOGICAL TESTING Specimen Preparation
and Processing
Simple Dilutions Compound Dilutions SUMMARY CASE STUDY EXERCISE: SERIAL DILUTION
S EXERCISE:
LABORATORY SAFETY REVIEW QUESTIONS REFERENCES Table 7-1. Types o
Sa
ety Hazards
TYPE SOURCE
POSSIBLE INJURY Biologic In
ectious agents Bacterial,
ungal, viral, or parasiti
c in
ections
Sharp Needles, lancets, and broken glass Cuts, punctures, or bloodborne pathogen
exposure
Chemical Preservatives and reagents Exposure to toxic, carcinogenic, or caustic
agents
Radioactive Equipment and radioisotopes Radiation exposure Electrical Ungrounded
or wet equipment

and
rayed cords Burns or shock Fire/explosive Bunsen burners and organic chemic
als Burns or
dismemberment Physical Wet oors, heavy boxes, and patients Falls, sprains, or str
ains
1814_Ch07_107-122.qxd 7/10/09 2:50 PM Page 109 110 SECTION 2 Basic Immunologi
cal Procedures
Handwashing Hand contact represents the number-one method o
in
ec- tion transmi
ssion. Hands
should always be washed at the
ollowing times: be
ore patient contact, when glo
ves are removed,
prior to leaving the work area, whenever the hands have been knowingly contamina
ted, be
ore going
to designated break areas, and be
ore and a
ter using bath- room
acilities. Cor
rect routine
handwashing technique includes the
ollowing: 1 1. Wet hands with warm water. 2.
Apply soap,
pre
erably antimicrobial. 3. Rub to
orm a lather, create
riction, and loosen d
ebris. 4. Clean
thoroughly between
ingers, thumbs, and under ngernails and rings
or at least 15
seconds and up
to the wrist. 5. Rinse hands in a downward position. 6. Dry hands with a paper t
owel. 7. Turn
o

aucets with a clean paper towel to prevent recontamination. Persona
l Protective
Equipment Personal protective equipment encountered by laboratorians includes gl
oves, gowns or
laboratory coats, masks, goggles,
ace shields, and Plexiglas countertop shields
. Gloves are worn
to protect the health-care workers hands
rom contamina- tion by patient body sub
stances and to
protect the patient
rom possible microorganisms on the health-care workers hands
. Wearing
gloves is not a substitute
or handwashing. Hands must always be washed when glo
ves are removed.
A variety o
gloves are available, including sterile and nonster- ile, powdered
and unpowdered,
and latex and nonlatex. Allergy to latex is increasing among health-care workers
. Laboratorians
should be alert
or symptoms o
reactions associated with latex contact, includi
ng irritant
contact der- matitis that produces patches o
dry, itchy irritation on the hands
; delayed
hypersensitivity reactions resembling poi- son ivy that appear 24 to 48 hours
o
llowing exposure;
and true immediate hypersensitivity reactions o
ten character- ized by
acial

lushing and
respiratory di

iculty (see Chapter 14). Handwashing immediately a

ter removal
o
gloves and
avoiding powdered gloves may aid in preventing the development o
latex allergy.
Replacing latex
gloves with nitrile or vinyl gloves provides an acceptable alternative. Any sign
s o
a latex
reaction should be reported to a super- visor, as true latex allergy can be li
e
-threatening. 2
In the immunology laboratory,
luid-resistant labora- tory coats with wrist cu

s are worn to
protect skin and clothing
rom contamination by patient specimens. Coats are wor
n at all times
when working with patient speci- mens. They must be completely buttoned with glo
ves pulled over

the cu

s. Both gloves and laboratory coats should be changed as soon as p

ossible i
they
become visibly soiled, and they must be removed when leaving the laboratory. Th
e mucous
membranes o
the eyes, nose, and mouth must be protected
rom specimen splashes
and aerosols. A
variety o
protective equipment is available, including gog- gles,
ull-
ace p
lastic shields,
and Plexiglas countertop shields (Fig. 72). Particular care should be taken to
avoid splashes
and aerosols when removing container tops and when trans
erring and centri
uging
specimens. Never
cen- tri
uge specimens in uncapped tubes or in uncovered centri
uges. Whe
n specimens are
received in containers with contaminated exteriors, the exterior o
the containe
r must be
disin
ected or, i
necessary, a new specimen may be requested. Procedure Precaut
ions Universal
precautions (UP) were instituted by the CDC in 1985 to protect health-care worke
rs
rom exposure
to bloodborne pathogens, primarily hepatitis B virus (HBV) and HIV. Under uni
versal
precautions, all patients are assumed to be possible carriers o
bloodborne p
athogens.
Transmission may occur by skin puncture
rom a contami- nated sharp object or by
passive contact
through open skin S O URC E T RAN S M I S S I O N H O S T Hand washing Biohazard
ous waste
disposal Decontamination Specimen bagging Hand washing Personal protective equip
ment Aerosol
prevention Sterile/disposable equipment Pest control Standard precautions Immuni
zation Healthy
li
estyle Exposure control plan Postexposure prophylaxis FIGURE 71. Chain o
in
e
ction and
sa
ety practices related to the biohazard symbol. (From Strasinger, SK, and DiLo
renzo, MS.
Urinalysis and Body Fluids, ed. 5. FA Davis, Philadelphia, 2008, with permission
.)
1814_Ch07_107-122.qxd 7/10/09 2:50 PM Page 110 CHAPTER 7 Sa
ety and Specimen
Preparation 111
lesions or mucous membranes. The guideline recommends wearing gloves when collec
ting or handling
blood and body uids contaminated with blood, wearing
ace shields when there is d
anger o
blood
splashing on mucous membranes, and disposing o
all needles and sharp objects in
punctureresistant containers without recapping. A modi cation o
universal precautions, bo
dy substance
isolation (BSI) is not limited to bloodborne pathogens and considers all body uid
s and moist
body substances to be poten- tially in
ectious. Personnel should wear gloves at
all times when
encountering moist body substances. A disadvantage o
the BSI guideline is that
it does not
recommend handwashing a
ter removing gloves unless visual contamination is prese
nt. The major

eatures o
UP and BSI have now been com- bined and are called standard pre
cautions.
Standard precautions should be used
or the care o
all patients and include the

ollowing: 3

Handwashing. Wash hands a

ter touching blood, body u- ids, secretions, excretio
ns, and
contaminated items, whether or not gloves are worn. Wash hands immedi- ately a

ter gloves are

removed, between patient contacts, and when otherwise indicated to avoid t
rans
er o

microorganisms to other patients or environments. It may be necessary to wash ha

nds between tasks
and pro- cedures on the same patient to prevent cross-contamination o
di

erent
body sites.
Gloves. Wear gloves (clean, nonsterile gloves are adequate) when touching blood,
body uids,
secretions, excretions, and contaminated items. Change gloves between tasks and
procedures on the
same patient a
ter contact with material that may contain a high concentra
tion o

microorganisms. Remove gloves promptly a

ter use, be
ore touching noncontam
inated items and
environ- mental sur
aces, and be
ore going to another patient. Wash hands immedi
ately to avoid
trans
er o
microorganisms to other patients or environments. Mask, Eye Protecti
on, and Face
Shield. Wear a mask and eye protection or a
ace shield to protect mucous mem- b
ranes o
the
eyes, nose, and mouth during procedures and patient-care activities that are lik
ely to generate
splashes or sprays o
blood, body uids, secretions, and excretions. Gown. Wear a
gown (a clean,
nonsterile gown is adequate) to protect skin and to prevent soiling o
clothing
during procedures
and patient-care activities that are likely to gen- erate splashes o
blood, bod
y uids,
secretions, or excretions. Select a gown that is appropriate
or the activity an
d amount o
uid
likely to be encountered. Remove a soiled gown as promptly as possible, and wash
hands to avoid
trans
er o
microorganisms to other patients or environments. Patient-Care Equip
ment. Handle used
patient-care equip- ment soiled with blood, body
luids, secretions, and
excretions in a
manner that prevents skin and mucous membrane exposures, contamination o
clothi
ng, and trans
er
o
microorganisms to other patients and envi- ronments. Ensure that reusable equ
ipment is not
used
or the care o
another patient until it has been cleaned and reprocessed a
ppropriately.
Ensure that single-use items are discarded properly. Environmental Control. Ensu
re that the
hospital has ade- quate procedures
or the routine care, cleaning, and di
sin
ection o

environmental sur
aces, beds, bed rails, bedside equipment, and other
requently
touched sur
aces, and ensure that these procedures are being
ollowed. Linen. Handle
, transport, and
process used linen soiled with blood, body uids, secretions, and excretions in a
man- ner that
prevents skin and mucous membrane exposure and contamination o
clothing and tha
t avoids trans
er
o
microorganisms to other patients and environments. Occupational Health and Bl
oodborne

Pathogens. Take care to prevent injuries when using needles, scalpels, and other
sharp
instruments or when handling sharp instru- ments a
ter procedures; when cleaning
used
instruments; and when disposing o
used needles. Never recap used needles or oth
erwise manipulate
them using both hands, and never use any technique that involves directing the p
oint o
a needle
toward any part o
the body; rather, use either a one-handed scoop technique or a
mechanical
device designed
or holding the needle sheath. Do not remove used needles
rom d
isposable
syringes by hand, and do not bend, break, or otherwise manipulate used needles b
y hand. Place
used disposable syringes and nee- dles, scalpel blades, and other sharp items in
appropriate
puncture-resistant containers, which are located as close as practical to the ar
ea in which the
items were used. Place reusable syringes and needles in a puncture-resistant con
- tainer
or
transport to the reprocessing area. Patient Placement. Place a patient who conta
minates the
environment or who does not (or cannot be expected to) assist in maintaining app
ropriate hygiene
or environmen- tal control in a private room. I
a private room is not available
, consult with
in
ection-control pro
essionals regarding patient placement or other alternative
s. FIGURE 72.
Personal protective equipment. (From Strasinger, SK, and DiLorenzo, MA. Phleboto
my Workbook
or
the Multiskilled Healthcare Pro
essional, ed. 2. FA Davis, Philadelphia, 2003, w
ith permission.)
1814_Ch07_107-122.qxd 7/10/09 2:50 PM Page 111 112 SECTION 2 Basic Immunologi
cal Procedures
Biological Waste Disposal All biological waste, except urine, must be placed in
appro- priate
containers labeled with the biohazard symbol. This includes not only specimens,
but also the
materials with which the specimens come in contact. Any supplies con- taminated
with blood and
body uids must also be disposed o
in containers clearly marked with the biohazar
d symbol or
with red or yellow color-coding. This includes alcohol pads, gauze, bandages,
disposable
tourniquets, gloves, masks, gowns, and plastic tubes and pipettes. Dispo
sal o
needles
and other sharp objects is discussed in the next section. Contaminated nondispos
able equipment,
blood spills, and blood and body uid processing areas must be disin
ec- ted. The
most commonly
used disin
ectant is a 1:10 dilution o
sodium hypochlorite (household bleach) p
repared weekly
and stored in a plastic, not a glass, bottle. The bleach should be allowed to ai
r-dry on the
contaminated area prior to removal. The National Committee
or Clinical Laborato
ry Standards
(NCCLS) states that a 1:100 dilution can be used
or routine cleaning. 4 Transpo
rting Patient
Specimens Depending on the scope o
testing per
ormed in an immunology l
aboratory,

transporting and receiving patient specimens must be considered. There

ore, regu
lations
or
packaging and labeling developed by the U.S. Department o
Transportation (DOT),
the
International Air Transport Association (IATA), and the United Nations mus
t be
ollowed.
Courier-Delivered Specimen Transport Specimens transported by a hospital courier
among clinics,
physicians o
ces, and the hospital laboratory are exempt
rom most DOT rules unles
s they are
suspected o
contain- ing an in
ectious substance. The transport vehicle should
be used
exclusively
or transport o
these materials and should be equipped to sec
ure the transport
containers. 5 Minimum shipping standards
or this type o
transportation include
6 1. Leak-proo
,
water-tight specimen containers. 2. Tightly capped tubes placed in a rack to mai
ntain an upright
position. 3. Leak-proo
inner packaging surrounded by enough absorbent mate
rial to
completely absorb all the liquid present. 4. A leak-proo
plastic or metal trans
port box with a
secure, tight- tting cover. 5. Properly labeled transport boxes accompanied by spe
c- imen data
and identi cation
orms. Specimens picked up by a courier that are to be shipped t
o an
out-o
-the-area laboratory, such as a re
erence labo- ratory, must
ollow DOT
regulations.
Many o
these laboratories supply shipping containers to their clients. DOT an
d IATA Specimen
Transport Under DOT and IATA regulations, all diagnostic specimens require trip
le packaging
(Fig. 73). This includes the
ollowing: 1. Watertight primary containers made
o
glass,
metal, or plastic with a positive (screw-on) cap. 2. The primary container must
be wrapped with
enough absorbent material to be capable o
absorbing all o
its contents. Multip
le specimens must
be wrapped individ- ually prior to placing them in the leak-proo
secondary cont
ainer. 3. The
secondary container is placed in a sturdy outer con- tainer made o
corrugated

iberboard, wood,
metal, or rigid plastic. An itemized list o
contents in a sealed plas- tic bag
is also placed in
the outer container. Ice packs are placed between the secondary and the outer co
n- tainer.
Additional measures must be taken when using ice and dry ice. In January 2007, l
abeling o
the
outer container changed. The terms clinical specimen and diagnostic specimen hav
e been replaced
with biological substances, Category B. This wording is placed next to the label
UN 3373. 7,8
SHARPS HAZARDS Sharp objects in the laboratory, including needles, lancets, and
broken glassware,
present a serious biological hazard
or possible exposure to bloodborne pathogen
s caused by
accidental puncture. Although bloodborne pathogens are also transmitted through
contact with
mucous membranes and nonintact skin, a needle or lancet used to collect blood ha
s the capability

to produce a very signi

icant exposure to bloodborne pathogens. It is essential
that sa
ety
precau- tions be
ollowed at all times when sharp hazards are present.
The number-one
personal sa
ety rule when handling nee- dles is to never manually recap one. Man
y sa
ety devices
are available
or needle disposal, and they provide a variety o
sa
eguards. The
se include needle
holders that become a sheath, needles that automatically resheath or become blun
t, and needles
with attached sheathes. All sharps must be dis- posed o
in puncture-resistant,
leak-proo

containers labeled with the biohazard symbol (Fig. 74). Containers should be loca
ted in close
proximity to the work area and must always be replaced when the sa
e capacity ma
rk is reached.
Government Regulations The
ederal government has enacted regulations to p
rotect
health-care workers
rom exposure to bloodborne pathogens. These regulations are
monitored and
en
orced by OSHA. The Occupational Exposure to Bloodborne Pathogens Standar
d became law in
1991. 9 It requires all employers to have a written Bloodborne Pathogen Exposure
Control Plan and
to provide necessary protection,
ree o
1814_Ch07_107-122.qxd 7/10/09 2:50 PM
Page 112
CHAPTER 7 Sa
ety and Specimen Preparation 113 charge,
or employees. Speci
ics
o
the OSHA
standard include the
ollowing: 1. Requiring all employees to practice universal
(standard)
precautions. 2. Providing lab coats, gowns,
ace shields, and gloves to employee
s and providing
laundry
acilities
or non disposable protective clothing. In
ectious substa
nce Package mark
Name and telephone number o
a person responsible. (This in
ormation may instead
be provided on a
written document such as an air waybill.) Rigid outer packaging Cross section o

closed package
Cushioning material Absorbent packing material (
or liquids) B io lo g ical S u
b stan ce C aterg
o ry B
U N 3373 Secondary packing leakproo
or si
tproo
(e.g., sealed plastic
bag) Rigid outer
packaging Absorbent material Secondary packing leakproo
or si
tproo
(e.g., sea
led plastic bag
or other intermediate packaging) Primary receptacle leakproo
or si
tproo
Prima
ry receptacle
leakproo
or si
tproo
FIGURE 73. Packing and labeling o
Category B in
ectious s
ubstances. I

multiple
ragile primary receptacles are placed in a single secondary package, t
hey must be
either individually wrapped or separated to prevent contact. (From Pipeline and
Hazardous
Materials Sa
ety Administration, U.S. Department o
Transportation.) FIGURE 74. E
xamples o

puncture-resistant containers. (From Strasinger, SK, and DiLorenzo, MA. Phleboto

my Workbook
or
the Multiskilled Healthcare Pro
essional, ed. 2. FA Davis, Philadelphia, 2003, w
ith permission.)

3. Providing sharps disposal containers and prohibiting recapping o

needles. 4.
Prohibiting
eating, drinking, smoking, and applying cos- metics in the work area. 5. Labelin
g all
biohazardous materials and containers. 6. Providing immunization
or the hepatit
is B virus
ree
o
charge. 7. Establishing a daily work sur
ace disin
ection protocol. The disin

ectant o
choice

or bloodborne pathogens is sodium hypochlorite (household bleach

reshly dilute
d 1:10). 8.
Providing medical
ollow-up to employees who have been accidentally exposed to b
loodborne
pathogens. 9. Documenting regular training o
employees in sa
ety standards. The
exposure control
plan must be available to employees. It must be updated annually and must identi

y procedures and
individuals at risk o
exposure to bloodborne pathogens. The plan must also iden
ti
y the
engineering controls (i.e., sharps containers) and the procedures in place to pr
event exposure
incidents. In 1999, OSHA issued a new compliance directive called En
orcement Pr
ocedures
or the
Occupational Exposure to Bloodborne Pathogens Standard. 10 The new directive pla
ced more emphasis
on using engineering controls to prevent accidental exposure to bloodborne patho
gens. Additional
1814_Ch07_107-122.qxd 7/10/09 2:50 PM Page 113 114 SECTION 2 Basic Immunologi
cal Procedures
changes to the directive were mandated by passage o
the Needlestick Sa
ety and
Prevention Act,
signed into law in 2001. 11 Under this law, employers must 1. Document their eva
luations and
implementation o
sa
er needle devices. 2. Involve employees in the selection an
d evaluation o

new devices. 3. Maintain a log o

all injuries
rom contaminated sharps. In June
2002, OSHA
issued a revision to the Bloodborne Pathogens Standard compliance directive. 12
In the revised
directive, the agency requires that all blood holders (adapters) with nee
dles attached be
immediately discarded into a sharps container a
ter the devices sa
ety
eature is
activated.
Rationale
or the new directive is based on the exposure o
workers to the unpro
tected
stopper-puncturing end o
evacuated tube needles, the increased needle manip- ul
ation required
to remove it
rom the holder, and the possible worker exposure
rom the u
se o

contaminated holders. Occupational Exposure to Bloodborne Pathogens Any acciden

tal exposure to
blood through needlestick, mucous membranes, or nonintact skin must be report
ed to a
supervisor, and a con dential medical examination must be immediately started. Eva
luation o
the
incident must begin right away to ensure appropriate postexposure pro- phylaxis
(PEP).
Needlesticks are the most
requently encountered exposure and place the labo
ratorian in
danger o
contracting HIV, HBV, and hepatitis C virus (HCV). Each health-care in
stitution is

responsible
or designing and implementing its own exposure control plan. 13 CHE
MICAL HAZARDS
General Precautions Serological testing may involve use o
chemical reagents tha
t must be
handled in a sa
e manner to avoid injury. General rules
or sa
e handling
o
chemicals
include taking precautions to avoid getting chemicals on your body, clothes, and
work area;
wearing PPE such as sa
ety goggles when pouring chemicals; observing strict labe
ling practices;
and
ollowing instructions care
ully. Preparing reagents under a
ume hood is
also a
recommended sa
ety precaution. Chemicals should never be mixed together, unles
s speci c
instructions are
ollowed, and they must be added in the order speci ed. This is p
articularly
important when com- bining acid and water, as acid should always be added to wat
er to avoid the
possibility o
sudden splashing. When skin or eye contact occurs, the best rst ai
d is to
immediately ush the area with water
or at least 15 minutes and then seek medical
attention.
Laboratorians must know the location o
the emergency shower and eyewash station
in the
laboratory. Do not try to neutralize chemicals spilled on the skin. Material Sa

ety Data Sheets

All chemicals and reagents containing hazardous ingredi- ents in a concentration
greater than 1
percent are required to have a material sa
ety data sheet (MSDS) on
ile in the
work area. By
law, vendors must provide these sheets to purchasers; however, it is the respons
ibility o
the

acility to obtain and keep them available to employees. An MSDS contains in

orm
ation on physical
and chemical characteris- tics, re, explosion reactivity, health hazards, primary
routes o

entry, exposure limits and carcinogenic potential, precau- tions

or sa
e handli
ng, spill
cleanup, and emergency
irst aid. Containers o
chemicals that pose a high risk
must be labeled
with a chemical hazard symbol representing the pos- sible hazard, such as ammable
, poisonous,
corrosive, and so on. State and
ederal regulations should be consulted
or the
disposal o

chemicals. Chemical Hygiene Plan OSHA requires that all

acilities that use haza
rdous chemicals
have a written Chemical Hygiene Plan available to employ- ees. 14 The purpose o

the plan is to
detail the
ollowing: 1. Appropriate work practices 2. Standard operating proced
ures 3. Personal
protective equipment 4. Engineering controls, such as
ume hoods and amma- bles s
a
ety cabinets
5. Employee training requirements 6. Medical consultation guidelines Each
acili
ty must appoint a
chemical hygiene o

icer, who is responsible

or implementing and documenting co
m- pliance with
the plan. Examples o
chemical sa
ety equipment and in
ormation are shown i
n Figure 75.
Chemical Waste Disposal Hazardous chemical waste should be disposed o
per current EPA

regulations. Local regulations and the Department o

Transportation also track d
isposal o

hazardous chemical waste. Many kits used in testing contain sodium azide, which
can be disposed
o
by ushing down the drain with plenty o
water to avoid buildup in plumbing. RA
DIOACTIVE
HAZARDS General Precautions Radioactivity is encountered in the clinical laborat
ory when
procedures using radioisotopes, such as radioimmunoassay, are per
ormed. The amo
unt o

radioactivity present in most medical situations is very small and represents li

ttle danger.
1814_Ch07_107-122.qxd 7/10/09 2:50 PM Page 114 CHAPTER 7 Sa
ety and Specimen
Preparation 115
However, the e

ects o
radiation are related to the length o
exposure and are
cumulative.
Exposure to radiation is dependent on the combination o
time, distance, and shi
eld- ing.
Persons working in a radioactive environment are required to wear measurin
g devices to
determine the amount o
radiation they are accumulating. Laboratorians should be

amiliar with
the radioactive symbol shown in Figure 76. This symbol must be dis- played on the
doors o
all
areas where radioactive material is present. Exposure to radiation during pregna
ncy pres- ents a
danger to the
etus, and personnel who are or who think they may be pregnant sho
uld avoid areas
with this symbol. Radioactive Waste Disposal Disposal o
radioactive waste is re
gulated by the
Nuclear Regulatory Commission (NRC). Such waste must be sep- arated
rom other w
aste materials in
the laboratory and may be disposed o
by storing in a locked, labeled room until
the background
count is reduced by a speci ed number o
hal
-lives. Typically, 125 I is the most

requently
encountered radiolabel, and this can be disposed o
in this manner. SEROLOGICAL
TESTING Specimen
Preparation and Processing The most
requently encountered specimen in immunolog
- ical testing
is serum. Blood is collected aseptically by venipuncture into a clean, dry
, sterile tube.
Care must be taken to avoid hemolysis, since this may produce a
alse- positive
test. The blood
specimen is allowed to clot at room temperature or at 4C and then centri
uged. Se
rum should be
promptly separated into another tube without trans
er- ring any cellular element
s. Fresh, nonheat
inactivated serum is usually recommended
or testing. However, i
testing can- n
ot be per
ormed
immediately, serum may be stored between 2C and 8C
or up to 72 hours. I
the
re is any
additional delay in testing, the serum should be
rozen at 20C or below. S
imple
Dilutions For many tests, a measured amount o
a serum sample is used directly

or detection o

antibodies. However, in order

or a visible end point to occur in a serological
reaction, the
relative proportions o
antigen and antibody present are important. Sometimes in
a serological

test, too much antibody may be present, and an end point may not be reached. In
this case, serum
that contains antibody must be diluted. There
ore, knowledge o
serial dilutions
is essential to
understanding all serological testing in the clin- ical laboratory. A dilution i
nvolves two
entities: the solute, which is the material being diluted, and the diluent, whic
h is the medium
making up the rest o
the solution. The relationship between these two is expres
sed as a

raction. For example, a 1:20 dilu- tion implies 1 part o

solute and 19 parts o

diluent. The
number on the bottom o
the
raction is the total volume, reached by adding the
volumes o
the
solute and diluent together. 1/Dilution
Amount o
Solute/Total Volume To create
a certain
volume o
a speci
ied dilution, it is help
ul to know how to manipulate this rel
ationship. An
FIGURE 76. Radioactive symbol. FIGURE 75. Examples o
chemical sa
ety equipment an
d in
ormation. (From Strasinger, SK, and DiLorenzo, MA. Urinalysis and Body Fluids, ed. 4
. FA Davis,
Philadelphia, 2001, with permission.) 1814_Ch07_107-122.qxd 7/10/09 2:50 PM P
age 115 Compound
Dilutions The previous examples represent simple dilutions. Occasionally in
the laboratory
it is necessary to make a very large dilution, and it is more accurate and less
costly to do this
in several steps rather than all at once. Such a process is known as a compound
dilution. The
same approach is used, but the dilution occurs in several stages. For example, i

a 1:500
dilution is necessary, it would take 49.9 mL o
dilu- ent to accomplish this in
one step with 0.1
mL o
serum. I
only a small amount o
solution is needed to run the test, this
is waste
ul;

urthermore, inaccuracy may occur i

the solution is not properly mixed. There
o
re, it is help
ul
to make several smaller dilutions. To calculate a compound dilution problem, the
rst step is to
plan the number and sizes o
simple dilutions necessary to reach the desired end
point. To use
the preceding exam- ple, a 1:500 dilution can be achieved by making a 1:5 diluti
on o
the
original serum, a 1:10 dilution
rom the rst dilution, and another 1:10 dilution.
This can be
shown as
ollows: Serum: 1:5 dilution 1:10 dilution 1:10 dilution 0.1 mL serum 0
.1 mL o
1:5
dilution 0.1 mL o
1:10 dilution 0.4 mL diluent 0.9 mL diluent 0.9 mL diluent Mu
ltiplying 5
10
10 equals 500, or the total dilu- tion. Each o
the simple dilutions is calculat
ed individually
by doing mental arithmetic or by using the
ormula given
or simple dilutions. I
n this example,
the 1:500 dilution was made using very little diluent in a series o
test tubes,
rather than
having to use a larger volume in a ask. The volumes were kept small enough so tha
t mixing could
take place easily, and the nal volume o
1.0 mL is all that is necessary to per
o
rm a test. I
,

in each step o
the dilution, the dilution
actor is exactly the same,
this is known as
a serial dilution. Serial dilu- tions are o
ten used to obtain a titer, or indic
ator o
an
antibodys strength. A series o
test tubes is set up with exactly the same amount
o
diluent in
each (Fig. 77). The most common serial dilution is a doubling dilution, in which
the amount o

serum is cut in hal

with each dilution. For example, six test tubes can be set
up with 0.2 mL o

diluent in each. I
0.2 mL o
serum is added to the
irst tube, this becomes a 1
:2 dilution.
algebraic equation can be set up to
ind the total volume, the amount o
solute,
or the amount o

diluent needed to make a dilution. Consider the

ollowing example: 2 mL o
a 1:2
0 dilution is
needed to run a speci c serological test. How much serum and how much diluent are
needed to make
this dilution? The equation is set up using the
raction
or the dilution, indic
ating the
relationship between the total volume and the solute, or the amount o
serum nee
ded: 1/20
x/2
mL Note that the 20 represents the total number o
parts in the solution and tha
t 2 mL is the
total volume desired. Solving this equation
or x gives 0.1 mL
or the amount o

serum needed to
make this dilution. The amount o
dilu- ent is obtained by subtracting 0.1 mL
r
om 2.0 mL to give
1.9 mL o
diluent. To check the answer, simply set up a pro- portion between the
amount o
solute
over the total volume. This should equal the dilution desired. Thus, the correct
answer has been
obtained. I
, on the other hand, one knows the amount o
serum to be used, a pro
blem can be set
up in the
ollowing manner: A 1:5 dilution o
patient serum is necessary to run
a serological
test. There is 0.1 mL o
serum that can be used. What amount o
diluent is neces
sary to make this
dilution using all o
the serum? A slightly di

erent
ormula can be used to sol
ve this problem:
1/Dilution 1
Amount o
Solute/Amount o
Diluent 1 4 0.1 mL/x x
0.4 mL o
diluent
Note
that the nal volume is obtained by adding 0.1 mL o
solute to the 0.4 mL o
dilue
nt. Dividing
the volume o
the solute by the total volume o
0.5 mL yields the desired 1:5 ra
tio. Depending on
the unknown being solved
or, either o
these
ormulas can be used. To calculate
the total
volume, the total dilution
actor must be used. I
, however, the amount o
dilue
nt is to be
calculated, the
ormula using dilution 21 can be used. Further problems are give
n at the end o

the chapter to allow practice with dilution calculations. 116 SECTION 2 B

asic
Immunological Procedures 1 1:2 1:4 1:8 1:16 1:32 1:64 Add 0.2 mL serum
Mix Remove 0.2
mL Mix Remove 0.2 mL Mix Remove 0.2 mL Mix Remove 0.2 mL Mix Remove 0.2 mL 2 3
4 5 6
Mix Discard 0.2 mL FIGURE 77. Serial dilution. Each tube contains 0.2 mL o
dilue
nt.

Patient serum (0.2 mL) is added to tube one. This is care-

ully mixed, and then
0.2 mL is
withdrawn and added to tube two. The process is continued until the last tube is
reached. The
sample is mixed, and 0.2 mL is discarded. Note that in this dilu- tion, the amou
nt o
antibody is
cut in hal
in each successive tube. 1814_Ch07_107-122.qxd 7/10/09 2:50 PM Pa
ge 116 source, a
method o
transmission, and a host. Handwashing and wearing PPE are essential to
prevent
transmission o
in
ec- tious organisms. Standard precautions should be
ollowed
at all times.
Specimens, except urine, and contaminated supplies must be disposed o
in a
biohazard
container. Sodium hypochlorite is the recommended disin
ectant
or blood and bo
dy uid
contamination o
countertops and nondisposables. All sharps, including needles a
nd adaptors, must
be disposed o
in puncture-proo
containers. Recapping o
needles is pro- hibite
d. Accidental
exposure to blood and body uids must be reported immediately to a supervisor. Whe
n transporting
bio- logical specimens, speci c packaging requirements must be
ollowed. Follow sp
eci
ic
directions when mixing chemicals, and always add acid to water, rather than wate
r to acid. When
chemical contact with skin or eyes occurs, immediately ush the area with water. A
n MSDS and a
Chemical Hygiene Plan must be available to employees. Dispose o
chemicals per E
PA guidelines.
Laboratorians who are pregnant should avoid areas with a radioactive symbol. Dis
pose o
radioactive material
ollowing NRC guidelines. Serum is typically the specimen used in
serological
testing to look
or the presence or absence o
antibody. In order
or a visible
end point to
occur in antigenantibody reactions, o
ten a dilution needs to be made. Patient se
rum, the
solute, is made weaker by adding diluent so that the antibody pres- ent is not a
s concentrated.
The relationship between the serum and the total volume is expressed as a
racti
on
or example,
1:20. When several dilutions are made in which the dilution
actor is the same i
n each case, this
is called a serial dilution. Serial dilutions are used to determine the titer, o
r strength, o
an
antibody. The last tube in which a visible reac- tion is seen is considered the
end point. 0.2 mL
serum/0.2 mL serum 0.2 mL diluent
0.2 mL/0.4 mL
1/2 Then when 0.2 mL o
the 1:2
dilution is
added to 0.2 mL o
diluent, a 1:4 dilution is obtained. The
inal dilution is ob
tained by
counting the number o
tubes and setting up a multiplication series in which the
original
dilution
actor is raised to a power equal to the number o
tubes. In this example, i
the rst
tube contains a 1:2 dilution, the dilution in tube number six is 1 2
1 2 1 2
1 2
1 2
1/64 I
, in this instance, an end point was reached at tube num- ber ve, the a
ctual
titer would be 1:32. To avoid con
usion, this is customarily written as the reci

procal o
the
dilution that is, 32. Serial dilutions do not always have to be doubling dilution
s. Consider the

ollowing set o
test tube dilutions: 1:51:251:1251:6251:3125 For each successive tu
be, the
dilution is increased by a
actor o
5, so this would indeed be considered a ser
ial dilution.
Having the ability to work with simple and com- pound dilutions and interpret
serial
dilutions is a necessary skill
or laboratory work. The laboratory exer- cise
at the end o

this chapter illustrates the principle o

serial dilutions. SUMMARY Laboratory p
ersonnel are most

requently exposed to biolog- ical, sharp, chemical, and radiation hazards. Tran
smission o

biological hazards that are encountered when testing patient specimens requires
a chain o

in
ection, which consists o
a CHAPTER 7 Sa
ety and Specimen Preparation 117
1814_Ch07_107-122.qxd 7/10/09 2:50 PM Page 117 118 SECTION 2 Basic Immunologi
cal Procedures
The serology supervisor who has been working
or the last 20 years in a small ru
ral hospital is
training a new employee. A dilution o
a patients serum must be made to run a par
ticular test.
The supervisor was having di
- culty using a serological pipette, so she r
emoved one
glove. In uncapping the serum tube, a small amount o
serum splashed onto the wo
rkbench. She
cleaned this up with a paper towel, which she discarded in the regular paper tra
sh. She also
spilled a small amount onto her dis- posable lab coat. She told the new employee
that since it
was such a small amount, she wasnt going to worry about it, and she continued on
to pipette the
specimen. She then replaced the glove onto her ungloved hand and said that since
it was almost
break time, she would wait to wash her hands until then. Please identi
y all the
sa
ety
violations involved. CASE STUDY 1814_Ch07_107-122.qxd 7/10/09 2:50 PM Page 11
8 CHAPTER 7
Sa
ety and Specimen Preparation 119 SERIAL DILUTIONS Principle Serial dilution
s are a set o

dilutions in which the dilution

actor is exactly the same at each step. These a
re used to make
high dilutions with a small number o
test tubes and a minimal amount o
diluent
. This is
commonly done to determine the strength or titer o
a particular antibody in pat
ient serum as a
part o
the diagnosis o
a disease state. Traditionally, serological pipettes
have been used
in this process, but now it is more common to employ micropipettes
or
this purpose. In
this experiment, a series o
doubling dilutions will be made with both serologic
al pipettes and
micropipettes, and the results will be compared. Sample Preparation None Reagent
s, Materials, and
Equipment Anti-A antiserum Serological pipettes, 1 mL Micropipettes Disposable p
lastic microtiter
plates Disposable glass test tubes, 12
75 mm Saline solution (0.85 percent) Type
A red blood

cells (3 to 4 percent solution) Centri

uge Procedure Macrotiter 1. Make a 1:10 d
ilution o

reagent anti-A antiserum by adding 1 mL o

anti-A to 9 mL o
saline
o
r every 10 mL o

reagent desired. Allow approximately 1.5 mL o

antiserum per student. This will
be enough to run
the dilutions with a little extra
or repeat testing. 2. I
type A red blood cel
ls are not
purchased, a 4 percent solution o
red blood cells can be made using type A bloo
d collected in
ethylenediaminetetraacetic acid (EDTA). Spin the collection tube in a centri
ug
e
or
approximately 10 minutes at 1500 revolutions per minute (rpm). Remove the
serum and add
saline to the remaining red blood cells. Trans
er the solution to a disposable c
onical centri
uge
tube that has graduations marked on it. Spin again
or 10 minutes and note the c
olor o
the
saline wash on top. Remove saline wash and resuspend in additional saline. Make
sure all cells
are uni
ormly resuspended. Repeat this procedure
or three washes or until the s
aline supernatant
is clear. Note the
inal volume o
the packed red blood cells. Make a 4 perce
nt solution by
suspending 4 mL o
packed red cells in 96 mL o
saline. Each student needs ap
proximately 4 mL
o
the nal solution. 3. Label eight 12 75 mm test tubes as
ollows: 10, 20, 40, 8
0, 160, 320,
640, 1280. 4. Using a 1 mL serological pipette, add 0.2 mL o
saline to tubes tw
o through eight.
5. Add 0.2 mL o
anti-A antiserum to tubes one and two, using a clean serologica
l pipette. 6.
With a new serological pipette, mix tube two by draw- ing uid up and down ve to te
n times. 7.
Trans
er 0.2 mL
rom tube two to tube three and mix. 8. Repeat the trans
er and
mixing process
with tubes three and
our, and so on through tube eight. A
ter tube eight is mix
ed, discard the
last 0.2 mL o
the dilution. 9. Using a 1 mL serological pipette, add 0.2 mL o

4 per- cent type

A red blood cells to each tube. 10. Centri
uge
or 30 to 45 seconds. 11. Observe

or
agglutination by gently shaking the red blood cell button loose
rom the side o

each test tube.

A positive reaction is indicated by cells that remain clumped together a
ter sha
king. Note that
the size o
the clumps decreases with
urther dilution, but any visible clumping
is considered
positive. 12. Record the titer. This is the last tube in which visible agglutina
tion can be
discerned. The titer is written as the reciprocal o
the dilutionthat is, i
the
1:160 tube is
the last positive one, the titer is written as 160. Microtiter 1. Label one row
o
a microtiter
plate as
ollows: 10, 20, 40, 80, 160, 320, 640, 1280. 2. Using a micropipette,
add 20 L o

saline to wells two through eight on the microtiter plate. 3. With a new pipette
tip, add 20 L
o
anti-A antiserum to wells one and two. 4. Using the micropipette set to 20 L a
nd a new

pipette tip, mix well number two by drawing up and down in the pipette several t
imes. Wipe the
outside o
the pipette tip, and trans
er 20
L to well number three. 5. Repeat th
is process with
successive wells through well number eight. 6. Add 20
L o
4 percent type A red
blood cells to
all wells. 7. Rotate plate on the lab bench
or 1 minute, making a concentric ci
rcular pattern.
Let the plate sit
or 30 min- utes at room temperature. EXERCISE 1814_Ch07_107-1
22.qxd 7/10/09
2:50 PM Page 119 120 SECTION 2 Basic Immunological Procedures Negative Positi
ve A B FIGURE
78. Microtiter agglutination patterns. (A) A negative result is indicated by a sm
ooth button.
(B) Agglutination is a posi- tive result and is indicated by a ragged edge as th
e cells settle
out. 8. Observe
or agglutination with a microtiter plate reader, or care
ully h
old up to the
light. A smooth button on the bottom o
a well indicates that the red blood cell
s have settled
out with no agglutination. I
agglutination has occurred, an irregular or crenul
ated pattern is
seen at the bottom o
the well (Fig. 78). 9. Report the titer as the last well in
which
agglutination can be seen. Compare the results with those
rom the macrotiter. T
he titers
obtained should be within one dilution o
each other (plus or minus). Interpreta
tion o
Results
This experiment introduces the student to the concept and techniques involved in
making serial
dilutions. This is a pro- cedure o
ten used to determine the titer o
an antibod
y. The titer is
de ned as the reciprocal o
the last tube in which a positive reaction is seen. Th
e titer
indicates the concentra- tion o
an antibody, and this is important in
diagnostic
testing. In some diseases, the mere presence o
the antibody is enough to con rm t
he diagnosis.
For other diseases, how- ever, it is necessary to nd a titer that is signi cantly e
levated
beyond what might be
ound in the normal population in order to link the ant
ibody to an
active disease state. Whenever a titer is reported, the accuracy is assumed to
be plus or minus
one tube. Explore the student laboratory or the area designated by the instructo
r and per
orm the

ollowing: 1. Locate the re extinguishers 2. State the instructions

or operating
the re
extinguisher 3. Locate the eyewash station 4. Locate the emergency shower 5. Loc
ate the rst-aid
kit 6. Locate the master electrical panel 7. Locate the re alarm 8. Identi
y the
emergency exit
route 9. Locate the MSDS pertaining to serology 10. Locate the emergency spill k
it 11. Locate the
Bloodborne Pathogen Exposure Control Plan 12. Identi
y the type o
disin
ectant
available
or
cleaning work areas ACKNOWLEDGMENT This experiment was originally designed by Da
n Southern o
the
Clinical Laboratory Sciences Program, Western Carolina University, Cullowhee, N.
C. LABORATORY

SAFETY EXERCISE 1814_Ch07_107-122.qxd 7/10/09 2:50 PM Page 120 CHAPTER 7 Sa

e
ty and Specimen
Preparation 121 1. A technologist who observes a red rash on her hands a
ter r
emoving her
gloves a. should apply antimicrobial lotion to the hands. b. may be washing the
hands too

requently. c. may have developed a latex allergy. d. should not create

riction
when washing the
hands. 2. In the chain o
in
ection, a contaminated work area would serv
e as which o
the

ollowing? a. Source b. Method o

transmission c. Host d. All o
the above 3. Th
e only biological
waste that does not have to be dis- carded in a container with a biohazard symbo
l is a. urine. b.
serum. c.
eces. d. none o
the above 4. Patient specimens transported by the De
partment o

Transportation must be labeled as a. diagnostic specimen. b. clinical specimen.

c. biological
specimen, category b. d. All o
the above 5. A technician places tightly capped
serum tubes in a
rack and places the rack and the specimen data in a labeled metal courier box. I
s there anything
wrong with this scenario? a. Yes, DOT requirements are not met. b. No, the tubes
are placed in a
rack. c. Yes, absorbent material is missing. d. No, the box contains the specime
n data. 6. The
Occupational Exposure to Bloodborne Pathogens Standard developed by OSHA require
s employers to
provide all o
the
ollowing except a. hepatitis B immunization. b. sa
ety train
ing. c. hepatitis
C immunization. d. laundry
acilities
or nondisposable lab coats. 7. An employ
ee who receives
an accidental needlestick should immediately a. apply sodium hypochlorite to
the area. b.
noti
y a supervisor. c. receive HIV prophylaxis. d. receive a hepatitis B booste
r shot. 8. The
rst thing to do when acid is spilled on the skin is to a. noti
y a supervisor. b.
neutralize the
area with a base. c. apply burn ointment. d. ush the area with water. 9. When com
bining acid and
water, a. acid is added to water. b. water is added to acid. c. water is slowly
added to acid. d.
both solutions are added simultaneously. 10. To determine the chemical character
istics o
sodium
azide, an employee would consult the a. chemical hygiene plan. b. Merck manual.
c. MSDS. d. NRC
guidelines. 11. A technician who is pregnant should avoid working with a. organi
c chemicals. b.
radioisotopes. c. HIV-positive serum. d. needles and lancets. 12. A 1:750 diluti
on o
serum is
needed to per
orm a sero- logical test. Which o
the
ollowing series o
dilutio
ns would be
correct to use in this situation? a. 1:5, 1:15, 1:10 b. 1:5, 1:10, 1:5 c. 1:15,
1:10, 1:3 d.
1:15, 1:3, 1:5 13. How much diluent needs to be added to 0.2 ml o
serum to make
a 1:20 dilution?
a. 19.8 mL b. 4.0 mL c. 3.8 mL d. 10.0 mL REVIEW QUESTIONS 1814_Ch07_107-122.qxd
7/10/09 2:50
PM Page 121 122 SECTION 2 Basic Immunological Procedures Re
erences 1. Centers

or Disease

Control and Prevention. Guideline

or handwashing hygiene in health-care
settings. MMWR
2002:51(rr16); 148. http://www.cdc.gov/mmwr/PDF/rr/ rr5116.pd
. Accessed Septem
ber 8, 2008. 2.
NIOSH Alert. Preventing allergic reactions to natural rubber latex in the workpl
ace. DHHS (NIOSH)
Publication 97135. National Institute
or Occupational Sa
ety and Health, C
incinnati, OH,
1997. 3. Preventing transmission o
in
ectious agents in healthcare settin
gs 2007.
http://www.cdc.gov/ncidod/dhqp/pd
/guidelines/ Isolation2007.pd
. Accessed Septe
mber 8, 2008. 4.
Clinical Laboratory Standards Institute,
ormerly NACCLSs. Protection o
laborat
ory workers
rom
instrument biohazards and in
ectious disease transmitted by blood, body
luids,
and tissue:
Approved guideline M29-A, NCCLS, Wayne, PA, 1997. 5. Hazardous materials: In
ect
ious Substances;
harmonizing with the United Nations recommendations. Federal Register, 70 (
May 19), 2005. 6.
Baer, DM. Standards
or transporting specimens. MLO, 37(11):38, Nokomis, F
L, 2005. 7. U.S.
Department o
Transportation. Transporting in
ectious substances sa
ely.
https://hazmatonline.phmsa.dot.gov/services/
publication_documents/Transporting%20In
ectious%20Substa nces%20Sa
ely.pd
. Acce
ssed September 8,
2008. 8. International Air Transport Association. 2005 Diagnostic Specimen
Transport
Regulations. http://www.qicstat.com/ Media/IATA changes_2005.pd
. 9. Occupationa
l Exposure to
Bloodborne Pathogens, Final Rule. Federal Register, 29 (Dec 6), 1991. 10. OSHA.
En
orcement
Procedures
or the Occupational Exposure to Bloodborne Pathogens Standard
. Directive
22.44D. Washington, DC, 1999. http://www.asha.slc.gov/ oshdoc/Directive data/cpl_
22_69.html.
11. OSHA. Needlestick requirements take e

ect April 18. OSHA, Washington, DC,

2001.
http://www.osha.sle.gov/medic/ oshnews/apr01/national-20010412.html. 12. OSHA. O
SHA clari es
position on the removal o
contami- nated needles. OSHA, Washington, DC, 20
02. http://
www.osha.gov/media/oshnews/june02/trade-20020612A.html. 13. CDC, Updated U.S. Pu
blic Health
Service guidelines
or the management o
occupational exposures to HBV, HCV, and
HIV and
recommendations
or post-exposure prophylaxis. MMWR 2001:50(RR11), 142. 14. Occup
ational
Exposure to Hazardous Chemical in Laboratories, Final Rule. Federal Register 55
(January 31),
1990. 1814_Ch07_107-122.qxd 7/10/09 2:50 PM Page 122 8 Precipitation Reaction
s 123 LEARNING
OBJECTIVES A
ter nishing this chapter, the reader will be able to: 1. Describe ho
w the law o

mass action relates to antigenantibody binding. 2. Distinguish precipitation and

agglutination.
3. Discuss a
nity and avidity and their in uence on antigenantibody reactions. 4. Ex
plain how
the zone o
equivalence is related to the lattice hypothesis. 5. Di

erentiate b
etween turbidity

and nephelometry and discuss the role o

each in measurement o
precipitation re
actions. 6.
Explain the di

erence between single di

usion and double di

usion. 7. Give th
e principle o

the end-point method o

radial immunodi

usion. 8. Determine the relationship be

tween two
antigens by looking at the pattern o
precipitation resulting
rom Ouchterlony i
mmunodi

usion.
9. Describe how rocket immunoelectrophoresis di

ers
rom radial immunodi

usion
. 10. Compare
immunoelectrophoresis and immuno xation electrophoresis regarding placement o
rea
gents, time to
obtain results, and limitations o
each method. KEY TERMS A
nity Agglutination Av
idity
Cross-reactivity Electrophoresis Immunoelectrophoresis Immuno xation electrophores
is Law o
mass
action Nephelometry Ouchterlony double di

usion Passive immunodi

usion Postzon
e phenomenon
Precipitation Prozone phenomenon Radial immunodi

usion Rocket immunoelec- troph

oresis
Turbidimetry Zone o
equivalence 1814_Ch08_123-136.qxd 7/10/09 2:52 PM Page 1
23 o
noncovalent
bonds hold them together. These include ionic bonds, hydrogen bonds, hydrophobic
bonds, and van
der Waals
orces. 1,2 Ionic bonds occur between oppositely charged particles. Hy
drogen bonds
involve an attraction between polar molecules that have a slight charge separation and in which
the positive charge resides on a hydrogen atom. Hydrophobic bonds occur between
nonpolar molecules that associate with one another and exclude molecules o
water as they do
so. Van der Waals

orces occur because o

the interaction between the electron clouds o
oscillating dipoles. All
o
these are rather weak bonds that can occur only over a short distance o
appr
oximately 1
10
7 mm, so there must be a very close
it between antigen and antibody. 1
The strength
o
attraction depends on the speci city o
antibody
or a particular antigen. One
antibody
molecule may initially attract numerous di

erent antigens, but it is the epitop

es shape and the
way it ts together with the bind- ing sites on an antibody molecule that determin
es whether the
bonding will be stable. Antibodies are capable o
react- ing with antigens that
are structurally
similar to the original antigen that induced antibody production. This is known
as
cross-reactivity. The more the cross-reacting antigen resembles the original ant
igen, the
stronger the bond will be between the antigen and the binding site. However, i

the epitope and

the binding site have a per
ect lock-and-key relationship, as is the case w
ith the
original antigen, the a
nity will be maximal, because there is a very close t (Fi
g. 81).
Avidity Avidity represents the sum o
all the attractive
orces between
an antigen and
an antibody. This involves the strength with which a multivalent antibody bin
ds a mul-

tivalent antigen, and it is a measure o

the overall stability o
an antigenantib
ody complex.
1,3 In other words, once The combination o
antigen with speci
ic antibody plays
an important
role in the laboratory in diagnosing many di
-
erent diseases. Immunoassays
have been
developed to detect either antigen or antibody, and they vary
rom eas- ily per

ormed manual
tests to highly complex automated assays. Many such assays are based on the prin
ciples o

precipitation or agglutination. Precipitation involves com- bining soluble antig

en with soluble
antibody to produce insoluble complexes that are visible. Agglutination is the p
rocess by which
particulate antigens such as cells aggre- gate to
orm larger complexes when a s
peci
ic antibody
is present. This chapter
ocuses on precipitation, and the
ollowing chapter dis
cusses
agglutination. Precipitation was
irst noted in 1897 by Kraus, who
ound that cu
lture
iltrates
o
enteric bacteria would pre- cipitate when they were mixed with speci
ic antib
ody. For such
reactions to occur, both antigen and antibody must have multiple binding sites

or one another,
and the rela- tive concentration o
each must be equal. Binding character
istics o

antibodies, called a

inity and avidity, also play a major role. These are discu
ssed, along with
theo- retical considerations o
binding, including the law o
mass action and th
e principle o

lattice
ormation. Such charac- teristics relate to the sensitivity and speci
ic
ity o
testing in
the clinical laboratory. ANTIGENANTIBODY BINDING A
nity The primary union o
bindi
ng sites on
antibody with spe- ci c epitopes on an antigen depends on two characteristics o
a
ntibody known
as a
nity and avidity. A
nity is the ini- tial
orce o
attraction that exists bet
ween a single
Fab site on an antibody molecule and a single epitope or determinant site on the
corresponding
antigen. 1 As epitope and binding site come into close proximity to each other,
several types
CHAPTER OUTLINE ANTIGENANTIBODY BINDING A
nity Avidity Law o
Mass Action PRECIPIT
ATION CURVE
Zone o
Equivalence Prozone and Postzone MEASUREMENT OF PRECIPITATION BY LIGHT S
CATTERING
Turbidimetry Nephelometry PASSIVE IMMUNODIFFUSION TECHNIQUES Radial Immunodi

us
ion Ouchterlony
Double Di

usion ELECTROPHORETIC TECHNIQUES Rocket Immunoelectrophoresis Immunoe

lectrophoresis
Immuno xation Electrophoresis Sources o
Error in Electrophoresis COMPARISON OF PR
ECIPITATION
TECHNIQUES SUMMARY CASE STUDY EXERCISE: RADIAL IMMUNODIFFUSION REVIEW QUESTIONS
REFERENCES 124
124 SECTION 2 Basic Immunological Procedures 1814_Ch08_123-136.qxd 7/10/09 2:5
2 PM Page 124
CHAPTER 8 Precipitation Reactions 125 binding has occurred, it is the
orce th
at keeps the
mole- cules together. A high avidity can actually compensate
or a low a
nity. St
ability o
the

antigenantibody complex is essential to detecting the presence o

an unknown, whe
ther it is
antigen or antibody. Law o
Mass Action All antigenantibody binding is reversible
and is
governed by the law o
mass action. This law states that
ree reac- tants are
in equilibrium
with bound reactants. 2 The equilibrium constant represents the di

erence in
the rates o
the

orward and reverse reactions according to the

ollowing equation: The eq
uilibrium
constant is thus K K 1
/K
2
[AgAb]/[Ab][Ag], where [AgAb]
concentration o
the antigenantibody comp
lex (mol/L)
[Ab] concentration o
antibody (mol/L) [Ag]
concentration o
antigen (mol/L) Thi
s constant
can be seen as a measure o
the goodness o
t. 3 Its value depends on the strengt
h o
binding
between antibody and antigen. As the strength o
binding, or avidity, in
creases, the
tendency o
the antigenantibody com- plexes to dissociate decreases, and the valu
e o
K 2
decreases. This increases the value o
K 1 . The higher the value o
K, the larg
er the amount o

antigenantibody complex and the more visible or easily detectable the reaction is
. The ideal
conditions in the clinical laboratory would be to have an anti- body with a high
a
nity, or
initial
orce o
attraction, and a high avidity, or strength o
binding. The hig
her the values
are
or both o
these and the more antigenantibody com- plexes that are
ormed, t
he more
sensitive the test will be. PRECIPITATION CURVE Zone o
Equivalence In addition
to the
a

inity and avidity o

the antibody involved, precipitation depends on the
relative
proportions o
antigen and antibody present. Optimum precipitation occurs in the
zone o

equivalence, in which the number o

multivalent sites o
antigen and antibody ar
e approximately
equal. In this zone, precipitation is the result o
random, reversible reactions
whereby each
antibody binds to more than one antigen and vice versa,
orming a stable network
or lattice. 4
The lattice hypothesis, as
ormulated by Marrack, is based on the assumptions th
at each antibody
molecule must have at least two binding sites, and antigen must be multivalent.
As they combine,
this results in a multimolec- ular lattice that increases in size until it preci
pitates out o

solution. Heidelberger and Kendall per

ormed the classic quantitative precipitat
ion reactions
that established proo

or this theory. As illustrated by the precipitin curve s
hown in Figure
82, when increasing amounts o
soluble antigen are added to xed amounts o
speci c a
ntibody,
the amount o
precipita- tion increases up to the zone o
equivalence. Then when
the amount o

antigen overwhelms the number o

antibody- combining sites present, precipitatio
n begins to

decline. Prozone and Postzone As can be seen on the precipitation curve, precipi
tation declines
on either side o
the equivalence zone due to an excess o
either antigen or ant
ibody. In the
case o
antibody excess, the prozone phenomenon occurs, in which antigen co
mbines with only
one or two antibody molecules, A B FAB site Intermolecular bonding Antigenic det
erminant o

stimulating antigen- strong a

inity Cross-reacting antigen- a

inity not as str

ong Bivalent
antibody Multivalent antigen Multivalent antigen Multivalent antigen FIGURE 81. A

nity and
avidity. (A) A
nity is the t o
one anti- genic determinant with one antibody bind
ing site. The
original stimulating antigen is a better t than cross-reacting antigen. (B) Avidi
ty is the sum
o
the
orces binding multivalent antigens to multivalent antibodies. Ag + Ab A
gAb K 1 K 2 Where
Ag = antigen Ab = antibody K 1 = rate constant
or the
orward reaction K 2 = ra
te constant
or
the reverse reaction 1814_Ch08_123-136.qxd 7/10/09 2:52 PM Page 125 126 SECTI
ON 2 Basic
Immunological Procedures and no cross-linkages are
ormed. This is because usual
ly only one site
on an antibody molecule is used, and many
ree antibody molecules remain in solu
tion. At the
other side o
the zone, where there is antigen excess, the postzone phenomenon o
ccurs, in which
small aggregates are surrounded by excess antigen, and again no lattice network
is
ormed. 1 In
this case, every available anti- body site is bound to a single antigen, and no
cross-links are

ormed. 4 Thus,
or precipitation reactions to be detectable, they must be run i
n the zone o

equivalence. The prozone and postzone phenomena must be consid- ered in the clin
ical setting,
because negative reactions occur in both. A
alse-negative reaction may take pla
ce in the prozone due to high antibody concentration. I
it is suspected that the reaction is
a
alse
negative, diluting out antibody and per
orming the test again may produce a posi
tive result. In
the postzone, excess antigen may obscure the presence o
a small amount o

antibody.
Typically, such a test is repeated with an additional patient specimen taken
about a week
later. This would give time
or the
urther production o
antibody. I
the test
is negative on
this occasion, it is unlikely that the patient has that particular antibody. MEA
SUREMENT OF
PRECIPITATION BY LIGHT SCATTERING Turbidimetry Precipitation is one o
the simpl
est methods o

detecting antigenantibody reactions, because most antigens are mul- tivalent and
thus capable
o

orming aggregates in the presence o
the corresponding antibody. When a
ntigen and
antibody solutions are mixed, the initial turbidity is
ollowed by precipitation
. Precipitates in
uids can be measured by means o
turbidimetry or nephelometry. Turbidimetry is a
measure o
the

turbidity or cloudiness o
a solution. A detection device is placed in direct li
ne with the
incident light, collecting light a
ter it has passed through the solu- tion. It
thus measures the
reduction in light intensity due to re
lection, absorption, or scatter. 5 Scatte
ring o
light
occurs in proportion to the size, shape, and concentration o
molecules presen
t in solution.
It is recorded in absorbance units, a measure o
the ratio o
incident light
to that o

transmitted light. Measurements are made using a spectrophotometer or an automat

ed clinical
chemistry analyzer. Nephelometry Nephelometry measures the light that is scatter
ed at a particular angle
rom the incident beam as it passes through a suspension 5 (Fig. 83
). The amount
o
light scattered is an index o
the solutions concentration. Beginning with a c
on- stant
amount o
antibody, increasing amounts o
antigen result in an increase in antig
enantibody
complexes. Thus, the relationship between antigen concentrations, as indi- cated
by
antigenantibody complex
ormation, and light scattering approaches linearity. 5
Light scatter
may be recorded in arbitrary units o
relative light scatter, or it may be direc
tly
extrapolated by a computer to give actual concentrations in milligrams per decil
iter (mg/dL) or
inter- national units per milliliter (IU/mL), based on established values o
sta
ndards.
Nephelometers measure light scatter at angles ranging
rom 10 degrees to about 9
0 degrees. I
a
laser beam is used, light de ected only a
ew degrees
rom Prozone Zone o
equival
ence Postzone
Increasing antigen concentration (antibody concentration is constant) = antibody
= soluble
antigen Concentration o
Ag-Ab complexes FIGURE 82. Precipitin curve. The precipi
tin curve shows
how the amount o
precipitation varies with varying antigen concentration when t
he antibody
concentration is kept constant. Excess antibody is called the prozone, and exces
s antigen
concentration is called the postzone. Antigen + antibody Lens Detector Light sou
rce Lens
ocusing
light rays 70 FIGURE 83. Principles o
nephelometry. The light detection device i
s at an angle
to the incident light, in contrast to turbidity, which measures light rays passi
ng directly
through the solution. (From Stites, DP, et al. Clinical laboratory methods
or d
etection o
antigens and antibodies. In Stites, DP, Terr, AI, and Parslow, TG (eds): Medical Imm
unology, ed. 9.
Appleton & Lange, Stam
ord, CT, 1997, p. 230, with permission.) 1814_Ch08_123-13
6.qxd 7/10/09
2:52 PM Page 126 CHAPTER 8 Precipitation Reactions 127 the original path can
be measured.
Although the sensitivity o
turbidity has increased, nephelometry is more sensit
ive, with a lower
limit o
detection o
1 to 10 mg/L. 2 Nephelometry can be used to detect either
antigen or

antibody, but it is usually run with antibody as the reagent and the patient ant
igen as the
unknown. In end point neph- elometry, the reaction is allowed to run esse
ntially to
completion, but large particles tend to
all out o
solution and decrease the am
ount o
scatter.
Thus, another method called kinetic or rate nephelometry was devised, in w
hich the rate
o
scattering increase is measured immediately a
ter the reagent is added.
This rate change
is directly related to antigen concentration i
the concentration o
antibody is
kept constant.
6 Many automated instruments utilize this principle
or the measurement
o
serum
proteins. Quanti cation o
immunoglobulins such as IgG, IgA, IgM, and IgE, and kap
pa and lambda
light chains is done almost exclusively by nephelometry, because other methods a
re more
labor-intensive. 6 Other serum proteins quanti
ied include complement components
, C-reactive
protein, and several clotting
actors. Nephelometry provides accurate and preci
se quantitation
o
serum proteins, and due to automation, the cost per test is typically lo
wer than other
methods. 6,7 PASSIVE IMMUNODIFFUSION TECHNIQUES The precipitation o
antigenantib
ody complexes
can also be determined in a support medium such as a gel. Agar, a high-molecular
-weight complex
polysaccharide derived
rom seaweed, and agarose, a puri ed agar, are used
or thi
s pur- pose.
Agar and agarose help stabilize the di

usion process and allow visualization o

the precipitin
bands. 2 Reactants are added to the gel, and antigenantibody combination occurs b
y means o

di

usion. When no electri- cal current is used to speed up this process, it is

known as passive
immunodi

usion. The rate o

di

usion is a

ected by the size o

the particles,
the temperature,
the gel viscos- ity, and the amount o
hydration. An agar concentration ranging

rom 0.3 percent

to 1.5 percent allows
or di

usion o
most reactants. Agarose is pre
erred to a
gar, because agar
has a strong negative charge, while agarose has almost none, so interactions b
etween the gel
and the reagents are minimized. Immunodi

usion reactions can be classi

ied a
ccording to the
number o
reactants di

using and the direc- tion o

di

usion. Radial Immunodi

usion James
Oudin was the
irst to use gels
or precipitation reactions, and he pioneered th
e technique known
as single di

usion. 4 In single di

usion, antibody was incorporated into agaro

se in a test
tube. The antigen was layered on top, and as the antigen moved down into the gel
, precip- itation
occurred and moved down the tube in proportion to the amount o
antigen present.
4 A modi
ication
o
the single-di

usion technique, radial immunodi

usion (RID), has been com

monly used in the
clinical laboratory. In this technique, antibody is uni
ormly distributed in the
support gel, and

antigen is applied to a well cut into the gel. As the antigen di

uses out
rom
the well,
antigen antibody combination occurs in changing proportions until the zone o
equ
ivalence is
reached and a stable lattice network is
ormed in the gel. The area o

the ring
obtained is a measure o
antigen concentration, and this can be compared to a st
andard curve
obtained by using antigens o
known concentration. 1,6 Figure 84 depicts some typ
ical results.
There are two techniques
or the measurement o
radial immunodi

usion. The rst w

as developed by
Mancini and is known as the end-point method. In this technique, antigen is allo
wed to di

use to
completion, and when equivalence is reached, there is no
urther change in the r
ing diameter. 8
This occurs between 24 and 72 hours. The square o
the diame- ter is then direct
ly proportional
to the concentration o
the antigen. A graph is obtained by plotting concentrati
ons o
standards
on the x-axis versus the diameter squared on the y-axis, and a smooth curve is

it to the points.
The major drawback to this method is the time it takes to obtain results. The Fa
hey and McKelvey
method, also called the kinetic method, uses measurements taken be
ore the point
o
equiv- alence
is reached. In this case, the diameter is proportional to the log o
the concent
ration. 9 A graph
is drawn on semi- log paper by plotting the antigen concentration on the log axi
s and the
diameter on the arithmetic axis. Readings are taken at about 18 hours. For eithe
r the end-point
method or the kinetic method, it is essential that monospeci c antiserum with a
a
irly high D i a
m e t e r
o
p r e c i p t i n
r i n g
( m m ) 2 15 20 25 30 Concentration o
test reactant (mg/dL) 0 10 20 30
40 50 std 1 std 2 std
3 Std 1 Std 2 Std 3 Unknown
Glass slide coated with agar containing speci
ic ant
ibody 60
FIGURE 84. Radial immunodi

usion. The amount o

precipitate
ormed is in proport
ion to the
antigen present in the sample. In the Mancini end-point method, concentration is
in proportion to
the diameter squared. (From Nakamura, RM, Tucker, ES III, and Carlson, IH. Immun
oassays in the
clinical laboratory. In Henry, JB: Clinical Diagnosis and Management by Laborato
ry Methods, ed.
18. WB Saunders, Philadelphia, 1991, p. 858, with permission.) 1814_Ch08_123-136
.qxd 7/10/09
2:52 PM Page 127 a
nity be used. This increases the clarity o
the precipita- ti
on reaction.
In addition, the precision o
the assay is directly related to accurate m
easurement o

samples and standards. Sources o

error include over lling or under ll- ing the well
s, nicking

the side o
the wells when
illing, spilling sample outside the wells, improper
incubation time
and temperature, and incorrect measurement. Radial immunodi

usion has been

used to measure
IgG, IgM, IgA, and complement components. It is simple to per
orm and requires n
o
instrumentation, but it is
airly expensive to run. 6 Thus, immunodi

usion has
largely been
replaced by more sensitive and automated methods such as nephelometry and enzyme
-linked
immunosorbent assays except
or low- volume analytes such as IgD or IgG subc
lasses. 6
Ouchterlony Double Di

usion One o
the older, classic immunochemical techniques
is Ouchterlony
double di

usion. In this technique, both antigen and antibody di

use independe
ntly through a
semi- solid medium in two dimensions, horizontally and vertically. Wells are cut
in a gel, and
reactants are added to the wells. A
ter an incubation period o
between 12 and 4
8 hours in a
moist chamber, precipitin lines
orm where the moving
ront o
antigen meets tha
t o
antibody.
The density o
the lines re ects the amount o
immune complex
ormed. Most Ouchter
lony plates are
set up with a central well surrounded by
our to six equidistant outer wells. An
tibody that is
multispeci c is placed in the central well, and di

er- ent antigens are placed

in the
surrounding wells to determine i
the antigens share identical epitopes. 4 The
posi- tion o

the precipitin bands between wells allows

or the antigens to be compared with o
ne another.
Several patterns are possible: (1) Fusion o
the lines at their junction to
orm
an arc
represents serological identity or the presence o
a common epitope, (2) a patte
rn o
crossed
lines demonstrates two separate reactions and indicates that the compared antigens share no
common epitopes, and (3)
usion o
two lines with a spur indicates partial ident
ity. In this last
case, the two antigens share a common epitope, but some antibody molecules are n
ot captured by
antigen and travel through the initial precipitin line to combine with additiona
l epi- topes

ound in the more complex antigen. There

ore, the spur always points to the simp
ler antigen 1
(Fig. 85). While o
more limited use because it is labor-intensive and requires e
xperience to
read, Ouchterlony double di

usion is still used to identi

y
ungal antigens suc
h as Aspergillus,
Blastomyces, Coccidioides, and Candida. 10 In addition, this has been a cl
assic technique
to detect antibodies to extractable nuclear antigens that occur in several
autoimmune
diseases. 11,12 (Re
er to Chapter 14
or a discussion o
autoimmune dise
ases.) It is
important to per
orm this technique with care, because several problems ma
y arise.
Irregular patterns o
precipitation may occur
rom over lling the wells, irregu- l
ar hole

punching, or nonlevel incubation. Other

actors a

ecting the accuracy o

result
s include drying
out o
the gels; inadequate time
or di

usion, resulting in weakness o

band in
tensity; and

ungal or bacterial contamination o

the gel. ELECTROPHORETIC TECHNIQUES Di

usi
on can be
combined with electrophoresis to speed up or sharpen the results. Electrophoresi
s separates molecules according to di

erences in their electric charge when they are placed in

an electric
eld. A direct current is
orced through the gel, causing antigen, antibody,
or both to
migrate. As di

usion takes place, distinct precipitin bands are

ormed. This te
chnique can be
applied to both single and double di

usion. Rocket Immunoelectrophoresis One-di

mension
electroimmunodi

usion, an adaptation o
radial immunodi

usion, was developed b

y Laurell in the
early 1960s. 13 Antibody is distributed in the gel, and antigen is placed in wel
ls cut in the
gel, just as in RID. However, instead o
allowing di

usion to take place at its

own rate,
electrophoresis is used to
acilitate migration o
the antigen 128 SECTION 2 Bas
ic Immunological
Procedures A B C Serological identity Ag = Ag1, Ab = anti-1 Nonidentity Ag = A
g1, Ag2 Ab =
anti-1, anti-2 Partial identity Ag = 1, 1a; Ab = anti-1 Ag1a is a part o
Ag1 bu
t is the simpler
Ag Ab 1 2 Ab 1 1 a Ab 1 1 FIGURE 85. Ouchterlony di

usion patterns. Antibody tha

t is a mixture
o
anti-1 and anti-2 is placed in the central well. Unknown antigens are placed
in the outside
wells. (A) Serological identity. The arc indicates that the two antigens are ide
ntical. (B)
Nonidentity. Two crossed lines represent two di

erent precipitation reactions.

The antigens
share no identical determinants. (C) Partial identity. Antigen 1a shares a deter
minant that is
part o
antigen 1, but it is not as complex. The spur
ormed always points to th
e simpler
antigen. 1814_Ch08_123-136.qxd 7/10/09 2:52 PM Page 128 CHAPTER 8 Precipitati
on Reactions
129 into the agar. When the antigen di

uses out o
the well, pre- cipitation be
gins. As the
concentration o
antigen changes, there is dissolution and re
ormation o
the pr
ecipitate at
ever-increasing distances
rom the well. The end result is a precipitin line tha
t is conical in
shape, resembling a rocket, hence the name rocket immunoelectrophoresis. The
height o
the
rocket, measured
rom the well to the apex, is directly in proportion to the
amount o

antigen in the sample. I

standards are run, a curve can be constructed to de
termine
concentrations o
unknown specimens 2 (Fig. 86). Rocket immunoelectrophoresis
is much more
rapid than RID, with results available in just a
ew hours. 6 It is essen- tial,
however, to
determine the net charge o
the molecules at the pH used
or the test, because t
his determines

the direction o
migration within the gel. Typically, a pH is selected so that a
ntibodies in the
gel do not move, but the antigens become negatively charged. Antigens then migra
te toward the
positive anode. 4 This technique has been used to quantitate immunoglobulins, us
ing a bu

er o

pH 8.6. Immunoelectrophoresis Immunoelectrophoresis is a double-di

usion techni
que that
incorporates electrophoresis current to enhance results. Introduced by Grabar an
d Williams in
1953, this is per-
ormed as a two-step process and can be used
or sem
iquantitation o

a wide range o
antigens. 2,14 Typically, the source o
the antigens is serum, w
hich is
electrophoresed to separate out the main protein
ractions; then a trough is cut
in the gel
parallel to the line o
separation. Antiserum is placed in the trough, and the g
el is incubated

or 18 to 24 hours. Double di

usion occurs at right angles to the elec- trophor

etic separation,
and precipitin lines develop where speci c antigenantibody combination takes place.
These lines
or arcs can be compared in shape, intensity, and location to that o
a normal se
rum control to
detect abnormalities. 5 Changes include bowing or thickening o
bands, or
changed
mobility. 14 Interpretation may take considerable experience. This procedure has
been used as a
screening tool
or the di

erentiation o
many serum proteins, including the maj
or classes o

immunoglobulins. It is both a qualitative and a semiquantitative technique and h

as been used in
clinical laboratories
or the detection o
myelomas, Waldenstrms macroglobulinemia
, malignant
lymphomas, and other lym- phoproli
erative disorders. 14 (See Chapter 15
or
more details on
lymphoproli
erative diseases.) In addition, immun- ode ciencies can be detected in
this manner,
i
no precipitin band is
ormed
or a particular immunoglobulin. De ciencies o
co
mplement
components can also be identi ed. Figure 87 shows examples o
a normal pattern and
an IgM
monoclonal gammopathy, an immunoproli
erative disorder. However, this technique
is relatively
insensitive, with a detection limit o
approximately 3 to 20 uL concentration. 4
Interpretation
can also be di
cult and may take considerable experience. There
ore, it ha
s largely been
replaced by immuno xation electrophoresis, which gives quicker results and is easi
er to
interpret. 2,14 Immuno xation Electrophoresis Immuno
ixation electrophoresis, as

irst described
by Alper and Johnson, 15 is similar to immunoelectrophoresis except that a
ter e
lectrophoresis
takes place, antiserum is applied directly to the gels sur
ace rather than placed
in a trough.
Agarose or cellulose acetate can be used
or this pur- pose. Immunodi

usion tak
es place in a
shorter time and results in a higher resolution than when antibody di

uses
rom
a trough.

Because di

usion is only across the thick- ness o

the gel, approximately 1 mm,
the reaction
usually takes place in less than 1 hour. Most o
ten, an antibody o
known speci

icity is used to
determine whether patient antigen is present. The unknown antigen is pl
aced on the
gel, electrophoretic separation takes place, and then the reagent antibody is a
pplied.
Immunoprecipitates
orm only where speci
ic antigenantibody combination has t
aken place, and
the + Antigen migration 1 2 3 4 5 6 FIGURE 86. Rocket immunoelectrophoresis. Stan
dards are
in wells 1 to 3. Patient samples are in wells 4 to 6. Note that well 4 contains
no antigen,
because no ring is
ormed. Well 5 has a low concentra- tion o
antigen, and well
6 has a high
concentration o
antigen. FIGURE 87. Immunoelectrophoresis lm showing normal contr
ols
(odd-numbered wells) and patient serum (even-numbered wells). Trough A contains
antitotal
immunoglobulin, and troughs B through F contain the
ollowing monospeci c antisera
: B, anti-; C,
anti-; D, nti-; E, nti-; and F, anti-. The restricted eectrphretic mbiity see
n in we
1 with antitta immungbuin and in we 5 with anti- and anti- immungbuins
indicate
that the patient has an IgM mncna gammpathy with ight chains. (Frm Harr,
R. Cinica
Labratry Science Review, ed. 2. FA Davis, Phiadephia, 2000, Cr Pate 4, w
ith permissin.)
See Cr Pate 8. 1814_Ch08_123-136.qxd 7/10/09 2:52 PM Page 129 cmpexes
have becme
trapped in the ge. The ge is washed t remve any nnprecipitating prte
ins and can then
be stained fr easier visibiity. Typicay, patient serum is appied t six an
es f the ge,
and after eectrphresis, ve anes are veraid with ne each f the fwing a
nti- bdies:
antibdy t gamma, apha, r mu heavy chains and t appa r ambda ight chains
. 16 The sixth
ane is ver- aid with antibdy t a serum prteins and serves as the referen
ce ane.
Reactins in each f the ve anes are cm- pared t the reference ane. Hypgamma
gbuinemias
wi exhibit fainty staining bands, whie pycna hypergam- magbuinemias
shw dar y
staining bands in the gamma regin. Mncna bands, such as fund in Wadenstrms
macrgbuinemia r mutipe myema, have dar and narrw bands in speci c anes
16 (Fig. 88).
This methd is especiay usefu in demnstrating thse antigens present in seru
m, urine, r
spina fuid in w cncentratins. Cerebrspina fuid is the specimen f
chice fr
diagnsing mutipe scersis, and urine is used t detect the presence f Bence
-Jnes prteins
that are fund in mutipe myema. 16,17 Athugh immun xatin is mre sensitive
than
immuneectrphresis, diutins may be necessary t avid the znes f
antigen excess,
which may ccur if cncentratins f mncna antibdy are very high. 14 Perha

ps ne f the
best- nwn adaptatins f this technique is the Western bt, used as a cnfirma
- try test t
detect antibdies t human immunde ciency virus 1 (HIV-1). A mixture f HIV antig
ens is paced
n a ge and eectrphresed t separate the individua cmp- nents. The cmpn
ents are then
transferred t nitrceuse paper by means f btting r aying the nitrce
use ver the
ge s that the eectrphresis pattern is preserved. Patient serum is appied t
 the
nitrceuse and awed t react. The strip is then washed and stained t det
ect pre- cipitin
bands. It is simper t visuaize the reactin n the nitrceuse, and in thi
s manner,
antibdies t severa antigens can be detected. Refer t Figure 23-1 fr a specific exampe
f a Western bt used t determine the presence f antibdy t HIV-1. Th
is technique is
character- isticay used t determine the presence f antibdies t rganisms 
f cmpex
antigenic cmpsitin. If antibdies t mre than ne disease-assciated antigen
are identi ed in
patient serum, this usuay cn rms presence f the sus- pected disease. Surces 
f Errr in
Eectrphresis Many f the surces f errr are simiar fr a the eec- trph
retic
techniques, s they are discussed tgether here. One prbem that can arise is a
ppying the
current in the wrng directin. If this ccurs, sampes may either run ff the g
e r nt be
separated. Incrrect pH f the buffer and incrrect eectrphresis time as hi
nder prper
separa- tin. Cncentratins f antigen and antibdy must be carefuy ch
sen s that
attice frmatin and precipitatin is pssibe. If either is t cncentrated,
n visibe
reactin wi resut. The amunt f current appied as in uences the ef ciency f
the
separatin. If the current is nt strng enugh, separatin may be incmpete. O
n the ther hand,
if the current is t strng, heat wi be generated and may denature prteins.
As in diffusin
techniques, wes must be carefuy ed. COMPARISON OF PRECIPITATION TECHNIQUES
Each type f
precipitatin technique has its wn distinct advantages and disadvantages. Sme
techniques are
techni- cay mre demanding, and thers are mre autmated. Each type f precip
itatin testing
has particuar appicatins fr which it is best suited. Tabe 81 presents a cmp
ari- sn f the
techniques discussed in this chapter. 130 SECTION 2 Basic Immungica Prcedur
es FIGURE 88.
Immun xatin eectrphresis. A cmpex antigen mixture such as serum prteins is
separated by
eectrphresis. An antiserum tempate is aigned ver the ge. Then prtein xati
ve and
mnspeci c antisera, IgG, IgA, IgM, , and are appied t the ge. After incubating
fr 30
minutes, the ge is stained and examined fr the presence f paraprteins. Preci

pitates frm
where speci c antigenantibdy cmbinatin has ta en pace. In this case, the patien
t has an IgG
mncna antibdy with chains. (Curtesy f Heena Labratries, Beaumnt, TX
). See Cr
Pate 9. Tabe 8-1. Cmparisn f Precipitatin Techniques TECHNIQUE APPLICATI
ON SENSITIVITY
(g ab/mL) COMMENT Nephemetry Immungbuins, cmpement, 110 Autmated, sens
itive,
expensive C-reactive prtein, ther serum equipment needed prteins Radia immu
ndiffusin
Immungbuins, cmpement 1050 Quantitative, sw, nt as sensitive as nephem
etry Cntinued
1814_Ch08_123-136.qxd 7/10/09 2:52 PM Page 130 CHAPTER 8 Precipitatin Reacti
ns 131 SUMMARY
Immunassays in which antigenantibdy cmbinatin ccurs have been deveped
t detect
either antigen r anti- bdy, and they pay an imprtant re in diagnsing dise
ases. Many such
assays are based n the principe f precipitatin, which invves cmbinatin 
f sube antigen
with sube antibdy t prduce insube cmpexes that are visibe. Unin f
antigen and
antibdy depends n affinity, r the frce f attractin that exists between ne
antibdybinding site and a singe epitpe n an antigen. Avidity is the sum f a attra
ctive frces
ccurring between mutipe binding sites n antigen and antibdy. Fr testing pu
rpses, it is
imprtant t have an antibdy with high affinity and high avidity fr the antige
n in questin.
Maximum binding f antigen and antibdy ccurs when the aggregate number f mut
ivaent sites f
antigen and antibdy are apprximatey equa. The cncentratins f antigen and
antibdy that
yied maximum binding represent the zne f equivaence. When antibdy is in exc
ess (the przne) r antigen is in excess (the pstzne), manifestatins f antigenantibdy c
mbinatin such
as precipitatin and aggutinatin are present t a much esser degree. A test
- ing shud ta e
pace in the zne f equivaence, where a reactin is mst visibe. Precipitati
n can be detected
in the abratry by severa different means. Light scatter prduced by immune c
m- pexes in
sutin can be measured as a reductin in ight intensity (turbidimetry) r as
the amunt f
ight scattered at a particuar ange (nephemetry). Severa autmated in
struments are
based n these principes. Other precipitatin techniques utiize a supprt medi
um such as a ge,
and antigenantibdy cmbinatin ta es pace by means f passive diffusin. In sin
ge diffusin,
ny ne f the reactants traves, whie the ther is incrprated in the ge. A
n exampe is
radia immundiffusin, in which antibdy is incrprated in a ge in a pate r
a petri dish.
The amunt f precipitate frmed is directy reated t the amunt f antigen pr
esent.
Ouchterny diffusin is a dube-diffusin technique in which bth antigen and

antibdy diffuse
frm wes and trave tward each ther. Precipitin ines may indicate iden- tit
y, nnidentity,
r partia identity, depending n the pattern frmed. Eectrphresis can be cm
bined with
diffusin t speed up the prcess and enhance the reactin patterns. Rc et immu
neectrphresis
appies an eectric charge t singe diffusin, with resutant precipitatin pat
terns that 
i e rc ets that prject upward frm the sampe wes. In immuneectrphresis
, the antigen is
rst eectrphresed by itsef t separate ut different cmpnents, and then anti
bdy is paced
in a trugh t aw diffusin t ta e pace. Specific reactin patterns ind
icate presence
r absence f certain antigens. A reated technique, immun x- atin eectrphres
is, differs in
that antibdy is appied directy t the ge after eectrphresis has ta en pa
ce. Cmpared t
immuneectrphresis, precipitatin ccurs in a shrter time, and bands with hi
gher resutin
are btained. Precipitatin is adaptabe t bth autmated techn- gies and in
dividuay
prcessed testing. Thus, it is a versatie and practica methd fr use in the c
inica
abratry. Tabe 8-1. Cmparisn f Precipitatin TechniquesCntd TECHNIQUE APP
LICATION
SENSITIVITY (g ab/mL) COMMENT Ouchterny dube Cmpex antigens such as fung
a 20200
Semiquantitative, sw, may be dif cut diffusin antigens t interpret Rc et ee
ctrphresis
Immungbuins, cmpement, 2 Fast, quantitative, technicay demanding aphafetprtein
Immuneectrphresis Differentiatin f serum prteins 20200 Sw, semiquantitat
ive, dif cut
t interpret Immun xatin HIV, Lyme disease, syphiis Variabe Fairy rapid, sem
iquantitative,
sensitive eectrphresis HIV = Human immunde ciency virus; Ab = antibdy Adapted
frm Kindt,
TJ, Gdsby, RA, and Osbrne, BA. Kuby Immungy, ed. 6. WH Freeman and C., Ne
w Yr , 2007, p.
152. 1814_Ch08_123-136.qxd 7/10/09 2:52 PM Page 131 132 SECTION 2 Basic Immun
gica
Prcedures A 4-year-d femae was hspitaized fr pneumnia. She has had a his
try f
upper-respiratry-tract infectins and severa buts f diarrhea since infancy.
Due t her recurring infectins, the physician decided t measure her immungbuin ev
es. The
fwing resuts were btained by nephemetry: Questins a. What d these re
suts indicate?
b. Hw d they expain the symptms? c. Hw d nephemetry measurements cmpare
with the use f
RID? CASE STUDY NORMAL LEVEL PATIENT LEVEL IMMUNOGLOBULIN (35 yrs) (mg/dL) (
mg/dL) IgG
5501700 800 IgA 50280 20 IgM 25120 75 1814_Ch08_123-136.qxd 7/10/09 2:52 PM Page
132
CHAPTER 8 Precipitatin Reactins 133 IMMUNOFIXATION ELECTROPHORESIS Principe
Immun xatin
eectrphresis (IFE) is a tw-stage prce- dure using agarse ge high-resuti

n
eectrphresis in the first stage and immunprecipitatin in the secnd.
Prteins are
rst separated by eectrphresis. Then, in the secnd stage, sube antigen in t
he ge reacts
with speci c antibdy, and the resutant antigenantibdy cmpexes becme insube
(as ng as
the antibdy is in sight excess r near equivaency) and precipitate. The preci
pitatin rate
depends n the prprtins f the reactants, temperature, sat cncentratin, an
d the sutins
pH. The unreacted prteins are remved by a washing step, and the antigen antibd
y cmpex is
visuaized by staining. The bands in the individua anes are cmpared with
the precipitin
bands btained with a nrma cntr serum. Sampe Preparatin Fresh serum r ur
ine is the
specimen f chice. Evapratin f uncvered specimens may cause inaccurate resu
ts, s specimens
must be handed carefuy. Pasma shud nt be used, because the bringen may ad
here t the ge
matrix, resuting in a band in a patterns acrss the ge. If strage is necess
ary, sampes may
be ept cvered at 2C t 8C fr up t 72 hurs. Reagents, Materias, and Equipment
Ge chamber
t eectrphrese, stain, destain, and then dry the ges IFE ges IFE prtein xat
ive Acid viet
stain Vias f anti-IgG, IgA, IgM appa, and ambda antisera Tris-buffered sain
e Citric acid
destain IFE tempates (20) Btters and btter cmbs NOTE: The abve are prvid
ed in its
avaiabe frm Heena, Sebia, r the Binding Site. Antisera tempate REP prep 31
00 Ge bc
remver 10 percent acetic acid 0.85 percent saine Pwer suppy capabe f prvi
ding at east 350
vts Precautins The ge cntains barbita, which, in suf cient quantity, can be
txic. In
additin, sdium azide is used as a preservative in certain reagents. T prevent
the frmatin f
txic vaprs, d nt mix with acidic sutins. When discarding, aways ush sin
with cpius
amunts f water. This wi prevent the frmatin f metaic azides, which, whe
n highy cncentrated in meta pumbing, are ptentiay expsive. In additin t pur
ging pipes with
water, pumbing shud ccasinay be decntaminated with 10 percent sdium hyd
rxide. The ges
must be stred in the prtective pac - aging in which they are shipped. D
nt
refrigerate r freeze. Prcedure 1. Prepare a reagents, incuding the tris bu
ffer, acid viet, and citric acid destain accrding t directins in the it. 2. Pug the ee
ctrphresis
chamber int a pwer suppy, and snap the eectrphresis id int pace n the
chamber. 3.
Diute patient serum sampes frm 1:3 t 1:10, fw- ing instructins fr the
individua
manufacturers. 4. Remve the ge frm the ge puch. Carefuy detach frm the p
astic md and
discard the md. 5. Dispense apprximatey 1 mL f REP prep nt the eft side

f the
eectrphresis chamber. 6. Pace the ge carefuy int the eectrphresis cha
m- ber, ma ing
sure that any ntches are aigned with pins n the chamber fr. Use a int-fre
e tissue t wipe
arund the edges f the ge bac ing t remve any excess REP prep. Ma e sure tha
t n bubbes
remain under the ge. 7. Using a btter, genty bt the entire ge usi
ng sight
fingertip pressure n the btter. Remve the btter. 8. Remve IFE tempates f
rm the pac age.
Hd the tempate s that the sma he in the crner is tward the frnt right
side f the
chamber. 9. Carefuy pace the tempate n the ge, aigning the tempate sits
with the mar s
n each side f the ge bac ing. The center he in the tempate shud aign wi
th the indentin
in the center f the ge. 10. Appy sight ngertip pressure t the tempate, ma i
ng sure there
are n bubbes under it. 11. Appy the apprpriate serum diutin t six tempat
e sits. Wait 2
minutes after the ast sampe appicatin t aw prper absrptin. EXERCISE
1814_Ch08_123-136.qxd 7/10/09 2:52 PM Page 133 134 SECTION 2 Basic Immungi
ca Prcedures
12. Genty bt the excess sampe frm the tempate. Then carefuy remve the t
empate. 13.
Cse the id f the chamber. Set the pwer t 350 vts and start the pwer sup
py.
Eectrphrese the ge fr 7 t 8 minutes, fwing instructins frm the its m
anufacturer.
14. Turn ff the pwer suppy and remve the id t the eectrphresis chamber.
15. Using the
ge bc remver, carefuy remve the tw ge bc s (ne n each end f the g
e). Use a
int-free tissue t wipe arund the edges f the ge bac ing t remve any exces
s misture. 16.
Repace the eectrphresis id with the drying id. 17. Hding the antisera te
mpate in the up
psitin, pace the tempate int the apprpriate sts in the chamber. 18. Gent
y wer the
antisera tempate nt the surface f the ge. N further pressure is needed. 19
. Quic y pipette
the xative and antisera int the sts at the right end (ande) f each antisera
channe in the
tempate. 20. After 2 minutes (1 t 3 minutes is acceptabe) incuba- tin time,
pace ne btter
cmb int the same sts where the antisera has been appied. After 2 minutes, r
emve the btter
cmb and the antisera tempate. 21. Genty bt the ge with a btter and remv
e it. Pace a
different btter n the surface f the ge. Pace the antisera tempate n tp
f the btter.
After 5 minutes, remve the antisera tempate and btter. Cse the id. 22. Dr
y the ge fr 8
minutes by turning n ny the cham- ber. After 8 minutes, turn the chamber ff
and remve the
ge. 23. Fi a cntainer with prepared stain. Fi anther cn- tainer with des
tain sutin.
Fi a third cntainer with tris-buffered saine (TBS). 24. Pace the ge in TBS
and sha e genty

n a rtatr fr 10 minutes. Remve the ge frm the TBS and aw it t drain 
n a btter. 25.
Pace the ge int the staining dish cntaining the pre- pared stain. Leave it f
r 4 minutes.
Remve the ge frm the stain and aw it t drain n a btter. 26. Destain th
e ge in tw
cnsecutive washes f destain sutin. Use a gente, aternatey rc ing
and swiring
technique. Aw the ge t remain in each wash fr 1 minute. The ge bac grund
shud be
cmpetey cear. Tap the ge t remve the excess destain sutin. 27. Ensure
that the chamber
r is cean. Repace the ge nt the chamber r. Set a timer fr 8 minutes, c
se the drying
id, and turn the chamber n. 28. Remve the ge frm the chamber, and pace it
int the destain
again fr 1 minute. Tap the ge t remve excess destain sutin. 29. Pace the
ge bac int
the chamber and dry fr 5 minutes. 30. Turn ff the chamber and remve the ge.
Resuts The
different anes f patient serum are cmpared t the nrma cntr. One ane wi
 have a the
serum prtein bands stained, and the remaining ve anes wi shw indi- vidua re
actins with
antisera t heavy chains (anti-IgG, anti-IgA, r anti-IgM) and t ight chains (
anti- appa and
anti-ambda). The presence f abnrma bands is indicated by an increase in size
and staining
activity in a particuar area. Pycna hypergammagbuinemia is indicated by
brad, dar
bands in the gamma regin. A mncna pr- tein band prduces a pattern t
hat is narrw
and dar . Cnversey, a faint band in the gamma regin indicates a hypgammag
buinemia.
Interpretatin f Resuts In the cinica abratry, immun xatin eectrphresi
s is primariy
used fr the detectin f mncna gam- mpathies. A mncna gammpathy is
a primary disease
state in which a singe cne f pasma ces prduces ee- vated eves f an i
mmungbuin f
a singe cass and type. Such immungbuins are referred t as mncna prt
eins, M-prteins,
r paraprteins. In sme cases they are indicative f a maignancy such as muti
pe myema r
Wadenstrms macrgbuinemia. A mncna gammpathy wi typi- cay shw a nar
rw dar band
in ne heavy-chain regin and in ne ight-chain regin. Differentiatin must be
made between
pycna and mncna gammpathies, because pycna gam- mpathies ar
e ny a
secndary disease state due t cinica disrders such as chrnic iver diseases
, cagen
disrders, rheumatid arthritis, and chrnic infectins. Pycna gammpathies
are indicated by
wide bands in ne r mre heavy- and ight-chain regins. If there is a very hig
h eve f
immungbuin in the patient sampe, antigen excess may ccur. This wi resut
in staining f
the margins, eaving the centra area with itte demnstrabe prtein stain. In
this case it

wi be nec- essary t adjust the prtein cntent f the sampe by diu
tin.
1814_Ch08_123-136.qxd 7/10/09 2:52 PM Page 134 CHAPTER 8 Precipitatin Reacti
ns 135 1. In a
precipitatin reactin, hw can the idea antibdy be characterized? a. Lw af nit
y and w
avidity b. High af nity and w avidity c. High af nity and high avidity d. Lw af nit
y and high
avidity 2. Precipitatin differs frm aggutinatin in which way? a. Precipitati
n can ny be
measured by an aut- mated instrument. b. Precipitatin ccurs with univaent an
tigen, whie
aggutinatin requires mutivaent antigen. c. Precipitatin des nt readiy c
cur, because few
antibdies can frm aggregates with antigen. d. Precipitatin invves a sube
antigen, whie
aggutinatin invves a particuate antigen. 3. When sube antigens diffuse i
n a ge that
cntains anti- bdy, in which zne des ptimum precipitatin ccur? a. Przne
b. Zne f
equivaence c. Pstzne d. Prezne 4. Which f the fwing statements app
y t rate
nephemetry? a. Readings are ta en befre equivaence is reached. b. It is mre
sensitive than
turbidity. c. Measurements are time dependent. d. A f the abve 5. Which f t
he fwing is
characteristic f the end-pint methd f RID? a. Readings are ta en befre equi
vaence. b.
Cncentratin is directy in prprtin t the square f the diameter. c. The di
ameter is ptted
against the g f the cn- centratin. d. It is primariy a quaitative rather
than a
quantitative methd. 6. Which statement is true f measurements f turbidity? a.
It indicates the
rati f incident ight t transmitted ight. b. Light that is scattered at an a
nge is detected.
c. It is recrded in units f reative ight scatter. d. It is nt affected by 
arge partices
faing ut f sutin. 7. Which f the fwing refers t the frce f attrac
tin between an
antibdy and a singe antigenic determinant? a. Af nity b. Avidity c. Van der Waa
s attractin d.
Cvaence 8. Which technique is typi ed by radia immundiffusin cmbined with e
ectrphresis?
a. Cuntercurrent eectrphresis b. Rc et eectrphresis c. Immuneectrphr
esis d. Suthern
btting 9. Immun xatin eectrphresis differs frm immun- eectrphresis in
which way? a.
Eectrphresis ta es pace after diffusin has ccurred. b. Better separatin 
f prteins with
the same eec- trphretic mbiities is btained. c. Antibdy is directy appi
ed t the ge
instead f being paced in a trugh. d. It is mainy used fr antigen detectin.
10. In which
zne might an antibdy-screening test be fase negative? a. Przne b. Zne f e
quivaence c.
Pstzne d. Nne f the abve 11. If crssed ines resut in an Ouchterny immu
ndiffu- sin
reactin with antigens 1 and 2, what des this indicate? a. Antigens 1 a
nd 2 are

identica. b. Antigen 2 is simper than antigen 1. c. Antigen 2 is mre cmpex

than antigen 1.
d. The tw antigens are unreated. 12. Which might affect the utcme f immund
iffusin
prcedures? a. Imprper diutin f antigen in the wes b. Over ing the wes c
. Nneve
incubatin f pates d. A f the abve REVIEW QUESTIONS 1814_Ch08_123-136.qxd
7/10/09 2:52 PM
Page 135 136 SECTION 2 Basic Immungica Prcedures 13. Which technique repre
sents a
singe-diffusin reactin? a. Radia immundiffusin b. Ouchterny diffusin c.
Immuneectrphresis d. A f the abve 14. Which best describes the aw f ma
ss actin? a.
Once antigenantibdy binding ta es pace, it is irreversibe. b. The equiibrium
cnstant
depends ny n the frward reactin. c. The equiibrium cnstant is reated t
strength f
antigenantibdy binding. d. If an antibdy has a high avidity, it wi dissciate
frm antigen
easiy. References 1. Kindt, TJ, Gdsby, RA, and Osbrne, BA. Kuby Immungy,
ed. 6. WH Freeman
and C., New Yr , 2007, pp. 145167. 2. Kric a, LJ. Principes f immunchemi
ca
techniques. In Burtis, CA, Ashwd, ER, and Bruns, DE (eds): Tietz Fund
amentas f
Cinica Chemistry, ed. 6. Saunders Esevier, St. Luis, 2008, pp. 155170. 3. Ash
ihara, Y,
Kasahara, Y, and Na amura, RM. Immunassays and immunchemistry. In McPhersn, R
A, and Pincus, MR
(eds): Henrys Cinica Diagnsis and Management by Labratry Methds, ed.
21. Saunders
Esevier, Phiadephia, 2007, pp. 793818. 4. Ma , T, and Saunders, ME. The Immune
Respnse:
Basic and Cinica Principes. Esevier Academic Press, Buringtn, MA, 2006, pp
. 147177. 5.
Kric a, LJ, and Par , JY. Optica techniques. In Burtis, CA, Ashwd, ER, and Br
uns, DE (eds):
Tietz Fundamentas f Cinica Chemistry, ed. 6. Saunders Esevier, St. Luis, 2
008, pp. 6383.
6. Warren, JS. Immungbuin quantitatin. In Detric , B, Hamitn, RG, and F
ds, JD (eds):
Manua f Mecuar and Cinica Labratry Immungy, ed. 7. American Sciety
fr Micrbigy
Press, Washingtn, D.C., 2006, pp. 6974. 7. Kang, S, Suh, J, Lee, H, Yn, H, et
a. Cinica
usefuness f free ight chain cncentratin as a tumr mar er in mutipe mye
ma. Ann Hemat
84:588593, 2005. 8. Mancini, G, Carbnara, AO, and Heremans, JF. Immunchemica q
uantitatin f
antigens by singe radia immundiffusin. Immunchem 2:235, 1965. 9. Fahey, JL,
and McKevey,
EM. Quantitative determinatin f serum immungbuins in antibdy-agar pates.
J Immun 94:84,
1965. 10. Lindsey, MD, Warnc , DW, and Mrrisn, CJ. Sergica and mecuar
diagnsis f
funga infectins. In Detric , B, Hamitn, RG, and Fds, JD (eds): Manua f M
ecuar and
Cinica Labratry Immungy, ed. 7. American Sciety fr Micrbigy Press,
Washingtn, D.C.,
2006, pp. 569605. 11. Reeves, WH, Sath, M, Lyns, R, Nichs, C, et a. Detecti

n f
autantibdies against prteins and ribnuceprteins by dube immundiffusi
n and
immunprecipitatin. In Detric , B, Hamitn, RG, and Fds, JD (eds): Manua 
f Mecuar and
Cinica Labratry Immungy, ed. 7. American Sciety fr Micrbigy Pre
ss, Washingtn,
D.C., 2006, pp. 10071018. 12. Ortn, SM, Peace-Brewer, A, Schmitz, JL, Freeman, K
, et a.
Practica evauatin f methds fr detectin and speci city f autantibdies t
extractabe
nucear antigens. Cin Vaccine Immun 11:297301, 2004. 13. Laure, CB. Antigenantibdy
crssed eectrphresis. Ana Bichem 10:35861, 1965. 14. McPhersn, RA, and Mass
ey, HD.
Labratry evauatin f immungbuin functin and humra immunity. In M
cPhersn, RA
and Pincus, MR (eds): Henrys Cinica Diagnsis and Management by Labratry
Methds, ed.
21. Saunders Esevier, Phiadephia, 2007, pp. 835848. 15. Aper, CA, and Jhnsn
, AM.
Immun xatin eectrphre- sis: A technique fr the study f prtein pymrphism
. Vx Sanguinis
17:445, 1969. 16. Katzman, JA, and Kye, RA. Immunchemica characteriza- tin 
f immungbuins
in serum, urine, and cerebrspina fuid. In Detric , B, Hamitn, RG, and
Fds, JD
(eds): Manua f Mecuar and Cinica Labratry Immungy, ed. 7. American S
ciety fr
Micrbigy Press, Washingtn, D.C., 2006, pp. 88100. 17. Keren, DF, and Hump
hrey, RL.
Cinica indicatins and appicatins f serum and urine prtein eectrphres
is. In Detric ,
B, Hamitn, RG, and Fds, JD (eds): Manua f Mecuar and Cinica Labra
try Immungy,
ed. 7. American Sciety fr Micrbigy Press, Washingtn, D.C., 2006, pp. 758
7.
1814_Ch08_123-136.qxd 7/10/09 2:52 PM Page 136 9 Aggutinatin 137 LEARNING O
BJECTIVES After
nishing this chapter, the reader wi be abe t: 1. Differentiate between aggut
inatin and
precipitatin. 2. Discuss hw IgM and IgG differ in abiity t participate in ag
gutinatin
reactins. 3. Describe physigica cnditins that can be atered t enhance a
ggutinatin. 4.
Describe and give an exampe f each f the fwing: a. Direct aggutinatin b
. Passive
aggutinatin c. Reverse passive aggutinatin d. Aggutinatin inhibitin e. He
maggutinatin
inhibitin f. Caggutinatin 5. Expain and give an appicatin fr the direct
Cmbs test. 6.
Discuss reasns fr the use f the indirect Cmbs test. 7. Describe the princip
e f
measurement used in partice-cunting immunassay (PACIA). 8. Discuss cnditins
that must be met
fr ptima resuts in aggutinatin testing. KEY TERMS Aggutinatin inhibitin
Aggutinin
Caggutinatin Direct aggutinatin Direct antigbuin test Hemaggutinatin H
emaggutinatin
inhibitin Indirect antigbuin test Lattice frmatin Lw inic strength sain

e
Partice-cunting immunassay (PACIA) Passive aggutinatin Reverse passive agg
utinatin
Sensitizatin 1814_Ch09_137-151.qxd 7/10/09 2:53 PM Page 137 138 SECTION 2 Ba
sic Immungica
Prcedures Whereas precipitatin reactins invve sube antigens, aggutinati
n is the visibe
aggregatin f partices caused by cmbinatin with speci c antibdy. Antibdies t
hat pr- duce
such reactins are ften caed aggutinins. Because this reactin ta es pace 
n the surface f
the partice, anti- gen must be expsed and abe t bind with antibdy.
Aggutinatin is
actuay a tw-step prcess, invving sen- sitizatin r initia binding fw
ed by attice
frmatin, r frmatin f arge aggregates. Types f partices participat- ing
in such reactins
incude erythrcytes, bacteria ces, and inert carriers such as atex partice
s. Each partice
must have mutipe antigenic r determinant sites, which are crss-in ed t sit
es n ther
partices thrugh the frma- tin f antibdy bridges. 1 In 1896, Gruber and Dur
ham pubished the
rst reprt abut the abiity f antibdy t cump ces, based n bser- vatins
f
aggutinatin f bacteria ces by serum. 2 This nding gave rise t the use f s
ergy as a
t in the diag- nsis f disease, and it as ed t the discvery f the ABO
bd grups.
Wida and Sicard deveped ne f the eari- est diagnstic tests in 1896 fr th
e detectin f
antibdies ccurring in typhid fever, brucesis, and tuaremia. 2 Aggu
tinatin
reactins nw have a wide variety f appica- tins in the detectin f bth ant
igens and
antibdies. Such testing is simpe t perfrm, and the end pints can easiy be
read visuay.
Aggutinatin reactins can be cassi ed int severa dis- tinct categries: direc
t, passive,
reverse passive, aggutinatin inhibitin, and caggutinatin. Principes f ea
ch f these types
f reactins are discussed, incuding their current use in tdays cinica abra
try. STEPS IN
AGGLUTINATION Sensitizatin Aggutinatin, i e precipitatin, is a tw-step pr
cess that resuts
in the frmatin f a stabe attice netwr . The rst reactin invves antigenant
ibdy
cmbinatin thrugh singe antigenic determinants n the partice surface and is
ften caed the
sensitizatin step. 3 This initia reactin f- ws the aw f mass actin (se
e Chapter 8) and
is rapid and reversibe. 4 The secnd step is the frmatin f crss-in s that
frm the visibe
aggregates. This represents the stabi- izatin f antigenantibdy cmpexes with
the binding
tgether f mutipe antigenic determinants. 4 Each stage f the prcess is affe
cted by different
factrs, and it is impr- tant t understand these in rder t manipuate and en
hance end pints
fr such reactins. Sensitizatin is affected by the nature f the antibdy me

cues themseves.
The af nity and avidity (discussed in Chapter 8) f an individua antibdy determi
ne hw much
antibdy remains attached. The cass f immungbuin is as imprtant; IgM wi
th a ptentia
vaence f 10 is ver 700 times mre ef cient in aggutinatin than is IgG with a
vaence f 2. 5
The nature f the antigen-bearing surface is as a ey factr in the initia se
nsitizatin
prcess. If epitpes are sparse r if they are bscured by ther surface mecu
es, they are ess
i ey t interact with antibdy. Lattice Frmatin The secnd stage, representi
ng the sum f
interactins between antibdy and mutipe antigenic determinants n a partice,
is dependent n
envirnmenta cnditins and the reative cncentratins f antigen and antibdy
. 3 Brdet
hypthesized that attice frmatin is gverned by physic- chemica factrs suc
h as the miieus
inic strength, pH, and temperature. 2 Antibdy must be abe t bridge the gap b
etween ces in
such a way that ne mecue can bind t a site n each f tw different ces.
The preceding
factrs can be manipuated t faciitate such attachment. Figure 91 depicts the t
w-stage
prcess. Erythrcytes and bacteria ces have a sight negative surface charge,
and because i e
charges tend t repe ne anther, it is dif cut t bring such ces tgether int
 at- tice
frmatin. Additinay, in an inic sutin, red ces surrund themseves wit
h catins t frm
an inic cud, which eeps them abut 25 nm apart. 3 The abiity t in ces
tgether depends
in part n the nature f the antibdy. CHAPTER OUTLINE STEPS IN AGGLUTINATION Se
nsitizatin
Lattice Frmatin Enhancement f Lattice Frmatin TYPES OF AGGLUTINATION REACTI
ONS Direct
Aggutinatin Passive Aggutinatin Reverse Passive Aggutinatin Aggutinatin
Inhibitin
Caggutinatin ANTIGLOBULIN-MEDIATED AGGLUTINATION Direct Antigbuin Test Ind
irect
Antigbuin Test INSTRUMENTATION QUALITY CONTROL AND QUALITY ASSURANCE SUMMARY
CASE STUDY
EXERCISE: LATEX AGGLUTINATION SLIDE TEST FOR RUBELLA REVIEW QUESTIONS REFERENCES
1814_Ch09_137-151.qxd 7/10/09 2:53 PM Page 138 CHAPTER 9 Aggutinatin 139 An
tibdies f the
IgG cass ften cannt bridge the distance between partices, because their sma
 size and
restricted exibiity at the hinge regin may prhibit mutivaent bind- ing. 1,3
IgM antibdies,
n the ther hand, have a diameter f abut 35nm, s they are strng aggutinins
. 3 Visibe reactins with IgG ften require the use f enhancement techniques, which va
ry
physicchemica cnditins. Enhancement f Lattice Frmatin The surface charge
must be
cntred fr attice frmatin, r a visibe aggutinatin reactin, t ta e p
ace. One means
f accmpishing this is by decreasing the buffers inic strength thrugh the use
f w inic

strength saine. 3,4 The additin f abumin in cncentratins f 5 t 30 per- c

ent as heps t
neutraize the surface charge and aws red ces t apprach each ther mre c
sey. 4 Other
techniques that enhance aggutinatin, especiay that f red bd ces, incu
de increasing the
viscsity, using enzymes, agitating centrifuging, and atering the tempera- ture
r the pH.
Viscsity can be increased by adding agents such as dextran r pyethyene gyc
 (PEG). These
agents reduce the water f hydratin arund ces and aw them t cme int c
ser prximity
fr antibdy t jin tgether. 3 Brmein, papain, trypsin, and cin are the enzym
es mst ften
used t enhance aggutinatin, and these are thught t wr by reducing the sur
face charge n
red bd ces thrugh ceaving f chemica grups and decreasing hydra- tin.
4 Ficin ceaves
siagycprteins frm the red bd ce surface; in additin t reducing the
charge, this may
change the externa cn guratin f the membrane t revea mre antigenic determin
ant sites. 3
Agitatin and centrifugatin prvide a physica means t increase cece cntac
t and thus
heighten aggutinatin. 4 A f these techniques pus the use f antigbuin r
eagents
(discussed in the sectin n antigbuin testing) are used in the bd ban t
 better detect
antigenantibdy reactins, especiay in seecting bd fr transfusin. The tem
perature at
which the antigenantibdy reactin ta es pace must as be cnsidered, because i
t has an in uence n the secndary, r aggregatin, phase. Antibdies benging t the IgG c
ass aggutinate
best at 30C t 37C, whie IgM antibdies react best at temperatures between 4C and
27C.
Because naturay ccurring antibdies against the ABO bd grups beng t
the IgM cass,
these reactins are best run at rm temperature. Antibdies t ther human b
d grups usuay
beng t the IgG cass, and reactins invving these must be run at 37C. These
atter
reactins are the mst imprtant t cnsider in seect- ing cmpatibe bd fr
a transfusin,
because these are the nes that wi actuay ccur in the bdy. An additina p
hysicchemica
factr that can be manip- uated when perfrming aggutinatin reactins is pH.
Mst reactins
prduce ptima antigenantibdy cmbinatin when the pH is between 6.5 and 7.5, b
ut there are
sme exceptins, such as human anti-M and antiP1, which react best at a wer pH
. 4 TYPES OF
AGGLUTINATION REACTIONS Aggutinatin reactins are easy t carry ut, require n
 cmpicated
equipment, and can be perfrmed as needed in the abratry withut having t ba
tch specimens.
Batching specimens is dne if a test is expensive r cmpicated; then a arge n
umber are run at
ne time, which may resut in a time deay. Many its are avaiabe fr standard
testing, s

reagent preparatin is minima, and aggutinatin reactins are a frequenty emp

yed sergica
test. They can be used t identify either antigen r antibdy. Typicay, mst a
ggu- tinatin
tests are quaitative, simpy indicating absence r presence f antigen r antib
dy, but
diutins can be made t btain semiquantitative resuts. Many variatins exist,
and these can be
categrized accrding t the type f par- tice used in the reactin and whether
antigen r
antibdy is attached t it. Direct Aggutinatin Direct aggutinatin ccurs whe
n antigens are
fund nat- uray n a partice. One f the best exampes f direct ag
gutinatin
testing invves nwn bacteria antigens used t test fr the presence f un n
wn antibdies in
the patient. Typicay, patient serum is diuted int a series f tubes r wes
n a side and
reacted with bacteria antigens speci c fr the suspected disease. Detectin f an
tibdies is
prima- riy used in diagnsis f diseases fr which the bacteria agents are ext
remey dif cut
t cutivate. One such exam- pe is the Wida test, a rapid screening test t he
p determine A
N visibe reactin B Rearrangement Visibe aggutinatin Antibdy Antigenantibd
y cmbinatin
Antigen with mutipe determinants + FIGURE 91. Phases f aggutinatin. (A) Sens
itizatin.
Antigen and antibdy unite thrugh antigenic determinant sites. (B) Lattice frmatin.
Rearrangement f antigen and antibdy bnds t frm stabe attice. 1814_Ch09_13
7-151.qxd
7/10/09 2:53 PM Page 139 140 SECTION 2 Basic Immungica Prcedures the pss
ibiity f
typhid fever. The antigens used in this prcedure incude Samnea O (smatic
) and H
( agear) antigens. A signi cant nding is a furfd increase in anti- bdy titer v
er time
when paired diutins f serum sampes are tested with any f these antigens. Wh
ie mre speci c
tests are nw avaiabe, this test is sti cnsidered usefu in diagnsing typh
id fever in
deveping cuntries, and it remains in use in many areas thrughut the wrd.
6,7 If an
aggutinatin reactin invves red bd ces, then it is caed hemaggutinat
in. The best
exampe f this ccurs in ABO bd grup typing f human red bd ces, ne 
f the wrds
mst frequenty used immunassays. 1 Antisera f the IgM type can be used t det
ermine the presence r absence f the A and B antigens, and this reactin is usuay perfrmed
at rm
temperature withut the need fr any enhancement techniques. This type f aggut
inatin reactin
is simpe t perfrm, is reativey sensitive, and is easy t read. A titer that
yieds
semiquantitative resuts can be perfrmed in test tubes r micrtiter pates by
ma ing seria
diutins f the antibdy. The reciprca f the ast diutin sti exhibiting
a visibe

reactin is the titer, indi- cating the antibdys strength. Interpretatin f the
test is dne
n the basis f the ce sedimentatin pattern. If there is a dar red, smth b
ut- tn at the
bttm f the micrtiter we, the resut is negative. A psitive resut
wi have ces
that are spread acrss the wes bttm, usuay in a jagged pattern with an irre
guar edge.
Test tubes as can be centrifuged and then sha en t see if the ce buttn can
be eveny
resuspended. If it is resuspended with n visibe cumping, then the resut is n
egative. Psitive
reactins can be graded t indicate the strength f the reactin (Fig. 92). Hemag
gutinatin
its are nw avaiabe fr detectin f antibdies t hepatitis A virus (HAV),
hepatitis B
virus (HBV), hepatitis C virus (HCV), and human immunde ciency virus (HIV) I
and II, t cite
just a few exampes. 810 Passive Aggutinatin Passive, r indirect, aggutinati
n empys
partices that are cated with antigens nt nrmay fund n their sur- faces.
A variety f
partices, incuding erythrcytes, atex, geatin, and siicates, are used fr t
his purpse. 8
The use f synthetic beads r partices prvides the advantage f cn- sistency,
unifrmity, and
stabiity. 1 Reactins are easy t read visuay and give quic resuts. Partic
e sizes vary frm
7 m fr red bd ces dwn t 0.8 m r ess fr fine atex partices. 11 Many an
tigens,
especiay pysaccharides, adsrb t red bd ces spntaneusy, s they are
reativey easy
t manipuate. Prbems encuntered with the use f erythr- cytes as carrier pa
rtices incude
the pssibiity f crss- reactivity, especiay with heterphie antibdy (see
Chapter 3) if the
ces used are nnhuman. In 1955, Singer and Ptz fund by happenstance that Ig
G was naturay
adsrbed t the surface f pystyrene atex partices. Whie ther substances s
uch as
pysaccha- rides and highy charged prteins are nt naturay adsrbed by thes
e partices,
manipuatin with certain chemicas is usuay successfu in cating atex parti
ces with these
sub- stances. Latex partices are inexpensive, are reativey stabe, and are n
t subject t
crss-reactivity with ther antibd- ies. A arge number f antibdy mecues c
an be bund t
the surface f atex partices, s the number f antigen- binding sites is arge
. 1 Additinay,
the arge partice size faciitates reading f the test. 5 Passive aggutinati
n tests have
been used t detect rheumatid factr; antinucear antibdy ccurring in the
disease upus
erythematsus; antibdies t grup A strep- tcccus antigens; antibdies t Tri
chinea
spirais; antibdies t Trepnema paidum; and antibdies t viruses such
as
cytmegavirus, rubea, varicea-zster, and HIV-1/ HIV-2. 1217 Because many 
f these its

are designed t detect A B Red ce buttn after centrifugatin 4+ One sid cu
mp 3+ Severa
arge cumps 2+ Numerus smaer cumps 1+ Barey discernabe cumps Negative sm
th suspensin
Sha e Strng aggutinatin arge cumps and cear bac grund Antigen and antibdy
are mixed
and rtated Wea aggutinatin sma cumps and cudy bac grund Negativeeven sus
pensin and
cudy bac grund FIGURE 92. Grading f agguti- natin reactins. (A) Tube meth
d. If tubes are
centrifuged and sha en t resuspend the buttn, reactins can be graded frm neg
ative t 4 ,
depending n the size f cumps bserved. (B) Side methd. Reactins can be gra
ded frm negative
t strngy reactive, depending n the size f the cumps in the suspensin.
1814_Ch09_137-151.qxd 7/10/09 2:53 PM Page 140 CHAPTER 9 Aggutinatin 141 Ig
M antibdy, and
there is aways the ris f nnspecific aggutinatin caused by the presence f
ther IgM
antibd- ies, reactins must be carefuy cntred and interpreted. Cmmercia
tests are
usuay perfrmed n dispsabe pas- tic r cardbard cards r gass sides. Ki
ts cntain
psitive and negative cntrs, and if the cntrs d nt give the expected res
uts, the test is
nt vaid. Such tests are typicay used as screening ts t be fwed by m
re extensive
testing if the resuts are psitive. Reverse Passive Aggutinatin In reverse pa
ssive
aggutinatin, antibdy rather than antigen is attached t a carrier partice. T
he antibdy must
sti be reactive and is jined in such a manner that the active sites are facin
g utward.
Adsrptin may be spntaneus, r it may require sme f the same manipuatin a
s is used fr
antigen attachment. This type f testing is ften used t detect micrbia antig
ens. Figure 93
shws the differences between passive and reverse passive aggutinatin. Numeru
s its are
avaiabe tday fr the rapid identi - catin f antigens frm such infectius age
nts as grup B
streptcccus, Staphycccus aureus, Neisseria meningitidis, streptccca gr
ups A and B,
Haemphius infuenzae, rtavirus, Cryptcccus nefrmans, Vibri chera
01, and
Leptspira. 7,1824 Rapid aggutinatin tests have fund the widest appicatin in
detecting
sube antigens in urine, spina fuid, and serum. 8 The principe is the same
fr a these
tests: Latex partices cated with antibdy are reacted with a patient sampe c
ntaining the
suspected antigen. In sme cases, an extractin step is necessary t isate ant
igen befre the
reagent atex partices are added. Organisms can be identi ed in a few minutes wit
h fairy high
sensitivity and speci city, athugh this varies fr different rganisms. Fr exam
pe, the
sensitivity f atex aggutinatin its fr the detectin f cryptccca antige
n has been
reprted t be as high as 99 percent, whie the specificity f testing fr Candi

da abicans is
much wer. 24 Use f mncna anti- bdies has greaty cut dwn n crss-reac
tivity, but there
is sti the pssibiity f interference r nnspeci c aggutina- tin. Such tests
are mst ften
used fr rganisms that are difficut t grw in the abratry r fr instances
when rapid
identi catin wi aw treatment t be initiated mre prmpty. Direct testing 
f specimens fr
the presence f vira antigens has sti nt reached the sensitivity f enzyme i
mmunassays, but
fr infectins in which a arge amunt f vira antigen is present, such as rta
virus and enteric
ade- nvirus in infants, atex aggutinatin tests are extremey usefu. 22,25 R
everse passive
aggutinatin testing has as been used t measure eves f certain therapeuti
c drugs, hrmnes, and pasma prteins such as haptgbin and C-reactive prtein. In
a f these
reactins, rheumatid fac- tr wi cause a fase psitive as it reacts
with any IgG
antibdy, s this must be ta en int accunt. Aggutinatin Inhibitin Aggutina
tin inhibitin
reactins are based n cmpe- titin between particuate and sube antigens f
r imited
antibdy-cmbining sites, and a ac f aggutinatin is an indicatr f a psit
ive reactin.
Typicay, this type f reac- tin invves haptens that are cmpexed t prtei
ns; the
haptenprtein cnjugate is then attached t a carrier par- tice. The patient sam
pe is first
reacted with a imited amunt f reagent antibdy that is specific fr the hapte
n being tested.
Indicatr partices that cntain the same hap- ten ne wishes t measure in the
patient are then
added. If the patient sampe has n free hapten, the reagent anti- bdy is ab
e t cmbine
with the carrier partices and prduce a visibe aggutinatin. In this case
, hwever, aggutinatin is a negative reactin, indicating that the patient did nt have suf cien
t hapten t
inhibit the secndary reac- tin (Fig. 94). Either antigen r antibdy can be att
ached t the
partices. The sensitivity f the reactin is gverned by the avidity f the
antibdy
itsef. It can be a highy sensitive assay capabe f detecting sma
quantities f
antigen. 1 Detectin f iicit drugs such as ccaine r herin A Cated partic
es
Antibdy
Visibe aggutinatin + 2 Carrier partice Sube antigen Cated partice + 1 B
Cated
partices
Antigen with mutipe determinants Visibe aggutinatin + 2 Carrier p
artice
Antibdy Cated partice 1
+ FIGURE 93. Passive and reverse passive aggutinatin
. (A) Passive
aggutinatin. Antigen is attached t the carrier partice, and aggu- tinatin
ccurs if patient
antibdy is present. (B) Reverse passive aggutinatin. Antibdy is attached t
the carrier
partice, and aggutinatin ccurs if patient antigen is present. 1814_Ch09_137-

151.qxd 7/10/09
2:53 PM Page 141 142 SECTION 2 Basic Immungica Prcedures are exampes f v
ery sensitive
aggutinatin inhibitin tests. Hemaggutinatin inhibitin reactins use the sa
me principe,
except red bd ces are the indicatr partices. This type f testing has bee
n used t detect
antibdies t certain viruses, such as rubea, mumps, meases, in uenza, parain uen
za, HBV,
herpesvirus, respiratry syncytia virus, and adenvirus. 1,8,10 Red bd ces
have naturay
ccur- ring vira receptrs. When virus is present, spntaneus aggutinatin c
curs, because the
virus partices in the red bd ces tgether. Presence f patient antibdy
inhibits the
aggutinatin reactin. T perfrm a hemaggutinatin inhibitin test, patient s
erum is rst
incubated with a vira preparatin. Then red bd ces that the virus is nwn
t aggutinate
are added t the mixture. If antibdy is present, this wi cmbine with vira p
artices and
prevent aggutinatin, s a ac f r reductin in aggutinatin indicates pres
ence f patient
anti- bdy. Cntrs are necessary, because there may be a factr in the serum t
hat causes
aggutinatin, r the virus may have st its abiity t aggutinate. Caggutin
atin
Caggutinatin is the name given t systems using bac- teria as the inert parti
ces t which
antibdy is attached. Staphycccus aureus is mst frequenty used, because it
has a prtein n
its uter surface, caed prtein A, which natu- ray adsrbs the fragment crys
taizabe (FC)
prtin f antibdy mecues. The active sites face utward and are capabe f
reacting with
speci c antigen (Fig. 95). These partices exhibit greater stabiity than atex par
tices and
are mre refractry t changes in inic strength. Hwever, because bacteria are
nt cred,
reactins are ften dif - cut t read. Such testing is highy speci c, but it may n
t be as
sensitive fr detecting sma quantities f antigen, as is atex aggutinatin.
11
Caggutinatin reagents have been used in identi catin f streptccci, Neisseri
a meningitidis,
Neisseria gnrrheae, Vibri chera 0139, and Haemphius in uenzae. 7,11 ANTIGL
OBULIN-MEDIATED
AGGLUTINATION The antihuman gbuin test, as nwn as the Cmbs test, is a te
chnique that
detects nnaggutinating antibdy by means f cuping with a secnd antibdy. I
t remains ne f
the mst widey used prcedures in bd ban ing. The ey cmpnent f the test
is antibdy t
human gbuin that is made in animas r by means f hybridma tech- niques. 3
Such antibdy
wi react with the FC prtin f the human antibdy attached t red bd ces
. Aggutinatin
ta es pace because the antihuman gbuin is abe t bridge the distance betwee
n ces that IgG
ane cannt d. The strength f the reactin is prprtina t the amunt f A

dd antigen-cated
atex partices + Incubate N patient antigen present Patient antigen present Ag
gutinatin =
negative test N aggutinatin = psitive test Patient sampe FIGURE 94. Aggutin
atin
inhibitin. Reagent antibdy is added t the patient sampe. If patient antigen
is present,
antigenantibdy cmbinatin resuts. When antigen-cated atex partices are adde
d, n
aggutinatin ccurs, which is a psitive test. If n patient anti- gen is there
, the reagent
antibdy cmbines with atex partices, and aggutinatin resuts, which is a ne
gative test. +
Partices nnspecificay absrb reagent antibdy thrugh prtein A
Prtein A Pa
tient antigen
(sube) Aggutinatin FIGURE 95. Caggutinatin with Staphycccus aureus. St
aphycccus
aureus partices nnspeci cay bind the FC prtin f immungbuin mecues. W
hen reagent
anti- bdy is used, cmbinatin with patient antigen prduces a visibe aggutin
atin reactin.
1814_Ch09_137-151.qxd 7/10/09 2:53 PM Page 142 CHAPTER 9 Aggutinatin 143 an
tibdy cating
the red bd ces. The Cmbs test can be divided int tw different types, dir
ect and
indirect, each f which has a different purpse. Direct Antigbuin Test The di
rect antigbuin
test is used t demnstrate in viv attachment f antibdy r cmpement t an i
ndividuas red
bd ces. This test serves as an indicatr f autim- mune hemytic anemia,
hemytic disease
f the newbrn, sensitizatin f red bd ces caused by the presence f drugs
, r a
transfusin reactin. 4 The test is caed direct, because red bd ces are t
ested directy as
they cme frm the bdy. A bd sampe is btained frm the patient, the red b
d ces are
washed t remve any antibdy that is nt speci cay attached, and then ces are
tested
directy with antibdy t IgG r cmpement cmpnents. If IgG r cmpement is
present n the
red bd ces, the anti- human gbuin (Cmbs reagent) is abe t bridge the
gap between red
bd ces and cause a visibe aggutinatin (Fig. 96A). Pyspeci c antiserum wi
 react with
IgG and with cm- pement cmpnent C3d. Antibdies t C3b, C4b, r C4d may as
be present. 3 If
such a reactin is psitive, then mnspeci c antibdy, which wi react with ny
ne cmpnent, is used. This wi differentiate between IgG and individua cmpement c
mpnents n the
patients red bd ces. A psitive test indicates that an immune reac- tin is
ta ing pace in
that individua. Indirect Antigbuin Test The indirect antigbuin test, r i
ndirect Cmbs
test, is used t determine the presence f a particuar antibdy in a patient, 
r it can be used
t type patient red bd ces fr speci c bd grup antigens. This is a tw-st
ep prcess, in
which washed red bd ces and antibdy are awed t cmbine at 37C, and the

ces are then

carefuy washed again t remve any unbund antibdy. When antihuman gbuin i
s added, a
visibe reactin ccurs where antibdy has been speci cay bund. This test is m
st ften used
t chec fr the presence f cinicay significant aantibdy in patient seru
m when perfrming
cmpatibiity testing fr a bd transfusin. 4 In this case, patient serum is
used t cmbine
with reagent red bd ces f nwn antigenicity. A reactins are run
at 37C t
detect cinicay significant antibdies. Ces are then washed, and antihuman g
buin is added.
Tubes are centrifuged and read fr aggutinatin (see Fig. 96B). Pssibe
surces f
errr in perfrming the Cmbs test incude faiure t wash ces, imprper centr
ifugatin,
fai- ure t add test serum r antihuman gbuin, and use f expired reagents
r thse that
have nt been prpery stred. 3 In additin, an imprper cncentratin f re
d ces may ater
the resuts. T heavy a red ce cncentratin may mas aggutinatin, whie t
 ight a
cncentratin wi ma e the reactin hard t read. 3 Thus, it is imprtant t us
e quaity
cntrs and t interpret resuts carefuy. INSTRUMENTATION Whie aggutinatin
reactins
require n cmpicated instru- mentatin t read, severa systems have been deve
ped using
autmatin t increase sensitivity. Many f these use turbidimetry, which is bas
ed n the
principe that as parti- ces cmbine, ight scatter increases, and the absrban
ce f the
sutin increases prprtinay. (Refer t Chapter 8 fr further discussin f
the principes
f turbidimetry.) Nephemetry has as been appied t the reading f aggu- ti
natin reactins,
and the term partice-enhanced immunassay is used t describe such reactins. N
ephemetry
withut the use f partices is capabe f detecting sube antigenantib
dy cmpexes
at a sensitivity f between 1 and 10 g/mL. 9 If partices are used, the sensitivi
ty can be
increased t nangrams/mL. 8,9 Fr this type f reactin, sma atex partices
with a diameter
f ess than 1 m are used. One such type f instrumentatin system is caed a PA
CIA. A
Antibdy-cated patient ces
Antihuman gbuin (IgG) Aggutinatin + B Antibd
y-cated ces
Antihuman gbuin (IgG) Aggutinatin + Reagent red ces Patient antibdy Anti
bdy-cated
ces + FIGURE 96. Direct and indirect antigbuin tests. (A) Direct antigbui
n test (DAT).
Antihuman gbuin is cmbined with patient ces that have becme cated with a
ntibdy in viv.
(B) Indirect antigbuin test (IAT). Reagent ces are reacted with patient ant
ibdy. These are
washed, and then antihuman gbuin is added t enhance aggutinatin. 1814_Ch09
_137-151.qxd
7/10/09 2:53 PM Page 143 144 SECTION 2 Basic Immungica Prcedures Partice

-cunting
immunassay (PACIA) invves measuring the number f residua nnaggutinating
parti- ces in a
specimen. These are cunted by means f a aser beam in an ptica partice cun
ter simiar t
ne that is designed t cunt bd ces. Nephemetric methds are used t mea
sure frward
ight scatter. Very arge and very sma partices are excuded by setting certa
in threshds n
the instrument. Latex partices are cated with whe antibdy mecues r with
F(ab) 2
fragments. Use f the atter reduces interfer- ence and nnspecific aggutinati
n. If antigen is
present, cmpexes wi frm and wi be screened ut by the cunter because f
their arge size.
An inverse reatinship exists between the number f unaggutinated partices c
unted and the
amunt f un nwn in the patient specimen. Measurements are made by  in
g at the rate at
which the number f unaggutinated partices decrease, caed a rate assay, r t
he tta number
f unaggutinated partices eft at the end, nwn as an end-pint assay. 9 PACI
As have been used
t measure severa serum prteins, therapeutic drugs, tumr mar ers, and vira a
ntigens, such as
thse assciated with meases, herpes simpex, and hepatitis B surface anti- gen
. 8 Partice
immunassays are apprximatey three rders f magnitude mre sensitive than sta
ndard manua
agguti- natin methds. 8 One disadvantage, hwever, is the need t invest in e
xpensive
equipment. QUALITY CONTROL AND QUALITY ASSURANCE Athugh aggutinatin reactin
s are simpe t
perfrm, interpretatin must be carefuy dne. Techniques must be standardized
as t
cncentratin f antigen, incubatin time, temperature, diuent, and the methd
f reading. The
pssibiity f crss-reactivity and interfering antibdy shud aways be cnsid
ered.
Crss-reactivity is caused by the presence f antigenic determinants that resemb
e ne anther s
csey that antibdy frmed against ne wi react with the ther. Mst crss-r
eactivity can be
avided thrugh the use f mncna antibdy directed against an antigenic det
erminant that is
unique t a particuar antigen. Heterphie antibdy and rheumatid factr
are tw
interfering antibdies that may prduce a fase-psitive resut. Heterphie ant
ibdies (see
Chapter 3) are mst ften a cn- sideratin when red bd ces are used
as the carrier
partice. Patients may have an antibdy that is capabe f reacting with an anti
gen n the red
bd ce ther than the antigen that is being tested fr. Such crss-reactins
can be
cntred by preadsrptin f test serum n red bd ces withut the test an
tigen r by
treating red bd ces t remve pssibe interfering antigens. Rheumatid fac
tr, as mentined
previusy, wi react with any IgG present and is especiay a prbem in rever

se passive
aggutinatin tests. If this is suspected, sampes can be treated with prnase 
r the reducing
agent 2-mercaptethan t reduce fase- psitive resuts due t the IgM rhe
umatid factr. 8
The use f psitive and negative cntr sera is essentia in aggutinatin test
ing, but it
cannt rue ut a fase-psitive reactins. Other cnsideratins incude prpe
r strage f
reagents and cse attentin t expiratin dates. Reagents shud never be used
beynd the
expiratin date. Each new t shud be eva- uated befre use, and the manufact
urers
instructins fr each it shud aways be fwed. The sensitivity and speci cit
y f different
its may vary and thus must be ta en int accunt. Advantages f aggutinatin r
eactins incude
rapidity; reative sensitivity; and the fact that if the sampe cntains a micr
rganism, it des
nt need t be viabe. In additin, mst tests are simpe t perfrm and require
n expensive
equipment. Test are cnducted n cards, tubes, and micrtiter pates, a
f which are
extremey prtabe. A wide variety f antigens and antibdies can be tested fr
in this manner.
It must be ept in mind, hwever, that aggutinatin tests are screening ts 
ny and that a
negative resut des nt rue ut presence f the disease r the antigen. The qu
antity f antigen
r antibdy may be bew the sensitiv- ity f the test system. Refer t T
abe 91 fr a
ist f fase-psitive and fase-negative reactins. Whie the num- ber f agg
utinatin tests
have decreased in recent years, they cntinue t pay an imprtant re in the i
denti catin f
rare pathgens such as Francisea and Brucea and mre cmmn rganisms such a
s rtavirus and
Cryptcccus, fr which ther testing is cmpex r unavaiabe. 8 1814_Ch09_137
-151.qxd 7/10/09
2:53 PM Page 144 CHAPTER 9 Aggutinatin 145 Tabe 9-1. Causes f Fase-Psiti
ve and
Fase-Negative Reactins in Aggutinatin Testing CAUSE CORRECTION Fase-Psit
ive Reactins
Overcentrifugatin: Buttn is pac ed t tight and is Reguate centrifuge t pr
per speed and
time. dif cut t resuspend. Cntaminated gassware, sides, r reagents: Cntamin
ants Keep
suppies cvered in strage and hande with care. (dust, dirt, r ngerprints) may
cause ces t
cump. Autaggutinatin: Test ces cump withut speci c antibdy Use a cntr
with saine
and n antibdy present. If the cntr is present; mainy a prbem with red ce
s. psitive,
test is invaidated. Saine stred in gass bttes: Cida siica may each
ut Stre saine
in pastic cntainers. and cause aggutinatin. Presence f crss-reactivity. Us
e puri ed antigen
preparatins and speci c mncna antibdy whenever pssibe. Presence f rheuma
tid factr.
Test speci cay fr rheumatid factr t rue ut its presence. If rheumatid fac

tr is present,
aggutinatin resuts must be interpreted carefuy. Presence f heterphie ant
ibdy: This
ccurs mainy when Preabsrb serum with red bd ces withut speci c antigen,
r red bd
ces are used as the carrier partice. pretreat red bd ces t remve ther
antigens. Deay
in reading a side test: Dried ut antigen may  Fw directins and read
reactins
immediatey after incubatin. i e aggutinatin. Fase-Negative Reactins Under
centrifugatin:
If sampe is undercentrifuged, ces Reguate centrifuge t prper speed and ti
me. may nt be
cse enugh t interact. Inadequate washing f ces, especiay in antigbui
n Wash ces
thrughy, accrding t the prcedure being fwed. Use testing: Unbund immu
ngbuins may
neutraize the cntr ces n negative reactins. antihuman gbuin. Reagent
s nt active:
This may be caused by imprper strage. Refrigerate antisera, but d nt freeze
because ss f
activity may ccur. Deays in testing prcedures: This especiay pertains t O
nce a prcedure
is begun, fw thrugh t the end withut deay. antigbuin testing. Antibd
y may be euted
frm red bd ces. Incrrect incubatin temperature: T w a temperature C
hec temperature
at which test is carried ut. may resut in the ac f assciatin f antigen a
nd antibdy.
Insuf cient incubatin time: Antigen and antibdy may Fw instructins carefu
y. nt have
time fr assciatin. Przne phenmenn: T much patient antibdy fr If this
is suspected,
diute antibdy and repeat the test. amunt f test. Faiure t add antigbuin
reagent: This
ccurs mainy Add chec ces that are antibdy-cated t see if aggutinatin
ccurs in direct
and indirect antigbuin testing. after a negative test. If there is sti n a
ggutinatin,
disregard the resuts and repeat. 1814_Ch09_137-151.qxd 7/10/09 2:53 PM Page
145 146 SECTION 2
Basic Immungica Prcedures SUMMARY Aggutinatin, first bserved in 1896 whe
n antibdy was
fund t react with bacteria ces, is a versatie technique that is simpe t
perfrm. The
prcess f aggutinatin can be divided int tw steps: (1) sensitizatin r ini
tia binding,
which depends n the nature f the antibdy and the antigen- bearing surface, an
d (2) attice
frmatin, which is gverned by such factrs as pH, inic strength, and temperat
ure. Decreasing
the inic strength f the miieu, running the reactin at a pH between 6.7
and 7.2, and
chsing the crrect temperature fr the particuar immungbuin type can a
enhance attice
frmatin. Many f these manipu- atins are necessary t vercme the fac
t that many
partices in sutin are charged, and i e charges tend t repe ne anther. D
ue t its arger
size, IgM is usuay abe t effect attice frmatin withut additina enhance

ment, whie fr

IgG, such measures are necessary t see a visibe reactin. Aggutinatin reacti
ns can be
divided int severa dis- tinct categries: (1) direct, (2) passive, (3) reverse
passive, (4)
aggutinatin inhibitin, and (5) caggutinatin. In direct aggutinatin, anti
gens are fund
naturay n the indicatr partice, but in passive aggutinatin, antigens are
arti ciay
attached t such a partice. Reverse passive aggu- tinatin is s caed becaus
e antibdy is
attached t the indicatr partice. Aggutinatin inhibitin is based n cm- pe
titin between
antigen-cated partices and sube patient antigen fr a imited number f ant
ibdy sites. This
is the ny instance in which aggutinatin represents a negative test. Caggut
inatin uses
bacteria as the carrier partice t which antibdy is attached. Cmbs tests inv
ve the use f
antihuman gbuin t enhance aggutinatin. Tw distinct techniques, direct and
indirect
testing, are frequenty used in the bd ban , and each has a speci c purpse. Th
e direct
Cmbs test is used t detect in viv cating f patient ces with antigen. The
indirect
Cmbs test ampifies in vitr antigenantibdy cmbinatin. Typicay, it is emp
yed t detect
unexpected patient antibdy t red bd ces intended fr a transfusin. Sever
a autmated
aggutinatin techniques have been empyed in the cinica abratry t increa
se the sensitivity f aggutinatin reactins. PACIA  s at residua nnaggutinating
partices by
means f nephemetry. As aggutinatin ccurs, cumps f antigens increase in s
ize, and these
arge cumps are nt cunted. The amunt f un nwn in a patient specimen is the
refre indirecty
pr- prtina t the number f unaggutinated partices, because an increase in
un nwn resuts
in a decrease in partices that d nt aggutinate. Quaity cntr is an imprt
ant aspect f the
perfrmance f aggutinatin reactins. Cncentratin f antigen, incu- batin t
ime, temperature,
diuent used, and the methd f reading shud be standardized fr each particu
ar test dne.
This heps t cut dwn n the ccurrence f fase-psitive and fase-negative re
suts.
Aggutinatin reactins are typi- cay used as screening tests; they are fast a
nd sensitive and
can yied vauabe infrmatin when interpreted crrecty. 1814_Ch09_137-151.qxd
7/10/09 2:53
PM Page 146 CHAPTER 9 Aggutinatin 147 A 25-year-d femae wh was 2 mnths p
regnant went t
her physician fr a prenata wr up. She had been vacci- nated against rubea,
but her titer was
never estabished. She was cncerned because a friend f hers wh had never been
vaccinated fr
rubea thught she might have the disease. The patient had previusy been n a
n a-day
shpping trip with her friend. The physician rdered a rubea test as a part f

the prenata
wr up. The resuts n an undiuted serum specimen were psitive. Questins a. W
hat des the
psitive rubea test indicate? b. Hw shud this be interpreted in the ight 
f the patients
cnditin? c. Is a semiquantitative test indicated? CASE STUDY 1814_Ch09_137-151
.qxd 7/10/09
2:53 PM Page 147 148 SECTION 2 Basic Immungica Prcedures LATEX AGGLUTINATI
ON SLIDE TEST FOR
RUBELLA Principe Rubea virus is the etigic agent f German meases. It gen
eray causes a
mid vira infectin with a sight rash. Hwever, it is recmmended that a wm
en f chidbearing age be tested fr the presence f rubea antibdies t determine their immu
ne status,
because vira infectin dur- ing the rst trimester f pregnancy can resut in bir
th defects r
stibirth. Manua card tests used t determine the pres- ence f antibdies t
rubea are based
n the principe f passive aggutinatin. Latex partices that are cated with
subiized
rubea virus aw a visibe bservatin f the antigenantibdy reactin. When p
atient serum is
mixed with the atex reagent, a visibe aggutinatin reactin wi resut if sp
eci c antibdy is
present. Specimen Cectin Cect bd asepticay by venipuncture int a c
ean, dry, sterie
tube and aw it t ct. Separate the serum withut transferring any ceua
r eements. D
nt use grssy hemyzed, excessivey ipemic, r bacteriay cntaminated sp
ecimens. Fresh
nnheat inactivated serum is recm- mended fr the test. Hwever, if the t
est cannt be
perfrmed immediatey, serum may be stred between 2C and 8C fr up t 48 hurs. I
f there is
any additina deay, freeze the serum at 20C. Fr diagnsis f current r re
cent rubea
infectin, paired sera (acute and cnvaescent) shud be btained. The acute s
era shud be
cected as sn after rash nset as pssibe r at the time f expsure. Cnva
escent sera
shud be btained 7 t 21 days after the nset f the rash r at east 30 days
after expsure if
n cinica symptms appear. Test acute and cnvaescent sera simutaneusy usi
ng the
semiquantitative prcedure. Reagents, Materias, and Equipment Kit cntaining th
e fwing:
Latex antigen Diutin buffer High reactive cntr Lw reactive cntr Negativ
e cntr Pastic
stirrers Dispsabe sides Materias required but nt prvided: A pipette r pip
ettes capabe f
prviding 10, 20, 35, and 50 L vumes. Timer Precautins Latex reagent cntr
s and buffer
cntain 0.1 percent sdium azide as a preservative. Sdium azide may react wit
h ead and cpper
pumbing t frm highy expsive meta azides. On dispsa, fush with a arge
vume f water
t prevent azide buidup. The virus strain used in the preparatin f th
e atex reagent
has been previusy inactivated. Hwever, it is rec- mmended that users fw

the same safety

precautins in effect fr the handing f ther types f ptentiay infec- tiu
s materia. Each
dnr unit used in the preparatin f cntr mate- ria was tested by an FDA-ap
prved methd fr
the presence f antibdies t HIV, HBV surface antigen, and HCV and was fund t
be negative.
Hwever, the cntrs shud be handed as recmmended fr any ptentiay
infectius human
serum r bd specimen. Prcedure* Quaitative Side Test 1. Aw reagents, c
ntrs, and
specimens t reach rm temperature. 2. Labe test side fr each sampe and cn
tr t be
tested. Avid tuching surface inside the circes. 3. Pipette 10 t 25 L f samp
e r cntr
(depending upn the particuar it) int the apprpriate circe. 4. Using a new
pastic stirrer
fr each circe, spread the serum t  the entire circe. 5. Resuspend the atex
reagent by
genty mixing the via unti the suspensin is hmgeneus. Using the drp- per
prvided, hd
the drpper perpendicuar t the side, and pace ne drp f atex reagent nt
each circe cntaining the serum r cntr. 6. Rtate the side bac and frth in a gure-eight
pattern, swy
and eveny, fr 2 minutes. Aternatey, pace the card n a rtatr under a mis
tened humidifying
cver fr the time speci ed in the it. 7. Immediatey fwing rtatin, pace t
he side n a
at surface and bserve fr aggutinatin using a direct ight surce. * This is a
generaized
prcedure that is used in its such as the RUBAscan, Sure-Vue Rubea, Reme C
r Side Rubea,
and thers. EXERCISE 1814_Ch09_137-151.qxd 7/10/09 2:53 PM Page 148 CHAPTER 9
Aggutinatin
149 Semiquantitative Side Test If a psitive reactin is btained, the specimen
may be seriay diuted with diuent t btain a semiquantitative estimate f the rubea a
ntibdy eve. 1.
Labe the test side using twfd seria diutins (e.g., 1:2, 1:4, 1:8) fr th
e high psitive
cntr and fr each psitive serum sampe t be tested. The w psitive cn- t
r and negative
cntr are nt subject t seria twfd diutins. 2. Prepare diutins accrd
ing t
instructins fr the par- ticuar it. 3. Using a separate pastic stirrer fr e
ach set f
sampes r cntrs, mix each specimen r cntr unti the entire area f each
va is ed.
Start at the highest diutin f each sampe r cntr, and prceed t the next
wer diutin
with the same stirrer unti a circes are spread. 4. Resuspend the atex reage
nt by genty
mixing the via unti the suspensin is hmgeneus. Using the drp- per prvide
d, hd it
perpendicuar t the side and pace ne drp f atex reagent nt each circe.
5. Rtate the
side bac and frth in a gure-eight pattern, swy and eveny, fr 2 minutes. A
ternatey,
pace the card n a rtatr under a mistened humidifying cver fr the time spe

ci ed in the it.
6. Immediatey fwing rtatin, pace the side n a at surface and bserve f
r aggutinatin
using a direct ight surce. Resuts The presence f any visibe aggutinatin s
igni canty different frm the negative cntr indicates the presence f antibdies against ru
bea virus in
the serum sampe. Bth the w and high psitive cntrs shud shw aggutinatin different
frm the unifrm appearance f the negative cntr. The negative cntr shud
shw n
aggutina- tin. In the semiquantitative test, the rubea titer wi c
rrespnd t the
highest serum diutin that prduces visibe aggutinatin. The high psitive c
ntr shud give
a titer within ne dubing diutin f that indicated n the abe. Interpretat
in f Resuts
Undiuted serum wi give a psitive reactin if at east 10  1 IU/mL f
antibdy is
present. This indicates that the individua has immunity t rubea. When a semi
quantitative test
is perfrmed with acute and cnvaescent sera frm the same patient, a furfd
increase in titer
is cnsidered t be signi cant. This typicay indicates infectin. Sme individuas previusy
expsed t rubea may demnstrate a rise in antibdy titer. Sercnversin can
as be seen
after a vaccinatin prcedure. A test resuts must be evauated by a physician
in ight f the
cinica symptms shwn by the patient. 1814_Ch09_137-151.qxd 7/10/09 2:53 PM
Page 149 150
SECTION 2 Basic Immungica Prcedures 1. Which f the fwing best describe
s aggutinatin?
a. A cmbinatin f sube antigen with sube antibdy b. A cmbinatin f pa
rticuate antigen
with sube antibdy c. A reactin that prduces n visibe end pint d. A reac
tin that
requires instrumentatin t read 2. A f the fwing cud be used t enhanc
e an aggutinatin reactin except a. increasing the viscsity f the medium. b. using ab
umin. c.
increasing the inic strength f the medium. d. centrifugatin. 3. Aggutinatin
f dyed
bacteria ces represents which type f reactin? a. Direct aggutinatin b. Pa
ssive
aggutinatin c. Reverse passive aggutinatin d. Aggutinatin inhibitin 4. In
which f the
fwing circumstances wud the indi- rect Cmbs test be empyed? a. Identi cat
in f the
ABO bd grups b. Identi catin f cd-reacting antibdy c. Identi catin f an u
nexpected IgG
antibdy d. Identi catin f hemytic disease f the newbrn 5. In an aggutinati
n reactin, if
ces are nt centrifuged ng enugh, which f the fwing might ccur? a. Fa
se-negative
resut b. Fase-psitive resut c. N effect d. Sight effect but can be ignred
6. Aggutinatin
inhibitin cud best be used fr which f the fwing types f antigens? a. L
arge ceuar
antigens such as erythrcytes b. Sube haptens c. Bacteria ces d. Antigen a

ttached t atex
partices 7. Which f the fwing crrecty describes reverse pas- sive aggut
inatin? a. It is
a negative test. b. It can be used t detect autantibdies. c. It is used fr i
denti catin f
bacteria antigens. d. It is used t detect sensitizatin f red bd ces. 8.
In which f the
fwing tests is patient antigen deter- mined by measuring the number f nnag
gutinating
partices eft after the reactin has ta en pace? a. Direct aggutinatin b. C
aggutinatin c.
PACIA d. Cmbs testing 9. Reactins invving IgG may need t be enhanced fr wh
ich reasn? a.
It is ny active at 25C. b. It may be t sma t prduce attice frmatin. c.
It has ny
ne antigen-binding site. d. It is nt abe t prduce visibe in vitr aggutin
atin. 10. Fr
which f the fwing tests is a ac f aggutina- tin a psitive reactin? a
.
Hemaggutinatin b. Passive aggutinatin c. Reverse passive aggutinatin d. Ag
gutinatin
inhibitin 11. A f the fwing wud be cnsidered gd quaity- cntr pr
cedures except
a. string reagents prpery. b. standardizing temperature f reactins. c. a
wing the reactin
t g as ng as pssibe. d. using mncna antibdy t avid crss-reactivit
y. 12. A psitive
direct Cmbs test cud ccur under which circumstances? a. Hemytic disease f
the newbrn b.
Autimmune hemytic anemia c. Antibdies t drugs that bind t red ces d. Any
f the abve
REVIEW QUESTIONS 1814_Ch09_137-151.qxd 7/10/09 2:53 PM Page 150 CHAPTER 9 Agg
utinatin 151
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infectins. In Detric , B, Hamitn, RG, and Fds, JD (eds): Manua f Mecua
r and Cinica
Labratry Immungy, ed. 7. ASM Press, Washingtn, DC, 2006, pp. 569605. 2
5. Landry, ML.
Rapid vira diagnsis. In Detric , B, Hamitn, RG, and Fds, JD (eds): Manua
f Mecuar and
Cinica Labratry Immungy, ed. 7. ASM Press, Washingtn, DC, 2006, pp. 61061
6.
1814_Ch09_137-151.qxd 7/10/09 2:53 PM Page 151 10 Labeed Immunassays LEARNI
NG OBJECTIVES
After nishing this chapter, the reader wi be abe t: 1. Describe the differenc
e between
cmpetitive and nncmpetitive immunassays. 2. Identify characteristics that an
antibdy must
have t be used fr immunassay. 3. Expain hw the principe f cmpetitive bin
ding is used in
radiim- munassays. 4. Distinguish between hetergeneus and hmgeneus enzyme
immunassay. 5.
Expain the principe f sandwich r capture immunassays. 6. Describe uses fr
rapid
immunassays. 7. Describe appicatins fr hmgeneus enzyme immunassay. 8. C
mpare and
cntrast enzyme immunassay and radiimmunassay regarding ease f perfrmance,
shef ife,
sensitivity, and cinica appicatin. 9. Describe the difference between direct
and indirect
immun urescence techniques. 10. Reate the principe f urescence parizatin i
mmunassay.
11. Discuss advantages and disadvantages f each type f immunassay. 12. Chse
an apprpriate
immunassay fr a particuar anayte. KEY TERMS Anayte Capture assay Chemiumin
escence
Cmpetitive immunassay Direct immun ures- cent assay Enzyme-in ed immunsrben
t assay (ELISA)
Furescence pariza- tin immunassay (FPIA) Hetergeneus enzyme immunassay
Hmgeneus
enzyme immunassay Immun urescent assay (IFA) Indirect immun ures- cent assay N
ncmpetitive
immunassay Radiimmunassay (RIA) Sandwich immunassays 152 1814_Ch10_152-165.q
xd 7/13/09 2:55
PM Page 152 CHAPTER 10 Labeed Immunassays 153 The need t devep rapid, spec
ific, and
sensitive assays t determine the presence f imprtant bigicay active me
cues ushered in

a new era f testing in the cinica abratry. Labeed immunassays are design
ed fr anti- gens
and antibdies that may be sma in size r present in very w cncentratins.
The presence f
such antigens r antibdies is determined indirecty by using a abeed reac- ta
nt t detect
whether r nt specific binding has ta en pace. The substance t be measured is
nwn as the
anayte. Exampes incude bacteria antigens, hrmnes, drugs, tumr mar ers, sp
ecific
immungbuins, and many ther substances. Anaytes are bund by mecues that
react
specificay with them. Typicay, this is antibdy. One reac- tant, either the
antigen r the
antibdy, is abeed with a mar er s that the amunt f binding can be mnitre
d. Labeed
immunassays have made pssibe rapid quanti- tative measurement f many imprta
nt entities such
as vira antigens in patients infected with HIV. This abiity t detect
very sma
quantities f antigen r antibdy has rev- utinized the diagnsis and mnitr
ing f numerus
diseases and has ed t mre prmpt treatment fr many such cnditins. CHARACTE
RISTICS OF
LABELED ASSAYS Cmpetitive versus Nncmpetitive Assays Current techniques incu
de the use f
urescent, radiac- tive, chemiuminescent, and enzyme abes. The underying pri
ncipes f a
these techniques are essentiay the same. There are tw majr frmats fr a 
abeed assays:
cmpet- itive and nncmpetitive. In a cmpetitive immunassay, a the reactant
s are mixed
tgether simutaneusy, and abeed antigen cmpetes with unabeed patient ant
igen fr a
imited number f antibdy-binding sites. The amunt f bund abe is inversey
prprtina t
the cncentratin f the abeed antigen. This means that the mre abe
 detected, the
ess there is f patient antigen. In a typica nncmpetitive immunassay, antib
dy, ften caed
capture antibdy, is rst passivey absrbed t a sid phase. Un nwn patient ant
igen is then
awed t react with and be captured by the antibdy. After washing t remve u
nbund antigen, a
secnd antibdy with a abe is added t the reactin. In this case, the amunt
f abe measured
is directy prprtina t the amunt f patient antigen. In bth types f assa
ys, the abe
must nt ater the reac- tivity f the mecue, and it shud remain stabe fr
the shef ife
f the reagent. Radiactivity, enzymes, urescent cmpunds, and chemiuminescent
substances
have a been used as abes. Each f these is discussed in a ater sectin. Ant
ibdies In any
assay, it is essentia fr the antibdy used t have a high affinity fr the ant
igen in questin.
As discussed in Chapter 8, af nity is the strength f the primary interactin betw
een a singe
antibdy-cmbining site and an antigenic determinant r epitpe. In cmpetitive
binding assays,

there is randm interactin between individua antigen and anti- bdy mecues
. Therefre,
the higher the affinity f antibdy fr antigen, the arger the amunt
f antigen
bund t antibdy and the mre accuratey specific bind- ing can be measured.
The utimate
sensitivity f the immunassay, in fact, depends argey n the magnitude f a
f nity. 1,2 The
antibdy used shud as be very specific fr the antigen invved in
the reactin.
The discvery f CHAPTER OUTLINE CHARACTERISTICS OF LABELED ASSAYS Cmpetitive
versus
Nncmpetitive Assays Antibdies Standards r Caibratrs Separatin Methds Det
ectin f the
Labe Quaity Cntr RADIOIMMUNOASSAY Cmpetitive Binding Assays Advantages and
Disadvantages f
Radiimmunassay ENZYME IMMUNOASSAY Hetergeneus Enzyme Immunassay Rapid Immun
assays
Hmgeneus Enzyme Immunassay Advantages and Disadvantages f Enzyme Immunassa
y FLUORESCENT
IMMUNOASSAY Direct Immun urescent Assays Indirect Immun urescent Assays Other F
urescent
Immunassays Advantages and Disadvantages f Furescent Immunassay CHEMILUMINE
SCENT
IMMUNOASSAYS Principe f Chemiuminescence Advantages and Disadvantages f Chem
iuminescent
Immunassays SUMMARY CASE STUDY EXERCISE: ENZYME IMMUNOASSAY DETERMINATION OF HU
MAN CHORIONIC
GONADOTROPIN REVIEW QUESTIONS REFERENCES 1814_Ch10_152-165.qxd 7/13/09 2:55 PM
Page 153 154
SECTION 2 Basic Immungica Prcedures mncna antibdies has made avaiab
e a cnstant
surce f highy speci c antibdy that has increased the abiity t detect sma a
munts f
anayte with great accuracy. 3 Standards r Caibratrs Standards, as nwn as
caibratrs, are
unabeed anaytes that are made up in nwn cncentratins f the substance t
be measured. They
are used t estabish a reatinship between the abeed anayte measured and an
y unabeed
anayte that might be present in patient specimens. 4 Differing amunts
f standards
are added t antibdy antigen mixtures t ascertain their effect n binding f
the abeed
reagent. Mst instruments then extrapate this infrmatin and d a best-fi
t curve (ne
that is nt absutey inear) t determine the cncentratin f the un nwn a
nayte.
Separatin Methds In mst assays, nce the reactin between antigen and anti- b
dy has ta en
pace, there must be a partitining step, r a way f separating reacted fr
m unreacted
anayte. Currenty, mst immunassays use a sid-phase vehice fr separatin.
Numerus
materias, such as pystyrene test tubes, micrtiter pates, gass r pystyre
ne beads, magnetic beads, and ceuse membranes, have been used fr this purpse. Antigen 
r antibdy is
attached by physica adsrptin, and when speci c binding ta es pace, cmpexes r
emain attached

t the sid phase. This prvides a simpe way t separate bund and free reacta
nts. If a
separatin step is empyed in an assay, the ef ciency f the separatin is critic
a t the
accuracy f the resuts. The bund and unbund fractins are usuay separated b
y physica means,
incuding decanting, centrifugatin, r - tratin. This is fwed by a washing
step t remve
any remaining unbund anayte. Great care must be ta en t perfrm this crrect
y, because
incmpete washing eads t incmpete remva f the abeed anayte and inaccu
rate resuts. 4
Detectin f the Labe The ast step cmmn t a immunassays is detectin f
the abeed
anayte. Fr radiimmunassays, this invves a system fr cunting radiactivit
y, whie fr
ther abes such as enzymes, urescence, r chemiuminescence, typicay a chang
e in absrbance
in a substrate is measured by spec- trphtmetry. A systems must use str
ingent quaity
cntrs. Quaity Cntr When measuring anaytes that are present in very imit
ed quantities, it
is essentia t estabish quaity-cntr prce- dures. Because the ga f test
ing is t
determine whether patient eves are increased r decreased ver nrma va- ues
, test
perfrmance must be mnitred t imit randm errrs. One means f ding this is
t run a ban
tube, usu- ay phsphate-buffered saine, with every test. This is nt expected
t have any
detectabe abe but serves as a chec fr nnspecific adsrptin and fr i
nadequate
washing between steps. Any readings indicative f abe in the ban are nwn a
s bac grund. If
the bac grund is t high, wash steps need t be made mre ef cient. 4 A negative
cntr and a
high and a w psitive cntr shud be run in additin. This serves as a chec
n the quaity f the reagents t ma e sure the abe is readiy detectabe under current t
esting
cnditins. A cntrs and the patient sampe are usuay run in dupicate. If
any cntrs are
ut f range, test vaues shud nt be reprted. Autmated pr- cedures have cu
t dwn n many
perfrmance variabes. Individua testing prcedures are nw cnsidered in the f
wing
sectins. RADIOIMMUNOASSAY Cmpetitive Binding Assays The first type f immunas
say deveped was
radiim- munassay (RIA), pineered by Yaw and Bersn in the ate 1950
s. It was used
t determine the eve f insuinanti-insuin cmpexes in diabetic pati
ents. 5,6 A
radiactive substance is used as a abe. Radiactive eements have nucei that
decay
spntaneusy, emitting matter and energy. Severa radiactive abes, incuding
131 I; 125 I;
and tritiated hydrgen, r 3 H, have been used, but 125 I is the mst ppuar. 5
It has a
haf-ife f 60 days, and because it has a higher cunting rate than that f 3 H
, the tta

cunting time is ess. It is easiy incrprated int prtein mecues, and it

emits gamma
radiatin, which is detected by a gamma cunter. Very w quantities f radiact
ivity can be
easiy measured. 2 RIA was riginay based n the principe f cmpetitive bind
ing. Thus, the
anayte being detected cmpetes with a radiabeed anayte fr a imited number
f binding sites
n a high-af nity antibdy. 1,5 The cncentratin f the radiac- tive anayte is
in excess, s
a binding sites n antibdy wi be ccupied. If patient antigen is present, s
me f the
binding sites wi be ed with unabeed anayte, thus decreasing the amunt f
bund
radiactive abe (Fig. 101). When bund and free radiabeed antigens are separ
ated and a
washing step has ccurred, the amunt f abe in the bund phase is indirecty
prprtina t
the amunt f patient antigen present. This can be iustrated by the fwing
equatin: 6Ag*
2Ag
4Ab 3Ag*Ab
1AgAb
3Ag* 1Ag In this exampe, abeed and unabeed antigens cc
ur in a
3:1 rati. Binding t a imited number f antibdy sites wi ta e pace in the
same rati. Thus,
n the right side f the equatin, three f the fur binding sites are ccupied
by abeed
antigen, whie ne site is fied by unabeed 1814_Ch10_152-165.qxd 7/1
3/09 2:55 PM
Page 154 CHAPTER 10 Labeed Immunassays 155 antigen. As the amunt f patient a
ntigen increases,
fewer binding sites wi be ccupied by abeed antigen, as demn- strated by th
e next equatin:
6Ag* 18Ag
4Ab 1Ag*Ab
3AgAb
5Ag* 15Ag In this case, the rati f abeed t unabe
ed
antigen is 1:3. Binding t antibdy sites ta es pace in the same rati, and the
amunt f bund
abe is greaty decreased in cm- parisn t the first equatin. In this type 
f RIA, use f a
cnstant amunt f radiabeed antigen with standards f nwn cncentratin wi
 resut in a
standard curve that can be used t extrapate the cncentratin f the un nwn
patient antigen.
5 The detectin imits f cmpetitive assays are argey determined by the af nity
f the
antibdy. 2 These imits have been cacuated t be as w as 10 fm/L, r 600,
000 mecues in
a sampe vume f 100 L. 1 Advantages and Disadvantages f Radiimmunassay Exam
pes f
substances that are measured by RIA incude thyrid-stimuating hrmne and tta
 serum IgE. 2,7
RIA is an extremey sensitive and precise technique fr determin- ing trace amu
nts f anaytes
that are sma in size. Hwever, chief amng the disadvantages f a RIA techniques is the
heath hazard invved in wr ing with radiactive substances. Labratries
have fund it
mre and mre difficut and expensive t maintain a icense and t cmpy with f
edera
reguatins. In additin, dispsa prb- ems, shrt shef ife, and the need f
r expensive

equipment has caused abratrians t expre ther techniques fr identi

fying anaytes
in w cncentratin. 2 Enzyme immunassays have argey repaced RIA because
f their
cmparabe sensitivity and the avaiabiity f autmated instrumentatin that a
ws fr
prcessing f a arge number f sampes in ess time. 3 ENZYME IMMUNOASSAY Enzym
es are naturay
ccurring mecues that catayze certain bichemica reactins. They react with
suitabe substrates t prduce brea dwn prducts that may be chrmgenic, furgeni
c, r
uminescent. Sme type f spectrscpy can then be used t measure the c
hanges invved.
As abes fr immunassay, they are cheap and readiy avaiabe, have a ng she
f ife, are
easiy adapted t autmatin, and cause changes that can be measured using inexp
ensive equipment.
2 Sensitivity can be achieved with- ut dispsa prbems r the heath hazards
f radiatin.
Because ne mecue f enzyme can generate many me- cues f prduct, itte
reagent is
necessary t prduce high sensitivity. 1,3 Enzyme abes can either be used qua
itativey t
determine the presence f an antigen r antibdy r quantitativey t determine
the actua
cncentratin f an anayte in an un nwn specimen. Assays based n the use f e
nzymes can be
fund in such diverse settings as cinica abratries, dctrs f ces, and at-hm
e testing.
Enzymes used as abes fr immunassay are typicay chsen accrding t the num
ber f substrate
mecues cn- verted per mecue f enzyme, ease and speed f detectin, and s
tabiity. 2 In
additin, avaiabiity and cst f enzyme and substrate pay a re in the chic
e f a particuar
enzyme as reagent. Typica enzymes that have been used as abes in crimetric
reactins
incude hrseradish perxidase, gucse-6-phsphate dehydrgenase, a aine phs
phatase, and
-D-galactosias. 1,4 Alkalin phosphatas an hors- raish proxias hav th
highst
turnovr (convrsion of su
strat) rats, high snsitivity, an ar asy to t
ct, so thy ar
most oftn us in such assays. 2 Enym assays ar classi  as ithr htrogn
ous or
homognous on th
asis of whthr a sparation stp is nc- ssary. Htrogn
ous nym
immunoassays rquir a stp to physically sparat fr from
oun analyt. In h
omo- gnous
immunoassays, on th othr han, no sparation stp is ncssary,
caus nym
activity
iminishs whn
ining of anti
oy an antign occurs. Th principls unr- ly
ing ach of ths
typs of assays ar iscuss nxt. Htrognous Enym Immunoassay Comptitiv
EIA Th first
nym immunoassays (EIAs) wr comptitiv assays
as on th principls of RI
A. Enym-la
l
anti- gn compts with unla
l patint antign for a limit num
r of
inin
g sits on

anti
oy molculs that ar attach to a soli phas. Aftr carfully washing t
o rmov
Soli-phas anti
oy Spcific
ining La
l an unla
l antign Wash Count
Spcific
ining
(lowr raioactivity) Soli-phas anti
oy La
l an unla
l antign Wash
Count A B FIGURE
101. Principl of RIA. La
l antign compts with patint antign for a limit
 num
r of

ining sits on soli-phas anti
oy. (A) Vry littl patint antign is prsn
t, making
raioac- tivity of th soli phas high. (B) Mor patint antign is prsnt, an
 th
raioactivity of th soli phas is ruc in proportion to th amount of pati
nt antign
oun.
1814_Ch10_152-165.qx 7/13/09 2:55 PM Pag 155 156 SECTION 2 Basic Immunologi
cal Procurs
any nonspci cally
oun antign, nym activity is tr- min. Enym activity
is invrsly
proportional to th concntration of th tst su
stanc, maning that th mor p
atint antign is

oun, th lss nym-la
l antign can attach. In this mannr, a snsi
tivity of
nanograms (10 -9 g)/mL can
 achiv. 3 This mtho is typically us for mas
uring small
antigns that ar rlativly pur, such as insulin an strogn. 3 Noncomptitiv
 EIA Although
comptitiv tsts hav a high spci city, th tn- ncy in th la
oratory to
ay is towar
th us of noncomptitiv assays,
caus thy hav a highr snsitiv- ity. 1
Many such assays
ar capa
l of tcting concntrations of lss than 1 pg/mL, achiving a snsit
ivity actually
highr than most RIAs. 3 Noncomptitiv assays ar oftn rfrr to as inir
ct nym-link
immunosor
nt assays (ELISA),
caus th nym-la
l ragnt os not participat in th
initial antignanti
oy
ining raction. This typ of assay is on of th most f
rquntly us
immunoas- says in th clinical la
oratory u to its snsitivity, spci city, simp
licity, an low
cost. 1,3,8 Eithr antign or anti
oy may

oun to soli phas. A varity of
soli-phas
supports ar us, incluing microtitr plats, nitrocllulos mm-
rans, an
magntic latx

as. Whn antign is
oun to soli phas, patint srum with unknown anti
oy
is a an
givn tim to ract. Aftr a wash stp, an nym-la
l antiglo
ulin is a.
This scon
anti
oy racts with any patint anti
oy that is
oun to soli phas. If no pa
tint anti
oy is

oun to th soli phas, th scon la
l anti-
oy will not

oun. Aftr
a scon wash
stp, th nym su
strat is a. Th amount of nym la
l tct is ir
ctly proportional
to th amount of anti
oy in th spc- imn (Fig. 102). This typ of assay has

n us to
masur anti
oy pro- uction to infctious agnts that ar if cult to isolat in
th la
oratory
an has
n us for autoanti
oy tsting. 2 Viral infctions spcially ar mo
r asily

iagnos
y this mtho than
y othr typs of tsting. 1 This tchniqu rmain
s th prfrr
scrning mtho for tcting anti
oy to HIV, hpatitis A, an hpatitis C. 912
ELISA-
as
tsts ar also us to intify Epstin-Barrspci c anti
ois prouc in infctio
us
mononuclosis. 13 Captur Assays If anti
oy is
oun to th soli phas, ths
assays ar oftn
call sanwich immunoassays, or captur assays. Antigns captur in ths
assays must hav
multipl pi- tops. Excss anti
oy attach to soli phas is allow to com
i
n with th tst
sampl to captur any antign prsnt. Aftr an appropriat incu
ation prio, 
nym-la
l
anti-
oy is a. This scon anti
oy rcognis a iffrnt pitop than th
 soli-phas
anti
oy an complts th sanwich. Enymatic activity is irctly proportional t
o th amount
of antign in th tst sampl (Fig. 103). Soli-phas antign Spcific
ining
Patint
anti
oy + Tu
 is wash to rmov un
oun anti
oy Bining to patint anti
oy
Enym-la
l
anti-immunoglo
ulin + FIGURE 102. Noncomptitiv ELISA. Patint anti
oy is incu

at with
soli-phas antign. Aftr a wash stp, nym-la
l anti- immunoglo
ulin is a
. This will

in to th patint anti
oy on soli phas. A scon wash stp is prform to
rmov any
un
oun anti-immunoglo
ulin, an su
strat for th nym is a. Color vlo
pmnt is irctly
proportional to th amount of patint anti-
oy prsnt. Soli-phas anti
oy P
atint antign
Spcific
ining Enym-la
l anti
oy Color Bining to patint antign +
+
FIGURE 103. Noncomptitiv ELISA: sanwich tchniqu with soli-phas anti
oy. P
atint antign
is incu
at with soli-phas anti
oy. Aftr washing, nym-la
l immunoglo

ulin is a,
which com
ins with aitional trminant sits on th
oun patint antign. A
ftr a scon
wash stp, su
strat for th nym is a. Color vlopmnt is irctly prop
ortional to th
amount of patint antign prsnt. 1814_Ch10_152-165.qx 7/13/09 2:55 PM Pag
156 CHAPTER 10
La
l Immunoassays 157 Captur assays ar
st suit to antigns that
hav multipl
trminants, such as anti
ois, polyppti hor- mons, protins, tumor mar
krs, an
microorganisms, spcially viruss. 2 Whn us with microorganisms, th pitop
must
 uniqu
to th organism
ing tst, an it must
 prsnt in all strains of that orga
nism. Us of
monoclonal anti
ois has ma this a vry snsitiv tst systm. Rotavirus in s
tool an
rspiratory syncytial virus in rspiratory tract scrtions ar two xampls of
captur assays.
1416 In aition, rcntly vlop ELISAs hav ma it much asir to tct pa
rasits such as
Giaria lam-
lia an Cryptosporiium in th stool. 1719 Antign-tction tsts
hav also

prov usful for intifying fungi such as Asprgillus, Cania, an Cryptococc
us. 17,2022
Anothr major us of captur assays is in th masur- mnt of immunoglo
ulins,
spcially thos
of crtain classs. For instanc, th prsnc of IgM can
 spci cally tr- mi
n, thus
inicating an acut infction. Masurmnt of IgE, incluing allrgn-spcific
IgE, which
appars in minut quantitis in srum, can also
 accomplish with this syst
m. 23 (Chaptr 13
provis a mor tail iscus- sion on tction of IgE.) Whn captur assays
ar us to
masur immunoglo
ulins, th spcific immunoglo
ulin class
ing tct is act
ually acting as
antign, an th anti
oy is antihuman immunoglo
ulin. Inirct ELISA tsts ar
mor snsitiv
than thir irct countrparts,
caus all patint antign has a chanc to participat in th
raction. Howvr, thr is mor manipulation than in irct tsts,
caus thr
 ar two
incu
ations an two wash stps. Htrognous nym assays, in gnral, achiv
a sn- sitivity
similar to that of RIA. 1 In sanwich assays, captur anti
oy on soli phas mu
st hav
oth a
high af nity an a high spcificity for this tst systm to
 ffctiv.
Howvr, thr
may
 pro
lms with nonspci c protin
ining or th prsnc of anti
ois to v
arious componnts of th tsting systm. 3 If this is suspct, srum can
 prtrat to
avoi this
pro
lm. Sanwich assays ar also su
jct to th hook ffct, an unxpct fall
in th amount of
masur analyt whn an xtrmly high con- cntration is prsnt. 24 This typi
cally occurs in
antign xcss, whr th majority of
ining sits ar fill, an th rmain
r of patint
antign has no plac to
in. If this conition is suspct, srum ilutions mu
st
 ma an
thn rtst. Rapi Immunoassays Mm
ran-Bas Casstt Assays Mm
ran-
as
casstt assays
ar a rlativly nw typ of nym immunoassay. Thy ar rapi, asy to pr
- form, an giv
rprouci
l rsults. 2 Although sign primarily for point-of-car or hom t
sting, many of
ths hav
n moifi for incras snsitivity an can
 ma smiquantitat
iv for us in a
clinical la
oratory. Typically ths ar sign as singl-us, isposa
l
 assays in a
plastic cartrig. Th mm
ran is usually nitrocllulos, which is a
l to asi
ly immo
ili
protins an nuclic acis. 3 Th rapi flow through th mm
ran an its larg
surfac ara
nhanc th sp an snsitiv- ity of ELISA ractions. 1 Eithr antign or anti

oy can

coupl to th mm
ran, an th raction is ra
y looking for th prsnc of
a color
raction prouct. Som tst vics rquir th sparat aition of patint sam
pl, wash
ragnt, la
l antign or anti
oy, an th su
strat. Anothr typ of rapi a
ssay, call

immunochromatogra- phy, com
ins all th prviously mntion stps into on. Th
 analyt is
appli at on n of th strip an migrats towar th istal n, whr thr
is an a
sor
nt
pa to maintain a constant capillary flow rat. 2 Th la
ling an tction on
s ar st

twn th two ns. As th sam- pl is loa, it rconstituts th la
l an
tign or
anti
oy, an th two form a complx that migrats towar th tc- tion on.
An antign or
anti
oy immo
ili in th tction on capturs th immun complx an f
orms a color
lin for a positiv tst (Fig. 104). This may
 in th form of a plus sign. Exc
ss la
l
immunoractant migrats to th a
sor
nt pa. This typ of tst vic has
n
us to intify
microorganisms such as Strptococcus pyogns an Strptococcus agalactia an h
as
n us to
tst for prgnancy, for troponin in a hart attack, an for hpa- titis B surfac
 antign, to
nam just a fw xampls. Tst rsults ar most oftn qualitativ rathr than qu
antitativ.
Homognous Enym Immunoassay A homognous nym immunoassay is any ant
ign anti
oy
systm in which no sparation stp is ncssary. Homognous assays ar gnr
ally lss
snsitiv than htrognous assays,
ut thy ar rapi, simpl to prform, an
aapt asily to
automation. 1,2 No washing stps ar nc- ssary. Thir chif us has
n in th
 trmination
of low-molcular-wight analyts such as hormons, thrapu- tic rugs, an rug
s of a
us in

oth srum an urin. 1,24 An C T Positiv Invali Ngativ C T C T C T C T C
X 3
T
FIGURE 104. Exampl of a mm
ran casstt assay. Invrnss Mical. Us

y prmission.
On lin rprsnts th positiv control, an th scon lin inicats a positi
v tst.
1814_Ch10_152-165.qx 7/13/09 2:55 PM Pag 157 158 SECTION 2 Basic Immunologi
cal Procurs
xampl of a homognous immunoassay is th nym- multipli immunoassay tchn
iqu (EMIT)
vlop
y th Syva Corporation. Homognous assays ar
as on th principl
of chang in
nym activity as spcific antignanti
oy com
ina- tion occurs. Ragnt antign
is la
l
with an nym tag. Whn anti
oy
ins to spcific trminant sits on th ant
ign, th activ
sit on th nym is
lock, rsulting in a masura
l loss of activity. Fr
analyt (antign)
com- pts with nym-la
l analyt for a limit num
r of anti
oy-
ining
sits, so this is
a comptitiv assay. Enym activity is irctly in proportion to th concntrat
ion of patint
antign or haptn prsnt in th tst solution. A physical sparation of
oun a
n fr analyt

is thus not ncssary. Th snsitivity of homognous assays is trmin

y th
following: (1) tcta
ility of nymatic activity; (2) chang in that acti
vity whn
anti
oy
ins to antign; (3) strngth of th anti
oys
ining; an (4) suscpt
i
ility of th
assay to intrfrnc from nognous nym activ- ity, cross-racting antigns
, or nym
inhi
itors. Typically, a snsitivity of micrograms pr millilitr is rach, fa
r lss than that
achiva
l
y htrognous nym assays,
caus th ampli cation proprtis of
nyms ar not
utili. 24 This tchniqu is usually appli only to tction of small molculs that coul
not
 asily masur
y othr mans. Othr consirations inclu th fact tha
t only crtain
nyms ar inhi
it in this mannr. Enymatic activity may
 altr
y stri
c xclusion of
th su
strat, or thr may also
 changs in th conformation structur
of th nym,
spcially in th rgion of th activ sit. Two nyms that ar fr
quntly us
in this typ of assay ar malat hyrognas an glucos-6-phosphat hyr
ognas. 2
Avantags an Disavantags of Enym Immunoassay Enym immunoassays hav achi
v a
snsitivity similar to that of RIA without crating a halth haar or causing 
is- posal
pro
lms. Th us of nonisotopic nym la
ls with high spci c activity in nonco
mptitiv
assays incrass sn- sitivity an os so using shortr incu
ation tims than t
h original RIAs.
2 Thr is no n for xpnsiv instrumnta- tion,
caus most assays can
 r
a
y
spctrophotomtry or
y simply noting th prsnc or a
snc of color.
Ragnts ar
inxpnsiv an hav a long shlf lif. Although homognous assays ar not as s
nsitiv as
htrognous assays, thy ar simpl an rquir no sparation stp. Disavanta
gs inclu th
fact that som spcimns may contain natural inhi
itors. Aitionally, th
si of th
nym la
l may
 a limiting factor in th sign of som assays. Nonspcific
protin
ining
is anothr ifficulty ncountr with th us of nym la
ls. 3 Howvr, this
tchniqu has

n succssfully appli to a wi rang of assays, an its us will continu t
o incras.
FLUORESCENT IMMUNOASSAY In 1941, Al
rt Coons monstrat that anti
ois coul

 la
l with
molculs that uorsc. 5,25 Ths uors- cnt compouns ar call uorophors or
uorochroms. Thy can a
sor
nrgy from an incint light sourc an con- vrt t
hat nrgy into
light of a longr wavlngth an lowr nrgy as th xcit lctrons rturn to
th groun
stat. Fluorophors ar typically organic molculs with a ring structur, an 
ach has a
charactristic optimum a
sorption rang. Th tim intrval
twn a
sorption of
nrgy an
mission of uorscnc is vry short an can
 masur in nanoscons. Ially,

a uorscnt
pro
 shoul xhi
it high intnsity, which can
 istinguish asily from
ack
groun uorscnc. 2 It shoul also
 sta
l. Th two compouns most oftn us ar u
orscin an
rhoamin, usually in th form of isothiocyanats,
caus ths can
 raily
coupl with
antign or anti
oy. Fluorscin a
sor
s maximally at 490 to 495 nm an
 mits a
grn color at 517 to 520 nm. It has a high intnsity, goo photosta
ility, an
 a high quantum
yil. Ttramthylrhoamin a
sor
s at 550 nm an mits r light at 580 to 585
nm. Bcaus thir
a
sor
anc an mission pattrns iffr, fluorscin an rhoamin can
 us t
ogthr. Othr
compouns us ar phycorythrin, uropium (-naphthyl tri uoroacton), an lucifr
yllow VS.
1,5 Fluorscnt tags or la
ls wr rst us for histochm- ical localiation of
antign in
tissus. This tchniqu is call immuno uorscnt assay (IFA), a trm rstrict
to qualitativ o
srvations involving th us of a fluorscnc microscop. In t
his mannr, many
typs of antigns can
 tct ithr in x tissu sctions or in liv cll s
uspn- sions
with a high gr of snsitivity an spci city. Th prsnc of a spci c antign
is trmin

y th apparanc of locali color against a ark
ackgroun. This mtho is u
s for rapi
inti cation of microorgan- isms in cll cultur or infct tissu, tumor-spci c
antigns on
noplastic tissu, transplantation antigns, an CD anti- gns on T an B c
lls through
th us of cll flow cytomtry. 26 (S Chaptr 12 for a mor complt iscu
s- sion of th
principls of cll ow cytomtry.) Dirct Immuno uorscnt Assays Fluorscnt staini
ng can

catgori as irct or ini- rct, pning on whthr th original anti
oy
has a uorscnt
tag attach. In a irct immuno uorscnt assay, anti
oy that is conjugat with
a uorscnt
tag is a irctly to unknown antign that is x to a micro- scop sli. Aft
r incu
ation
an a wash stp, th sli is ra using a uorscnc microscop. Antigns ar ty
pically
visuali as
right appl grn or orang-yllow o
jcts against a ark
ackgro
un. Dirct
immuno uorscnt assay is
st suit to antign tction in tissu or
oy uis,
whil
inirct assays can
 us for
oth antign an 1814_Ch10_152-165.qx
7/13/09 2:55 PM
Pag 158 CHAPTER 10 La
l Immunoassays 159 anti
oy inti cation. 3,25 Exampls
of antigns
tct
y this mtho inclu Lgionlla pnumophila, Pnumocystis carinii, Ch
lamyia
trachomatis, an rspiratory syncytial virus (RSV) 26, 27 (Fig. 105). Inirct Im
muno uorscnt
Assays Inirct immuno uorscnt assays involv two stps, th rst of which is incu

ation of
patint srum with a known antign attach to a soli phas. Th sli is wash

, an thn an
antihuman immunoglo
ulin containing a uors- cnt tag is a. This com
ins wit
h th rst
anti
oy to form a sanwich, which localis th fluorscnc. In this mannr, o
n anti
oy
conjugat can
 us for many if- frnt typs of ractions, liminating th n
 for numrous
purifi, la
l ragnt anti
ois. Inirct assays rsult in incras staini
ng,
caus
multipl molculs can
in to ach primary molcul, thus making this a mor s
nsi- tiv
tchniqu. 5 Such assays ar spcially usful in anti
oy inti cation an hav

n us to
tct trponma, anti- nuclar, chlamyial, an toxoplasma anti
ois, as wll
as anti
ois to
such viruss such as hrps simplx, Epstin- Barr, an cytomgalovirus. 3,25,28
,29 Figur 106
picts th iffrnc
twn th two tchniqus. Both tchniqus allow for a v
isual assssmnt
of th a- quacy of th spcimn. This is spcially hlpful in tsting for ch
lamyia an RSV
antigns. 26 Immunofluorscnt assays in gnral, howvr, fac th issu of su

jctivity in th
raing of slis. Only xprinc clinical la
oratori- ans shoul
 rsponsi

l for rporting
out sli rsults. Othr Fluorscnt Immunoassays Quantitativ fluorscnt imm
unoassays (FIAs)
can
 classifi as htrognous or homognous, corrspon- ing to similar
typs of nym
immunoassays. In this cas, th la
l is fluorscnt, an such a la
l can
 ap
pli to ithr
antign or anti
oy. Soli-phas htrognous flu- orscnt assays hav
n 
vlop for th
intification of anti
ois to nuclar antign, toxoplasma antign, ru
l
la virus, an
numrous othr virus antigns. 2 In ai- tion, fluorscnt assays ar us to 
tct such
important
iological compouns as cortisol, progstron, an srum thyroxin (T
4). 2 Howvr,
many of th nwr vlopmnts in fluors- cnt immunoassay hav
n rlat
 to
homognous immunoassays. Homognous FIA, just lik th corrspon- ing EIAs, r
quirs no
sparation procur, so it is rapi an simpl to prform. Thr is only on in
cu
ation stp an
no wash stp, an usually comptitiv
ining is involv. Th
asis for
this tchniqu
is th chang that occurs in th uorscnt la
l on antign whn it
ins to spc
i c anti
oy.
Such changs may
 rlat to wavlngth FIGURE 105. Dirct uorscnt anti
oy t
st for
Giaria an Cryptosporiium in stool. Largr oval
ois ar Giaria lam
lia cys
ts, an th
smallr roun
ois ar Cryptosporiium sp. cysts. (Courtsy of Mriian Biosci
nc, Cincinnati,
OH.) S Color Plat 10.
+ Soli-phas antign La
l anti
oy Antignanti
oy
com
ination
fluorscnc Soli-phas antign Unla
l anti
oy Antignanti
oy com
ination
+ La
l
anti-immunoglo
ulin Fluorscnc
B A
+ FIGURE 106. Dirct vrsus inirct immuno uo

rscnt
assays. (A) Dirct uorscnt assay. Soli- phas antign x to a microscop sli
is incu
at
irctly with a uorscnt-la
l anti
oy. Th sli is wash to rmov un
oun
anti
oy. If
spci c antign is prsnt in th patint sampl, uorscnc will
 o
srv. (B)
Inirct
uorscnc. Patint anti
oy is ract with spci c anti- gn x to a microscop s
li. A
wash stp is prform, an a la
l antihuman immunoglo
ulin is a. Aftr a
scon wash stp
to rmov any uncom
in anti-immunoglo
ulin, uorscnc of th sampl is trmi
n. Th amount
of uorscnc is irctly in proportion to th amount of patint anti
oy prsnt
.
1814_Ch10_152-165.qx 7/13/09 2:55 PM Pag 159 160 SECTION 2 Basic Immunologi
cal Procurs
mission, rotation from, polarity, or ilctric strngth. Thr is a irct
rlationship

twn th amount of uo- rscnc masur an th amount of antign in th pati
nt sampl. As

ining of patint antign incrass,
ining of th uorscnt analyt crass,
an hnc mor
uors- cnc is o
srv. On of th most popular tchniqus vlop is uo- rsc
nc
polariation immunoassay (FPIA), which is
as on th chang in polariation of
fluorscnt
light mitt from a la
l molcul whn it is
oun
y anti-
oy. 24 Incin
t light irct
at th spcimn is polari with a lns or prism so th wavs ar align in on
 plan. If a
molcul is small an rotats quickly nough, th mit- t light is unpolari
 aftr it
is xcit
y polari light. 1,3 If, howvr, th la
l molcul is
oun
to anti-
oy,
th molcul is una
l to tum
l as rapily, an it mits an incras amount of
polari light.
Thus, th gr of polari light r cts th amount of la
l ana- lyt that i
s
oun. In
FPIA, la
l antigns compt with unla
l anti- gn in th patint sampl fo
r a limit
num
r of anti
oy
ining sits. Th mor antign that is prsnt in th patin
t sampl, th
lss th uorscnc-la
l antign is
oun an th lss th polariation that w
ill

tct. Hnc, th gr of uorscnc polariation is invrsly propor- tiona
l to
concntration of th analyt (Fig. 107). This tchniqu is limit to small molc
uls that tum
l frly in solution, usually thos analyts with a molcular wight u
nr 2000 . 2,3
An aitional consir- ation is nonspcific
ining of th la
l conjugat to
othr protins
in srum. Bining to ths molculs woul incras polariation, thus falsly 
crasing valus.
FPIA has
n us mainly to trmin concntrations of thr- aputic rugs a
n hormons. It
rquirs sophisticat instrumntation an is th
asis for svral aut
omat analyrs
on th markt toay. Avantags an Disavantags of Fluorscnt Immunoassay In

principl, th
us of fluorscnc has th potntial for high snsitivity an vrsatility. 24 T
h mthoology is
fairly simpl, an thr is no n to al with an ispos of ha- arous su
s
tancs. Th main
pro
lm, howvr, has
n sparation of th signal on th la
l from auto uorsc
nc prouc
y
iffrnt organic su
stancs normally prsnt in srum. Anothr ifficulty ncou
ntr is th
fact that nonspcific
ining to su
stancs in srum can caus qunching
or iminishing
of th signal an chang th amount of uorscnc gnrat. Fluorscnc po
laria- tion
has
n vlop to ovrcom som of ths pro
lms, an this tchniqu
has sn mor
wispra us. 3 It os, howvr, rquir xpnsiv icat instrumnta- tio
n, which may
limit its us in smallr la
oratoris. CHEMILUMINESCENT IMMUNOASSAYS Principl o
f
Chmiluminscnc Chmiluminscnc is anothr tchniqu mploy to follow anti
gnanti
oy
com
ination. It is th mission of light caus
y a chmical raction, typicall
y an oxia- tion
raction, proucing an xcit molcul that cays
ack to its original gro
un stat. 3 A
larg num
r of molculs ar capa
l of chmiluminscnc,
ut som of th mo
st common
su
stancs us ar luminol, acri- inium strs, ruthnium rivativs, an
nitrophnyl
oxalats. 13 Whn ths su
stancs ar oxii, typically using hyrogn proxi
 an an nym
for a catalyst, intrmiats ar prouc that ar of a highr nrgy stat. Th
s intrmiats
spontanously rturn to thir original stat, giving off nrgy in th form of l
ight. 30 Light
missions rang from a rapi flash of light to a mor continuous glow that can l
ast for hours.
Acriinium strs, for xampl, mit a quick
urst or flash of light, whil th
light rmains for
a longr tim with luminol an iox- tan. 30 Diffrnt typs of instrumntatio
n ar ncssary
for ach kin of mission. This typ of la
ling can
 us for htrognous a
n homognous
assays,
caus la
ls can
 attach to ithr antign or anti
oy. In htrog
nous assays,
com- ptitiv an sanwich formats ar th ons most oftn us. Smallr analyt
s such as
thraputic rugs an stroi hor- mons ar masur using comptitiv assays,
whil th
sanwich format is us for largr analyts such as protin hormons.
Anti
oy
La
l an
patint antign Incras polariation
+ Anti
oy La
l an patint antign
Dcras
polariation B A F F P P P P P P F F F F F P F F P P P F F F F
F F F F F F
F + + P P FIGURE 107. Fluorscnc polariation immunoassay. Ragnt anti
oy
is com
in
with patint antign an uorscnt-la
l antign. (A) If littl or no patint a
ntign is
prsnt, anti
oy will
in to th la
l antign. Bining causs ths molcul

s to rotat mor
slowly in solution, incrasing th amount of polari light that thy rturn. (
B) Whn patint
antign is prsnt, mor la
l antign will
 un
oun. Ths molculs will r
otat mor
quickly in solution, giving off light in many irctions, an polaria- tion wil
l
 cras.
1814_Ch10_152-165.qx 7/13/09 2:55 PM Pag 160 CHAPTER 10 La
l Immunoassay
s 161 Avantags
an Disavantags of Chmiluminscnt Assays Chmiluminscnt assays hav an xc
llnt
snsitivity, com- para
l to EIA an RIA, an th ragnts ar sta
l an rlati
vly nontoxic.
2,30 Th snsitivity of som assays has
n rport to
 in th rang of atta
mols (10 -18
mol) to ptomols (10 -21 mol). 1,3 Bcaus vry littl ragnt is us, thy a
r also quit
inxpnsiv to prform. Th rlativly high sp of tction also mans a fast
r turnaroun
tim. Dtction systms
asically consist of photomultiplir tu
s, which ar si
mpl an
rlativly inxpnsiv. Howvr, fals rsults may
 o
tain if thr is lack
of prcision in
injction of th hyrogn proxi or if som
iological matrials such as urin
or plasma caus
qunch- ing of th light mission. This mtho has
gun to
 mor wily appli
 to immunologic
tsting an has grat potn- tial for th futur. SUMMARY La
l immunoassays w
r vlop to
masur antigns an anti
ois that may
 small in si or prsnt in vry low
concntrations.
Th su
stanc that is
ing masur is th analyt. La
ling tchniqus in
clu th us
of raioactivity, nyms, fluorscnc, an chmilumins- cnc. Thr ar t
wo major typs
of immunoassays: comptitiv an noncomptitiv. In comptitiv assays, all th
ractants ar
a at th sam tim, an la
l anti- gn compts with patint antign for
a limit num
r
of anti
oy-
ining sits. Noncomptitiv assays, on th othr han, allow any a
ntign prsnt to
com
in with an xcss of anti
oy attach to a soli phas. Thn a scon anti
-
oy
aring a
la
l is a an
ins whrvr thr is patint antign. Anti
ois us in i
mmunoassays must

 vry spci c an hav a high affinity for th antign in qustion. Sp
cificity hlps
to cut own on cross-ractivity, an th af nity trmins how sta
l th
ining
is
twn
anti- gn an anti
oy. Ths two factors hlp to trmin th snsitivity of s
uch assays. Boun
an un
oun la
l ar sp- arat
y using a sol-phas surfac, such as glass

as, cllulos
mm
rans, polystyrn tst tu
s, or microtitr plats, to attach ithr antig
n or anti
oy.
Raioimmunoassay was th rst typ of immunoassay to
 appli to quantitativ m
asurmnts of
analyts in th clinical la
oratory. Th original tchniqu was
as on
comptition

twn la
l an unla
l antign for a limit num
r of anti
oy-
ini

ng sits. Th
mor analyt that is prsnt in th patint sampl, th lowr th amount of rai
oactivity that is
tct. Enyms can
 us as la
ls in much th sam mannr as raioactivity
. Comptitiv
assays involv th us of la
l analyt an a limit num
r of anti
oy-
ini
ng sits. Onc
sparation of
oun an un
oun analyt has
n achiv, su
strat for th n
ym is a to
th raction mix, an th prsnc of th nym is tct
y a color chang i
n th su
strat.
Most nym assays us in th la
oratory toay ar non- comptitiv. Oftn, it
is antign that
is
oun to soli phas an patint anti
oy that is
ing tct. Aftr allow
ing suf cint
tim for
ining to occur, a scon nym-la
l anti
oy is a. Th snsit
ivity of
noncomptitiv assays is gratr than that of comptitiv assays. Simpl on-st
p formats hav

n vlop for htrognous nym assays. Rapi flow-through tst
vics ar a
l
to captur antign or anti
oy in a crtain spot on a mm
ran. Th rsults ar
asy to
intrprt. Homognous nym assays rquir no sparation stp. Thy ar
as
on th principl
that nym activity changs as spci c antignanti
oy
ining occurs. Whn anti
o
y
ins to
nym-la
l antign, stric hinranc rsults in a loss in nym activity. F
luorochroms ar
uorscnt compouns that ar us as markrs in immunologic ractions. Dirct imm
uno uo- rscnt
assays involv antign tction through a spci c anti
oy that is la
l with a
fluorscnt
tag. In inirct immunofluorscnt assays, th original anti
oy is unla-
l.
Incu
ation with
antign is follow
y aition of a scon la
l anti-immunoglo
ulin that t
cts antign
anti
oy complxs. Htrognous uorscnt immunoassays ar similar to thos s
cri
 for
othr la
ls. Rcnt rsarch has
n irct towar improvmnt of homognou
s uorscnt
immunoassays to ruc th
ackgroun uorscnc nor- mally foun in srum. FPIA
taks avantag
of th fact that whn an antign is
oun to anti
oy, polariation of light inc
rass.
Chmiluminscnc is th prouction of light nrgy
y crtain compouns whn th
y ar oxii.
Su
stancs that o this can
 us as markrs in ractions that ar similar to
RIA an EIA. Each
typ of systm has avantags an isavantags rlat to th particular natur
of th la
l.
Som ar
ttr for larg multivalnt antigns, whil othrs ar capa
l of t
cting only small
haptns. Ths limitations must
 kpt in min whn choosing th tchniqu that
is most
snsitiv an
st suit for a particular analyt. 1814_Ch10_152-165.qx 7/13/
09 2:55 PM Pag
161 162 SECTION 2 Basic Immunological Procurs A 2-yar-ol mal chil has sym
ptoms that
inclu fatigu, nausa, vomiting, an iarrha. Ths symptoms hav pr- sist

for svral
ays. Stool culturs for
actria pathogns such as Salmonlla an Shiglla wr
ngativ. Th
stool was also chck for ova an parasits, an th rsults wr ngativ. Th
ay-car cntr
that th chil attns has ha a prvious pro
lm with contaminat watr, an t
h physician is
suspicious that this infction might
 caus
y Cryptosporiium, a watr
orn
 pathogn.
Howvr,
caus no parasits wr foun, h is not crtain how to proc. Qus
tions a. Dos a
ngativ fining rul out th prsnc of a parasit?
. What othr typ of tst
ing coul

on? c. How os th snsitivity of tsting such as nym immunoassay compar
with visual
inspction of stain slis? . What ar othr avantags of nym immunoassa
y tsts? CASE
STUDY 1814_Ch10_152-165.qx 7/13/09 2:55 PM Pag 162 CHAPTER 10 La
l Immun
oassays 163
ENZYME IMMUNOASSAY DETERMINATION OF HUMAN CHORIONIC GONADOTROPIN Principl Mm
r
an casstt
tsts for prgnancy trmination ar on-stp soli-phas nym immunoassays 
sign to tct
th prsnc of hCG in urin or srum. hCG is a hor- mon scrt
y th tropho

last of th
vloping m
ryo; it rapily incrass in th urin or srum uring th arly s
tags of
prgnancy. In a normal prgnancy, hCG can
 tct in srum as arly as 7 ay
s following
concption, an th concntration ou
ls vry 1.3 to 2 ays. It is su
- squn
tly xcrt into
th urin. Lvls of hCG rach a pak of approximatly 200,000 mIU/mL at th n
of th rst
trimstr. Bcaus th tst casstt contains all ncssary ragnts, this is ca
ll
immunochromatography. Th tst
an rgion is prcoat with anti-alpha hCG anti

oy to trap hCG
as it movs through th mm
ran caus
y capillary action. Whn th patint sp
cimn is a,
it rconstituts an anti-
ta hCG anti
oy, which is complx to colloial gol
 particls. This
complx is trapp
y th anti-alpha hCG an forms a color complx in th tst
rgion. This may

 in th form of a straight lin or a plus sign. A positiv tst r
sults if a
minimum concntration of approximatly 25 mIU/mL is prsnt. Th control rgion
contains a scon
anti
oy irct against th anti-
ta hCG anti
oy. This scon anti
oy ract
s with th xcss
anti-
ta hCG anti-
oy gol particls to inicat that th tst is working cor
rctly. Sampl
Prparation Srum, plasma, or urin may
 us for tsting. Any urin spcimn
may
 us,
ut
th first morning spcimn is prfra
l,
caus it is th most highly c
oncntrat.
Plasma collct with thylniaminttra-actic aci, hparin, or citrat may

 us.
Spcimns may
 stor for up to 72 hours at 2C to 8C. If spcimns n to
 h
l longr,
fring at 220C is rcommn. Fring an thawing is not rcommn. Ragnt

s, Matrials,
an Equipmnt Tst kit containing tst casstts Mark piptts or sampl isp
nsrs Urin
control Srum control Timr NOTE: Thr ar numrous kits on th markt. Exampl
s ar SurStp,
QuickViw, Concis, an Clarviw. Procur 1. Rmov th tst vic from its
protctiv pouch.
2. Allow th tst vic to quili
rat to room tmpratur. 3. Draw sampl up t
o th mark on th
piptt. 4. Dispns a crtain num
r of rops or th ntir con- tnts of th p
iptt, pning
on kit instructions. 5. Wait for th sampl to compltly run through th mm-

ran. 6. Color

ans will usually appar in 1 to 3 minuts. 7. A color
an shoul also appa
r in th control
rgion. If it os not, th tst is invaliat. Rsults A sampl is positiv if
a lin forms in
th tst rgion. A ng- ativ spcimn will prouc a lin in th control rgion
only. If no
color vlops in th control rgion, ithr th ragnts ar not activ or th
tst procur
was incorrct. Intrprtation of Rsults Th hCG hormon consists of two su
unit
s, call alpha
an
ta. Th
ta su
unit is uniqu to hCG, whil th alpha su
unit can
 foun
 in othr
hormons. In this tst systm, as soon as a patint sampl containing hCG is a
, it
ins to
monoclonal anti-
ta hCG anti
oy, which is complx to an inicator such as co
lloi gol
particls. Th mono- clonal anti
oy to th alpha portion of hCG on th casstt
mm
ran
ins
th hCG complx as th patint sampl travls along th mm
ran. A scon anti

oy irct
against th anti-
ta hCG anti
oy is
oun in th control rgion. Fals ngativ
s caus
y
cross-ractivity with othr hor- mons such as lutiniing hormon an folliclstimulating
hormon ar avoi through th us of monoclonal anti-
oy that is irct ag
ainst th chain
of hCG. Not that th actual procur an ragnts prsnt will vary slightly w
ith iniviual
kits. This typ of tsting is a visual qualita- tiv tst only. NOTE: Thr ar
othr kits on th
markt that can
 us to monstrat EIA ractions raily. Som of ths ar
 availa
l for
infctious agnts such as Strptococcus pyogns an Strptococcus agalactia
. Anothr
altrnativ is an EIA kit that simulats HIV tst- ing, availa
l from Evotk,
Inc. (S Chaptr
23 for th procur.) EXERCISE 1814_Ch10_152-165.qx 7/13/09 2:55 PM Pag 16
3 164 SECTION 2
Basic Immunological Procurs 1. Which of th following statmnts accuratly 
scri
s
comptitiv
ining assays? a. Excss
ining sits for th analyt ar provi
.
. La
l an
unla
l analyt ar prsnt in qual amounts. c. Th concntration of patint
analyt is
invrsly proportional to
oun raioactiv la
l. . All th patint analyt is

oun in th
raction. 2. How o htrognous assays iffr from homognous assays? a. Ht

rognous assays
rquir a sparation stp.
. Htrognous assays ar asir to prform than ho
mognous assays.
c. Th concntration of patint analyt is irctly pro- portional to
oun la

l in homognous
assays. . Homognous assays ar mor snsitiv than htrognous ons. 3. In
th following
quation, what is th ratio of
oun raioactiv antign (Ag*) to
oun patint
antign (Ag)?
12Ag * 4Ag
4A
:___Ag * A

___AgA

Ag* ___Ag a. 1:4
. 1:3 c. 3:1 . 8:4 4. Which
of
th following charactris a captur or san- wich nym assay? a. Lss snsit
iv than
comptitiv nym assays
. Rquirs two wash stps c. Bst for small antigns
with a singl
trminant . A limit num
r of anti
oy sits on soli phas 5. Avantags o
f EIA ovr RIA
inclu all xcpt which on of th following? a. Dcras in haarous wast
.
Shortr shlf
lif of kit c. No n for xpnsiv quipmnt . Eas of aaptation to automat
 tchniqus 6.
Which of th following is charactristic of irct fluo- rscnt assays? a. Th
anti-immunoglo
ulin has th uorscnt tag.
. Anti
oy is attach to a soli pha
s. c.
Micro
ial antigns can
 rapily inti 
y this mtho. . Th amount of color
is in invrs
proportion to th amount of antign prsnt. 7. Which of th following is tru o
f fluorscnc
polar- iation immunoassay? a. Both antign an anti
oy ar la
l.
. Larg m
olculs polari
mor light than smallr molculs. c. Whn
ining occurs, thr is qunching of
th uorscnt
tag. . Th amount of uorscnc is irctly propor- tional to concntration of t
h analyt. 8.
All of th following ar sira
l charactristics of anti-
ois us in immun
oassays xcpt a.
high af nity.
. high spci city. c. high cross-ractivity. . not foun in th pati
nt sampl.
9. In a noncomptitiv nym immunoassay, if a nga- tiv control shows th pr
snc of color,
which of th following might
 a possi
l xplanation? a. No ragnt was a.

. Washing stps
wr incomplt. c. Th nym was inactivat. . No su
strat was prsnt. 10.
Which of th
following
st charactris chmilumi- nscnt assays? a. Only th antign can

 la
l.
.
Tsts can
 ra manually. c. Ths ar only homognous assays. . A chmical
is oxii to
prouc light. 11. Immuno uorscnt assays may
 if cult to intrprt for which r
ason? a.
Auto uorscnc of su
stancs in srum
. Nonspci c
ining to srum protins c. Su

jctivity in
raing rsults . Any of th a
ov 12. Which statmnt
st scri
s flowthrough
immunoassays? a. Rsults ar quantitativ.
. Ragnts must
 a sparatly.
c. Thy ar
if cult to intrprt. . Thy ar sign for point-of-car tsting. REVIEW QUES
TIONS
1814_Ch10_152-165.qx 7/13/09 2:55 PM Pag 164 CHAPTER 10 La
l Immunoassay
s 165 Rfrncs

1. Kricka, LJ. Principls of immunochmical tchniqus. In Burtis, CA, A

shwoo, ER, an
Bruns, DE (s): Tit Funamntals of Clinical Chmistry, . 6. Saunrs E
lsvir, St.
Louis, 2008, pp. 155170. 2. Ashihara, Y, Kasahara, Y, an Nakamura, RM. Immunoass
ays an
immunochmistry. In McPhrson, RA, an Pincus, MR (s): Hnrys Clinical Diagn
osis an
Managmnt
y La
oratory Mthos, . 21. Saunrs Elsvir, Philalphia, 2007
, pp. 793818. 3.
Carpntr, AB. Immunoassays for th iagnosis of infctious isass. In Murra
y, PR, t al.
(s): Manual of Clinical Micro
iology, . 9. ASM Prss, Washington, D
C, 2007, pp.
257269. 4. Mak, TW, an Saunrs, ME. Th Immun Rspons: Basic an Clinical P
rincipls.
Elsvir, Burlington, MA, 2006, pp. 147176. 5. Kint, TJ, Gols
y, RA, an Os

orn, BA. Ku
y
Immunology, . 6. WH Frman an Co., Nw York, 2007, pp. 145167. 6. Yalow, RS,
an Brson,
SA. Immunoassay of nognous plasma insulin in man. J Clin Invst 39:1157, 1
960. 7.
Flischr, DM, an Woo, RA. Tsts for immunological rac- tions to foos. In D
trick, B,
Hamilton, RG, an Fols, JD (s): Manual of Molcular an Clinical La
ora
tory Immunology,
. 7. ASM Prss, Washington, DC, 2006, pp. 975973. 8. Mriggioli, MN. Us
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in nurological iag- nosis an rsarch. Nurological Rs 27:734, 2005. 9. Dwa
r, R t al.
Principls an procurs of human immun- o cincy virus sroiagnosis. In Dtri
ck, B,
Hamilton, RG, an Fols, JD (s): Manual of Molcular an Clinical La
o
ratory
Immunology, . 7. ASM Prss, Washington, DC, 2006, pp. 834846. 10. Bninlli, M
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an Clinical
La
oratory Immunology, . 7. ASM Prss, Washington, DC, 2006, pp. 724745. 11. P
arlman, BL.
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SS. Hpatitis C:
A rviw for primary car physicians. CMAJ 174:649, 2006. 13. Jnson, HB. Epsti
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linical
La
oratory Immunology, . 7. ASM Prss, Washington, DC, 2006, pp. 637647. 14. F
ng, N, an
Matsui, SM. Rotaviruss an noroviruss. In Dtrick, B, Hamilton, RG, an Fols,
JD (s): Manual
of Molcular an Clinical La
oratory Immunology, . 7. ASM Prss, Washington, D
C, 2006, pp.
746755. 15. Pira, PA, an Boivin, G. Rspiratory syncytial virus, human mtapn
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an Clinical La
oratory Immunology, . 7. ASM Prss, Washington, DC, 200
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. Antivir Thr

12:659, 2007. 17. Nutman, TB. Mycotic an parasitic isass: Introuction. In D
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mmunology, . 7.
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r, Al, an Novak-Wkly, S.
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urognital am
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iology, . 9. ASM Prss, Washington, DC, 2007, pp. 20922112. 19. Xiao,
L, an
Cama, V. Cryptosporiium. In Murray, PR, t al. (s): Manual of Clinical M
icro
iology, .
9. ASM Prss, Washington, DC, 2007, pp. 17451761. 20. Linsly, MD, Warnock, DW,
an Morrison,
CJ. Srological an molcular iagnosis of fungal infctions. In Dtrick, B, Ham
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v M 59:361,
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oratory Mthos, . 21. Saunrs
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oratory Immunology, . 7. ASM Prss, Washington, DC, 2006, pp. 721.
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oratory Mthos, . 21. Saunrs Elsvir, Philalphia, 2007, pp. 916932. 29
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munoas- says.
La
M 29:555560, 1998. 1814_Ch10_152-165.qx 7/13/09 2:55 PM Pag 165 11 Mol
cular
Diagnostic Tchniqus Susan M Orton, PhD, MT(ASCP), D (ABMLI) LEARNING OBJECTIVE
S Aftr nishing

this chaptr, th rar will
 a
l to: 1. Dscri
 th structur an function
of
oxyri
onuclic aci (DNA) an ri
onuclic aci (RNA) within a cll. 2. Explain
what a nuclic
aci pro
 is an how it is us. 3. D n hy
riiation an iscuss th conition
s that in unc
its spci city. 4. Diffrntiat ot-
lot from a Southrn
lot. 5. Dscri
 th ro
l of
rstriction nonuclass in DNA analysis. 6. Explain th
asis of DNA chip tch
nology. 7.
Discuss th principls an major stps of th polymras chain raction (PCR). 8
. Discuss th
avantags of ral-tim PCR. 9. Dscri
 th major iffrnc
twn n-point
an ral-tim
PCR. 10. Diffrntiat targt ampli cation from pro
 ampli cation an giv xampls
of ach. 11.
Compar th rlativ avantags an isavantags of ampli cation systms in gnr
al. 12.
Corrlat iniviual nuclic aci tchniqus with actual us in clinical stting
s. 13. Dtrmin
whn nuclic aci tchnology is appropriat for clinical tsting. KEY TERMS Bran
ch DNA (
DNA)
signal ampli cation Doxyri
onuclic aci (DNA) DNA squncing Dot-
lot Gl lctr
ophorsis
Hy
riiation In situ hy
riiation Ligas chain raction (LCR) Northrn
lot Nu
clic aci pro

Nuclic aci squnc
as ampli cation (NASBA) Polymras chain raction (PCR) P
rimrs Purin
Pyrimiin Ral-tim PCR Rstriction nonuclas Rstriction fragmnt lngth po
lymorphisms
(RFLPs) Rvrs transcriptas Ri
onuclic aci (RNA) Sanwich hy
riiation Sout
hrn
lot Stran
isplacmnt ampli cation (SDA) Stringncy Transcription Transcription-miat am
pli cation
(TMA) Translation 166 1814_Ch11_166-185.qx 7/10/09 2:55 PM Pag 166 CHAPTER
11 Molcular
Diagnostic Tchniqus 167 CHARACTERISTICS OF NUCLEIC ACIDS Molcular iagnostic
assays ar
powrful nw tools us to gain information to ai in iagnosis an monitoring o
f is- as.
Ths tchniqus ar
as on th tction of spci c nuclic aci squncs in m
icroorganisms
or particular clls. Molcular tchniqus us in th clinical la
to intify u
niqu nuclic
aci squncs inclu nymatic clavag of nuclic acis, gl lctrophorsis,
nymatic
amplifica- tion of targt squncs, an hy
riiation with nuclic aci pro
s.
All of ths
tchniqus ar iscuss in this chaptr in aition to an ovrviw of th struc
tur an
functions of oxyri
onuclic aci (DNA) an ri
onuclic aci (RNA). Avantags
an isavantags
of ach typ of tction mtho will
 prsnt along with xampls of us in
actual clinical
sttings. Composition of DNA an RNA Th two main typs of nuclic acis ar o
xyri
onuclic
aci (DNA) an ri
onuclic aci (RNA). DNA carris th primary gntic informati
on within
chromosoms foun in ach cll. Gns ar squncs of DNA that nco infor- ma
tion for th

translation of nuclic aci squnc into amino aci squnc, rsulting in prot
in prouction.
Th ntir squnc of th human gnom is mor than 3
il- lion DNA
ass lo
ng. 1 Howvr,
th actual num
r of nco gns, approximatly 30,000, is much lowr than
originally
prict. DNA is packag into gns, which ar all packag within uplicat p
airs of
chromosoms. Humans hav 23 pairs of chromosoms an 1 pair of sx chromosoms,
giving us a total
of 46 chromosoms. RNA, on th othr han, is an intrmiat nuclic aci struc
tur that hlps
convrt th gntic information nco within DNA into protins that ar th pr
imary cllular
com- ponnt. DNA acts as th tmplat for synthsis of RNA. Both nuclic aci st
ructurs ar
polymrs ma up of rpat- ing nuclotis, or
ass, that ar link togthr
to form long
molculs. Each nucloti consists of a cyclic, 5-car
on sugar, with a phosphat
 group at th 5
C an on of four nitrognous
ass at th 1 C. Th sugar oxyri
os is prs- n
t in DNA,
whil ri
os is th primary sugar foun in RNA. DNA an RNA hav th sam
two purin
ass,
a- nin an guanin,
ut th pyrimiin
ass iffr. DNA uss cytosin an th
ymin, whil in
RNA, uracil rplacs thymin. DNA xists primarily as a ou
l-stran mol- cu
l with vry
spcific
as pair linkags: Anin pairs with thymin, an cytosin pairs with
guanin. Any
othr com
inations of
as pairs ar ithr too larg or too small an o not fi
t togthr. Th
nitrognous
ass ar foun on th insi of th molcul, an th sugars ar li
nk togthr
via altrnating phosphat groups to form th outsi
ack
on. Nuclotis
ar join
togthr
y phos- phoistr
ons that link th 5 phosphat group of on sugar t
o th 3
hyroxyl group of an ajacnt sugar. 2 Th two strans ar twist into an alpha
hlix (Fig.
111). Hyrogn
oning
twn ajacnt
ass hols th two chains togthr. Rpl
ication of DNA
DNA is a vry sta
l molcul, which maks it ial for a clinical spcimn. It
loss its
conformational structur only unr xtrms of hat, pH, or th prsnc of s
ta
ili- ing
agnts. 2 Rplication of th DNA molcul is vry straightforwar. It is c
haractri as a
smi-consrvativ procss,
caus on stran of th molcul acts as a tm- pla
t for cration
of a complmntary stran. Two aughtr molculs rsult, ach of which is an x
act copy of th
orig- inal molcul. This procss can
 rplicat in th la
oratory,

caus th
hyrogn
ons that hol th two CHAPTER OUTLINE CHARACTERISTICS OF NUCLEIC ACID
S Composition of
DNA an RNA Rplication of DNA Typs of RNA NUCLEIC ACID PROBES HYBRIDIZATION TE
CHNIQUES Soli
Support Hy
riiation Solution Hy
riiation In Situ Hy
riiation DNA Chip Tch
nology Draw
acks

of Hy
riiation Tchniqus DNA SEQUENCING TARGET AMPLIFICATION Polymras Chain

Raction (PCR)
Transcription-Bas Ampli cation Stran Displacmnt Ampli cation (SDA) PROBE AMPLI
FICATION
SIGNAL AMPLIFICATION DRAWBACKS OF AMPLIFICATION SYSTEMS FUTURE OF MOLECULAR DIAG
NOSTIC TECHNIQUES
SUMMARY CASE STUDY EXERCISE: GEL ELECTROPHORESIS OF DNA REVIEW QUESTIONS REFEREN
CES
1814_Ch11_166-185.qx 7/10/09 2:55 PM Pag 167 168 SECTION 2 Basic Immunologi
cal Procurs
strans togthr ar rlativly wak. 3 Th ou
l hlix can asily
 sparat
or natur
using hat or an alkalin solution. Whn hat is us to sparat th strans, t
h procss is
known as mlting. Whn DNA strans cool to normal physiological conitions, comp
lmntary strans
spontanously rjoin or annal. It is ths charactristics that ar xploit i
n molcular
tsting. Going from a gntic squnc nco within DNA into a functional prot
in involvs two
major procsss: transcription an translation. Transcription is th gnration
of a stran of
mssngr RNA (mRNA) that cos for th gn that is to
 xprss as a proti
n. In turn,
translation occurs whn th mRNA travls from th nuclus into th cytoplasm of
th cll to
ri
osoms, whr th mRNA squnc is translat into an amino aci squnc that
maks th
protin nco for
y DNA in th nuclus. Typs of RNA On stran of DNA, th c
oing stran,
srvs as th tm- plat for th prouction of mRNA. All forms of RNA ar singl
-stran
polymrs with an irrgular thr-imnsional structur an ar of much shor
tr lngths
than DNA. Mssngr RNA is us to translat th DNA co into making functiona
l protins. Th
two othr typs of RNA ar transfr RNA (tRNA), which transports iffrnt amino
acis to mak
protins, an ri
osomal RNA (rRNA), which acts as th sit for protin synthsis
irct
y th
mRNA. RNA is lss sta
l than DNA
caus it is singl stran; this maks it m
or suscpti
l
to alkalin hyrolysis. RNA is also rapily gra
y RNas nyms, which app
ar in a
unanc
vrywhr in th nvironmnt. 2 Howvr, RNA can asily
 rplicat an is al
so us in
molcular iagnostic tchniqus. HYBRIDIZATION TECHNIQUES Spontanous pairing of
complmntary
DNA strans forms th
asis for tchniqus that ar us to tct an charac- t
ri gns.
Pro
 tchnology is typically th
asis for intification of iniviual gns o
r DNA squncs.
A nuclic aci pro
 is a short stran of DNA or RNA of a known squnc that is
us to intify
th prsnc of its complmntary DNA/RNA in a patint spcimn. Bining of two
such
complmntary strans is known as hy
riia- tion an is vry spcific. Th two
strans shoul
shar at last 16 to 20 conscutiv
ass of prfct complmntar- ity to form a
sta
l hy
ri. 1

Th pro
a
ility of such a match occurring
y chanc is lss than 1 in 1
illion,

so thr is an
xtraorinary gr of spci city to this procss. 1 Pro
s C H H C O O P O O H H
C CH 2 CH
2 H H C H 2 C H 2 C H 2 P P P H Sugar Cytosine and Thymine Adenine and Guanine P
hosphate group
Bases DNA molecule H H H H H H H H H P P P P FIGURE 111. DNA molecule. The double
helix of DNA
is shown along with details of how the bases, sugars, and phosphates connect to
form the
structure of the molecule. DNA is a double stranded molecule twisted into a hel
ix (think of a
spiral staircase). Each spiraling strand, composed of a sugarphosphate backbone
and attached
bases, is connected to a complementary strand by noncovalent hydrogen bonding be
tween paired
bases. The bases are adenine (A), thymine (T), cyto sine (C), and guanine (G).
A and T are
connected by two hydrogen bonds. G and C are connected by three hydrogen bonds.
1814_Ch11_166185.qxd 7/10/09 2:55 PM Page 168 CHAPTER 11 Molecular Diagnosti
c Techniques 169
are labeled with a marker such as a radioisotope, a fluo rochrome, an enzyme, o
r a
chemiluminescent substrate to make detection of hybridization possible. Hybridiz
ation can take
place either in a solid support medium or in solution. Solid Support Hybridizati
on Dotblot and
sandwich hybridization assays are the simplest types of solid support hybridizat
ion assays. 1 In
the dotblot assay, clinical samples are applied directly to a membrane surface.
The membrane is
heated to denature or separate DNA strands, and then labeled probes are added. A
fter care ful
washing to remove any unhybridized probe, presence of remaining probe is detecte
d by
autoradiography or enzyme assays (Fig. 112). A positive result indicates presence
of a speci c
sequence of interest. This permits qualitative test ing of a clinical specimen,
because it only
indicates presence or absence of a particular genetic sequence. It is much eas
ier to handle
multiple samples in this manner. 4 However, there may be dif culty with the interp
retation of
weak positive reac tions, because there can be background interference. 5 Sandw
ich hybridization
is a modification of the dot blot procedure. It was designed to overcome some o
f the background
problems associated with the use of unpuri fied samples. 2 The technique uses t
wo probes, one of
which is bound to the membrane and serves to capture target sample DNA. The seco
nd probe anneals
to a different site on the target DNA, and it has a label for detection. The sam
ple nucleic acid
is thus sandwiched in between the cap ture probe on the membrane and the signal
generating
probe. 2 Because two hybridization events must take place, specificity is increa
sed. Sandwich
hybridization assays have been developed using microtiter plates instead of mem
branes, which
has made the procedure more adaptable to automation. 2 To characterize DNA prese

nt in a patient
sample in a more detailed fashion, enzymes called restriction endonu cleases ar
e often used.
These enzymes cleave both strands of a doublestranded DNA at speci c recognition
sites that are
approximately 4 to 6 base pairs long. 1 Human DNA may yield millions of unique f
ragments. 1 The
resulting fragments are separated from each other on the basis of size and charg
e by a procedure
known as gel electrophoresis. Digested cellular DNA from a patient blood or tiss
ue sample is
placed in wells in an agarose gel and covered with buffer. The molecules migrate
through the gel
under the influence of an electrical field, and they are separated on the basis
of molecular
weight. Smaller fragments migrate faster, while the larger fragments remain clos
er to the origin.
The gel can either be stained with ethidium bromide and viewed under ultraviolet
(UV) light to
see the entire pattern of frag ments, or speci c nucleic acid sequences can be id
enti ed through
the use of DNA probes (Fig. 113). Differences in restriction patterns are re
ferred to as
restriction fragment length polymorphisms (RFLPs). Specimen is applied directly
to the filter
Specimen fixed on membrane and denatured Labeled probe hybridizes to target Wash
and look for
color Add substrate Add probe FIGURE 112. Dotblot hybridization assay. BamHI No
Enzyme EcoRI
HindIII FIGURE 113. Restriction analysis of bacteriophage lambda DNA. Three enzym
esBamHI,
EcoRI, and HindIIIare used to cleave DNA at speci c sites. Each produces a differen
t pattern
when the DNA is electrophoresed. Ethidium bromide is used to help visualize the
DNA under UV
light. (From Restriction Enzyme and DNA Kit, Carolina Biological Supply Company,
Burlington, NC,
with permission.) 1814_Ch11_166185.qxd 7/10/09 2:55 PM Page 169 170 SECTION
2 Basic
Immunological Procedures This is caused by variations in nucleotides within gene
s that change
where the restriction enzymes cleave the DNA. When such a mutation occurs, diffe
rentsized pieces
of DNA are obtained. Such RFLP patterns can be used to identify specific microor
ganisms, to
establish strain relat edness, to detect polymorphisms in major histocompatibil
ity complex (MHC)
genes, or to obtain a DNA ngerprint of a particular individual. 6 Analysis of DNA
fragments
using probes is typically car ried out using a technique known as a Southern hy
bridization
assay, or Southern blot, named after its discoverer, E. M. Southern. After DNA f
ragments are
separated out by gel electrophoresis, the fragments are denatured using alkali a
nd then
transferred, or blotted, onto a nitrocellulose or nylon membrane for the hybridiza
tion reaction
to take place. The transfer is accomplished by placing the membrane on top of th
e gel and
allowing the buffer plus DNA to wick onto the membrane. Traditionally, this proc

edure takes
overnight, but newer methods using vacuum and pressure systems have signi cantly i
ncreased the
speed of the transfer. 2 Once the DNA is on the nitrocellulose membrane, heat i
ng or UV light is
used to crosslink the strands onto the membrane, thus immobilizing them. A labe
led probe, whose
sequence is complimentary to the sequence of interest, is then added to the memb
rane for
hybridization to take place. The membrane is then washed, and the amount of boun
d probe remaining
is determined by detection of the label (Fig. 114). Several nonradiolabeled p
robes,
including enzyme, chemiluminescent, and acridinium labels, have been developed f
or use, and these
appear to be as sensitive as the original radiolabeled probes. The use of these
probes avoids the
hazards associated with use of radioactivity. 7 The absence of any visible bands
indicates an
absence of the sequence of interest. Southern blots are useful to analyze altera
tions of large
spans of DNA, where ampli cation by the polymerase chain reaction (PCR) may not be
practical.
Southern blot analy sis can reveal polymorphisms in DNA sequence based upon the
RFLP pro le made
visible by the probes. Southern blots have been used to determine the clonal com
position of lym
phocyte populations. Only when a large number of cloned cells are present do rea
rranged genes
specific for Tcell receptors or immunoglobulins appear in suf cient quantity to p
roduce a
detectable band that is different from the nor mal or germline DNA. This is ty
pically an
indicator of a lymphoid malignancy such as Bcell lymphoma, chronic myelogenous
leukemia, or
hairy cell leukemia. 1,4 Detection of a malignant clone can help distinguish bet
ween reactive
lymphocytes seen in an inflammatory process and true malignancies. DNA te
sting is
especially helpful in both diag nosis and classi cation of Tcell malignancies. 4
It has had a
tremendous impact on diagnosis of nonHodgkins lym phomas. 8 Molecular testing c
an also be used
to monitor therapy and remission of lymphomas. If tissue from sepa rate lymphom
atous lesions are
from the same original source or clone, they usually show identical DNA rearrang
ements and
produce identical bands on a Southern blot. 1 In gen eral, however, Southern bl
ots are complex,
timeconsuming to perform, and require a relatively large sample, which can limi
t its clinical
usefulness. 5 Northern blots are performed in a similar manner, but in this tech
nique, RNA is
extracted and separated. This tool Sample DNA Digest with restriction enzymes Ge
l electrophoresis
Blot gel with nylon membrane Denature DNA and add labeled probe Patients 1 and 2
have two
different restriction patterns 1 2 1 2 1 2 FIGURE 114. Southern blot. 1814_Ch11_1
66185.qxd
7/10/09 2:55 PM Page 170 CHAPTER 11 Molecular Diagnostic Techniques 171 is use

d most often to
determine the level of expression of a particular messenger RNA species to see i
f a gene is
actually being expressed. 2 Because RNA is a short, singlestranded molecule, i
t does not
have to be digested before elec trophoresis, but it has to be denatured to
ensure that it
is in an unfolded, linear form. Probes are then used to identify the presence of
speci c bands.
Solution Hybridization Hybridization assays can also be performed in a solution
phase. In this
type of setting, both the target nucleic acid and the probe are free to interact
in a reaction
mixture, resulting in increased sensitivity compared to that of solid support hy
bridization. 5 It
also requires a smaller amount of sample, although the sensitivity is improved w
hen target DNA is
extracted and puri ed. 5 For solution hybridizations, the probe must be single st
randed and
incapable of selfannealing. 4 Several unique detection methods exist. In one of
these, an S1
nuclease is added to the reaction mix. This will digest only unannealed or singl
estranded DNA,
leaving the hybrids intact. Double stranded DNA can then be recovered by precip
itation with
trichloroacetic acid or by binding it to hydroxyapatite columns. 4,9 A se
cond and less
technically dif cult means of solution hybridization is the hybridization protecti
on assay (HPA).
For this assay, a chemiluminescent acridinium ester is attached to a prob
e. After the
hybridization reaction takes place, the solution is subjected to alkaline hydrol
ysis, which
hydrolyzes the chemiluminescent ester if the probe is not attached to the target
molecule. If the
probe is attached to the target DNA, the ester is protected from hydrolysis. Pro
bes that remain
bound to a speci c target sequence give off light when exposed to a chemical trigg
er such as
hydro gen peroxide at the end of the assay. An additional type of solution phas
e hybridization
assay uses an antibody specific for DNA/RNA hybrids to cap ture and detect the hy
brids that
are formed during the solution hybridization. Digenes Hybrid Capture 2 (Digene Co
rp.,
Gaithersburg, MD) assay, used to detect human papillomavirus (HPV), is an
example of this
type of assay. HPV DNA in the sample is hybridized with an unlabeled RNA probe,
and the viral
DNARNA hybrids are then cap tured by the antibody. Solution phase hybridizatio
n assays are
fairly adaptable to automation, especially those using chemiluminescent labels.
Assays can be
performed in a few hours. 4 However, low positive reactions are difficult to int
erpret because of
the possibility of crossreacting target molecules. 9 In Situ Hybridization In s
itu hybridization
represents a third type of hybridiza tion reaction in which the target nucleic
acid is found in
intact cells. It provides information about the presence of specific DNA targets

in relation to
its distribution within the tissues. This technique is the same as immunohisto
chemistry except
that nucleic acids instead of antibodies are used as probes. For probes to reach
the nucleic
acid, they must be small enough, usually limited to 500 bases or less, to penetr
ate the cells in
question. 4 Formalin xed and paraf n embedded tissue sections are typically u
sed for
this procedure. Because preparation of tissue specimens can vary greatly in each
lab, it is
recommended that an endogenous control probe be used. 5 This probe will react po
sitively with all
cells and is used to indicate that penetration of the sam ple has occurred. Pro
bes have
typically been labeled with radioactive sub stances, but the trend today is tow
ard using
uorescent or enzyme labels. If a uorescent tag is used, the procedure is called fl
uorescent in
situ hybridization, or FISH. After com pletion of the procedure, evaluation sho
uld be performed
by an experienced histopathologist. 5 This technique is used to detect a number
of malignancies
linked to chromosomal abnormalities, such as chronic myelogenous leukemia, in wh
ich there is a
translocation from chromosome 9 to chro mosome 22, and inherited disorders that
occur as a
result of chromosomal abnormalities. When FISH is used on metaphase chrom
osome spreads or
interphase nuclei, it can detect numerical alterations or translocation of chrom
oso mal
material. DNA Chip Technology Previously, genetic studies examined individual ge
ne expres sion
by northern blotting or examined polymorphisms by gelbased restriction digests
and sequence
analysis, but with advances in microchips and beadbased array technologies, hig
hthroughput
analysis of genetic variation is now possi ble. Biochips, also called microarra
ys, are very
small devices used to examine DNA, RNA, and other substances. These chips allow
thousands of
biological reactions to be per formed at once. 10 Typically, a biochip consists
of a small
rectangular solid surface that is made of glass or silicon with short DNA or RNA
probes anchored
to the surface. 11 The number of probes on a biochip surface can vary from 10 to
20 up to
hundreds of thousands. 10,11 Usually, the nucleic acid in the sample is ampli ed b
efore it is
analyzed. After ampli cation, the sample, labeled with a uorescent tag, is loaded
onto the
chip. Hybridization occurs on the chips surface, allowing thousands of hybridizat
ion reactions
to occur at one time. Unbound strands of the target sample are then washed away.
The
fluorescenttagged hybridized samples are detected using a uorescent detector 10
(Fig. 115).
The intensity of the uorescent signal at a particular location is proportional to
the sequence
homology at a particular locus. Complete sequence matches results in bright fluo

 rescence, while
singlebase mismatches result in a dimmer signal, indicative of a point mutation
. Since most
single nucleotide polymorphisms (SNPs) are silent and have no apparent fun
ctional
consequence, the challenge is to 1814_Ch11_166185.qxd 7/10/09 2:55 PM Page 1
71 172 SECTION 2
Basic Immunological Procedures identify the set of SNPs that are directly relate
d to or that
cause disease. Detection of point mutations can be used for classifying leukemia
s, molecular
staging of tumors, and characterizing microbial agents. 12 One prime example is
the determination
of genes associated with drug resist ance in HIV testing. Identification of suc
h genes guides
the physician in selecting a proper drug regimen for a particular patient. Drawb
acks of
Hybridization Techniques In any of the previously mentioned hybridization techni
ques, conditions
of the reaction itself may determine how sensi tive and speci c the technique is.
Under the
conditions in which most assays are conducted, two nucleic acid strands must sha
re at least 16 to
20 consecutive bases of perfect com plementarity to form a stable hybrid.
12 Stringency, or
correct pairing, is most affected by the salt concentration, the temperature, an
d the
concentration of destabilizing agents such as formamide or urea. Decreasing the
salt con
centration, increasing the temperature, and increasing the concentration of form
amide or urea all
help to ensure that only the most perfectly matched strands will remain paired i
n a stable
hybrid. If the conditions are not carefully con trolled, however, mismatches ca
n occur, and the
results may not be valid. In addition, even under carefully controlled condition
s, often the
amount of nucleic acid in a patient specimen may present in very low quantity, w
hich may be below
the thresh old for probe detection. The sensitivity of these assays can be incr
eased by
amplifying the sequence of interest. There are other ampli cation techniques as we
ll, and they
fall into three general categories: target ampli cation, probe ampli fication, a
nd signal
amplification. Each of these will be discussed and relevant clinical exampl
es given. DNA
SEQUENCING DNA sequencing is considered the gold standard for many molecular appli
cations, from
mutation detection to genotyping, but it requires proper methodology and i
nterpretation to
prevent misidentification. The sequence should be analyzed on both DNA strands t
o provide even
greater accuracy. Patient sequences are compared to known reference sequen
ces to detect
mutations. Most sequencing strategies include PCR amplification as the first st
ep to amplify
the region of interest to be sequenced. The sequencing reaction itself is b
ased upon the
dideoxy chain termination reaction developed by Sanger and colleagues in 19

77. 13 If any one

of the four dideoxynucleotides are incorporated into a growing DNA GeneChip con
taining probe
array Each probe cell contains millions of copies of a specific oligonucleotide
probe
Biotinylated DNA target from experimental sample * Streptravidin phycoerythrin
conjugate
Fluorescence * * * * * FIGURE 115. Analysis of HIVI. Labeled patient DNA is plac
ed on the
biochip or microarray. In this case, the label is biotin. Hybridi zation takes
place, followed
by a washing step. Strepavidin conju gated to a uorescent dye is added. This wil
l complex with
any patient DNA present. The resulting uorescent pattern can pick up mutations as
sociated with
drug resistance. 1814_Ch11_166185.qxd 7/10/09 2:55 PM Page 172 CHAPTER 11 Mo
lecular
Diagnostic Techniques 173 chain, synthesis is halted. This results in fragments
of varying
lengths depending upon where incorporation of the dideoxynucleotide bases ha
s occurred.
These nucleotides lack the 3 and 2 hydroxyl (OH) group on the pentose ring, and be
cause DNA
chain elongation requires the addition of deoxynucleotides to the 3OH grou
p,
incorporation of the dideoxynucleotide terminates chain length. The most commo
nly used form
of this sequencing method in the clinical lab uses cycle sequencing, which is si
milar to a
PCR reaction in that the steps involved include denaturing, annealing of a prime
r, chain
extending, and ter minating by varying the temperature of the reaction. The new
ly generated
fragments are tagged with a fluorescent dye and separated, based upon size, by d
enaturing gel or
capillary electrophoresis and are spotted by fluorescence detectors as the fr
agments pass
through the detector (Fig. 116). Using this automated method with capillary ele
ctrophoresis,
about 600 base pairs can be sequenced in 2.5 hours. 6 DNA sequencing is most com
monly used to
detect mutations. For example, in infectious disease testing such as sequencing
of HIV for drug
resistance, information about the particular strain is used to establish appropr
iate therapy and
make treatment decisions. TARGET AMPLIFICATION Target amplification systems are
in vitro methods
for the enzymatic replication of a target molecule to levels at which it can be
readily detected.
5 This allows the target sequence to be identi ed and further characterized. There
are numer ous
different types of target ampli cation. Examples include polymerase chain reaction
(PCR),
transcription mediated amplification (TMA), strand displacement amplification (S
DA), and
nucleic acid sequencebased amplification (NASBA). Of these, PCR is by far th
e bestknown and
most widely used technique in clinical laboratories. 6 However, the other nonPC
R methods have
become more popular in recent years. ddA PCR amplicon Denature strands Hybridize

to specific
primers Add DNA polymerase dNTPs Labeled dideoxynucleotides (ddA, ddG, ddT, ddC)
T C A T T
C A C T G G T A T C A T T C A C T G G T A A G T A A G T G A C C A T Primer exten
sion and
termination ddG ddT ddC Primer Primer FIGURE 116. DNA sequencing based upon the S
anger dideoxy
chain termination reaction. The region of interest is rst ampli ed using PCR. Seq
uencespeci c
primers then hybridize with the denatured amplicons followed by extension of the
new strand by
DNA polymerase. At various points in the DNA extension, a dideoxy base (ddA, ddG
, ddT, or ddC) is
incorporated, which stops further extension of DNA. This results in a mixture of
newly
synthesized products of various lengths. Each dideoxy terminator base is labeled
with a speci c
uorescent tag that can be detected by a uorescent detec tor. The fragments are se
parated based
on their size, and uores cence is read on each strand, resulting in the sequence
of the DNA.
1814_Ch11_166185.qxd 7/10/09 2:55 PM Page 173 174 SECTION 2 Basic Immunologi
cal Procedures
Polymerase Chain Reaction The polymerase chain reaction (PCR) is capable of ampl
i fying tiny
quantities of nucleic acid up to levels that can be later detected with various
strategies,
usually involving a hybridization reaction using nucleic acid probes. Kary Mulli
s was awarded the
Nobel Prize in 1993 for developing this technique. Cellular DNA is isolated
from the
clinical sample (either the patients own DNA or DNA from an infectious organism).
PCR requires
several components for the reaction to occur: (1) a thermostable DNA polymerase
(originally
isolated from the bacterium Thermus aquaticus, thus Taq polymerase), (2) deoxynucl
eotides of
each base (referred to as dNTPs), (3) the DNA of interest containing the target
sequence, and (4)
oligonucleotide primers, short segments of DNA that are between 20 and 30 nucleo
tides long, that
are complimentary to opposite strands that ank the sequence of interest to be amp
li ed and
detected. Each PCR cycle consists of three steps: denaturing, annealing,
and extending at
different temperatures. During denaturation, the doublestranded DNA is heated t
o 95C to
separate or denature the DNA into single strands. The mixture is then cooled sli
ghtly to 52C, at
which time the primers bind to or anneal to the complimentary sequences on the s
eparate DNA
strands. During the third step, elon gation, which is typically at 72C, the heat
stable DNA
polymerase binds to the 3 end of each primer and synthe sizes a new strand of DN
A using the
original sample DNA as a template. 12,14 The usual span between primers is betwe
en 50 and 1500
nucleotide bases. 5 Ampli cation of PCR prod ucts is an exponential process
in which,
under optimal conditions, each cycle results in a doubling of product (

Fig. 117). With

newer technology, one cycle takes approx imately 60 to 90 seconds, resulting in
a rapid process.
The resulting DNA fragments, referred to as amplicons, are detected using
various methods.
Initially, amplicons were detected by visualization on agarose gels. For most cl
inical
applications, amplicons are detected using labeled nucleic acid probes. In this
manner, a target
sequence present at fewer than 100 copies can be detected. 15,16 RNA can also be
amplified with
the addition of a step prior to the PCR reaction itself. The enzyme reverse tran
 scriptase is
used to generate copy DNA (cDNA) using the RNA as a template. This reverse trans
cription step
must be performed, since the DNA polymerase used in the PCR reaction recognizes
only DNA
sequences and not RNA. The need for quicker turnaround times and ways to
reduce
contamination led to the development of automated A. B. C. D. 5 5 5 3 5 5
5 5 5 3 3 3
5 3 95C 55C 72C Short product 3 3 3 3 3 3 3 3 5 3 3 3 3 3 5 5 5
5 5 5 5
3 Short product FIGURE 117. Principles of PCR. 1814_Ch11_166185.qxd 7/10/09 2
:55 PM Page
174 CHAPTER 11 Molecular Diagnostic Techniques 175 methods that use closedtube te
chnology to
prevent con tamination of unampli ed samples with ampli ed amplicons from other sam
ples. The
most recent development in PCR technology is the ability to measure the generati
on of ampli cons
in real time, which reduces the amount of time it takes to run a PCR reaction. Rea
ltime PCR is
facilitated by using uorescent dyes or probes and thermal cyclers that can take f
luorescence
readings during each cycle instead of waiting until all the cycles have been com
pleted before
analyzing all the amplicons (endpoint PCR). PCR is an extremely sensitive techn
ique that is used
to amplify a target sequence present in low amounts. It is often used for organi
sms such as
viruses that are hard to grow in the laboratory, and it is so sensitive that it
can actually
detect nonreplicating viral genomes. One of the major uses for PCR is in testing
for the presence
of HIV. Because HIV is an RNA virus, an initial enzyme, reverse transcriptase, m
ust be added to
convert the original RNA into DNA. This is known as reverse transcriptase PCR (R
TPCR). A blood
sample of less than 1 mL is suf cient to perform this test, making it ideal for te
sting neonates.
17 It is also useful in detecting infected indi viduals during the period b
efore antibody
appears, in predicting prognosis, and in monitoring treatment. 17,18 Curre
ntly, there are
several FDAapproved assays for quan titation of HIV1 in human plasma (Table 1
11). RTPCR is
also considered the gold standard for detect ing hepatitis C virus and mon
itoring therapy
in those infected. 19 Additional uses for PCR include identifying Mycobact

erium
tuberculosis and diagnosing early initial infec tion with cytomegalovirus (CMV)
. 19 Another
important use of PCR in immunology is the identi cation of human leukocyte antigen
s (HLAs) for
tis sue transplantation, especially bone marrow transplantation in which the na
ture of the match
determines the success or failure of the transplant. PCR is used to amplify HLA
gene sequences so
that donors and recipients can be matched at the allele level. 19,20 This is esp
ecially helpful
in identifying polymorphisms at the HLA DP locus, which is speci cally related to
graft versus
host reactivity in bone marrow trans plants. HLA typing for class I antigens ha
d traditionally
been done using serological methods, but this was not help ful in identificatio
n of HLA class II
antigens. It has been reported that at least 25 percent of serologically identi ed
class II
antigens were incorrectly identified. 4 Matching of class II antigens at the all
elic level has
proved to increase the success rate of bone marrow transplants. 20 HLA antigens
can be identi ed
by PCR using several dif ferent methods. Sequence speci c oligonucleotide probe h
ybridization
(SSOPH) creates millions of copies of HLA genes in a test tube using a generic p
rimer for the
whole region. 4,21 The ampli ed DNA is then tested with a series of probes. The le
vel of
resolution is controlled by the choice and number of probes used in testing. 21
Numerous probes
are required for clear res olution of HLA antigens to the allele level. This is
currently the
most commonly used method for DNAbased typing. 21 It is utilized for highresol
ution typing by
the National Marrow Donor Program registry. A second method, called sequence spe
cific primer
typing, uses only PCR to identify speci c HLA alleles. There are unique primer p
airs for each
gene locus, so when elec trophoresis is done, presence of a band in
dicates that a
particular gene is there. 4 The PCR product can also be sub jected to direct se
quence base
typing to detect exact alleles present. The latter is more complex and more expe
nsive. The
activity of cytokines (see Chapter 5) can also be measured using PCR. Because cy
tokines tend to
have a short halflife, identi cation of messenger RNA is a more sensi tive metho
d of
determining cytokine production. RTPCR is a highly sensitive method that requir
es only a few
hours to perform and needs only a nanogram of total RNA. Measurement of
cytokines is a
very new eld and has great potential for monitoring and treating numerous hemato
logic
diseases. However, the extreme sensitivity of PCR can be detri mental, because
carryover of
previously ampli ed DNA into unampli ed samples may cause falsepositive results. A
sin gle copy
can cause sample contamination if amplicon is aerosolized. 9 Automation has redu

ced the potential

for con tamination by decreasing the number of manual steps and developing close
d systems in
which amplified products Table 111. FDA Approved Quantitive HIV1 (Viral Load)
Assays*
MANUFACTURER TEST NAME METHOD LINEAR RANGE (copies/mL) Roche Molecular Diagnosti
cs Amplicor HIV1
Monitor RTPCR 1 400750,000 standard 50100,000 ultrasensitive BioMerieux, Inc. Nuc
lisens NASBA
2 1763.47
10 6 Siemens Health Care Diagnostics VERSANT HIV1 RNA 3.0 bDNA 3 75500,
000 Abbott
Molecular, Inc. Abbott RealTime HIV1 Realtime PCR 4010
10 6 1 RTPCR: reverse
transcriptase
PCR 2 NASBA: Nucleic Acid SequenceBased Ampli cation 3 bDNA: Branched DNA *The As
sociation for
Molecular Pathology maintains an uptodate listing of FDA Cleared/Approved Molec
ular Diagnostic
Tests. To access this list, please visit http://amp.org/, under Resources.
1814_Ch11_166185.qxd 7/10/09 2:55 PM Page 175 176 SECTION 2 Basic Immunologi
cal Procedures
are detected in the same tube as the PCR reaction. Automation has also
signi cantly
reduced turnaround time and costs per test. With the availability of reagents in
kit form and the
lower cost of oligonucleotide probes, many more labs are starting to perfor
m molecular
assays. 5 Although it may not be suitable for routine screening, when used as an
adjunct to
standard testing, PCR can be extremely helpful. It will play a more significant
role in diagnosis
as further developments and re nements are made. Falsepositives can be decreased
by physically
separating areas of sample preparation from amplification areas. UV light can be
used to
inactivate DNA within a hood and on pipettes, test tubes, and reaction mixe
s. 9
Additionally, 10 percent bleach (7 mM sodium hypochlorite) is also effec tive a
nd widely used to
decontaminate nonporous work surfaces, including bench tops, hoods, or other are
as that might
come into contact with nucleic acidcontaminated material. There is also a widely
used chemical
method to prevent contamination of unamplified samples with previously ampl
i ed PCR
products. This method is the dUTPUNG system in which dTTP is substituted with d
UTP in the PCR
reaction master mix. This results in dUTP being incorporated into the new
ly synthesized
amplicon. At the beginning of each PCR, the enzyme uracilNglycosylase (UNG) i
s added to the
reaction mixture. This enzyme degrades any nucleic acid containing uracil, s
uch as with a
sample contaminated with previously ampli ed products. UNG is added initially and
allowed to
react for 2 to 10 minutes at 50C prior to the PCR reaction. Upon the initial dena
tura tion
step, the UNG is degraded so that it will not affect subsequently synthesized
products that
incorporate the dUTP in the master mix. 22,23 The use of appropriate posi tive
and negative

controls is also essential. TranscriptionBased Ampli cation In 1989, Kwoh and col
leagues
developed the rst nonPCR nucleic acid ampli cation method, which was a transcripti
on based
amplification system (TAS) that amplified an RNA target. 24 The reaction was a t
wostep process
that involved generation of cDNA from the target RNA followed by reverse
transcription of
the cDNA template into multiple copies of RNA. Multiple cycles result in ampli cat
ion of the
target. From this system, two other nonPCR target ampli fication methods have
been developed
that are currently used in clinical assays: nucleic acid sequence based ampli  ca
tion (NASBA)
and transcription mediated ampli cation (TMA). The advantage of these two methods
is that they
are both isothermal reactions that do not require the use of a thermal cycler. N
ucleic acid
sequence based ampli cation (NASBA) uses the enzymes reverse transcriptase, RNase
H, and bac
teriophage T7 DNAdependent RNA polymerase to isothermally amplify an RN
A target.
Initially, cDNA is formed from target RNA using sequencespeci c primers, one of
which contains
a T7 RNA polymerase binding site at its 5 end. RNase H in the reaction degrades t
he initial
strands of target RNA in the RNA/DNA hybrids. A second primer binds to and exten
ds the newly
synthesized cDNA, resulting in the formation of doublestranded cDNAs with one s
trand containing
the template for the T7 RNA poly merase to bind and generate multiple RNA trans
cripts that can
then serve as target RNA to which the primers bind and synthesize more cDNA (Fig
. 118). Organon
Teknika (now BioMerieux, Durham, NC) developed an electrochemilu minescencebas
ed solidphase
sandwich hybridization system to automate the detection of ampli ed products gen
erated by
NASBA. This system can be adapted to detect various targets; the NucliSENS syste
m is FDAapproved
for quantitation of HIV1. Transcriptionmediated amplification (TMA) is anoth
er
transcriptionbased ampli cation method that, like NASBA, is also isothermal. 15 H
owever, TMA
uses only two enzymes to drive the reaction: an RNA polymerase and a reverse tr
anscriptase,
unlike NASBA, which uses three enzymes. 25 In addition, two primers are used
to target a par
ticular nucleic acid sequence. One of the primers contains a promotor sequence f
or RNA
polymerase. The first step is to produce a DNA template for RNA transcription by
hybridizing the
promotor primer to the target rRNA at a particular site. Reverse transcriptase c
re ates a DNA
copy, called cDNA, of the target rRNA. The original RNA strand is destroyed by t
he RNase activity
of the reverse transcriptase. A new strand of DNA is synthe sized from the DNA
copy, thus making
a doublestranded DNA molecule. RNA polymerase recognizes the promotor sequence
in the DNA

template and begins transcription of multiple copies. 15 Again, each newly synth
esized RNA
molecule serves as a template for a new round of replication. This initiates an
autocatalytic
cycle in which up to 10 billion RNA molecules can be transcribed from the DNA te
mplate in less
than 1 hour. 4,25,26 GenProbe (San Diego, CA) has developed and patented this t
echnique for
detection of Chlamydia tra chomatis, Neisseria gonorrhoeae, Mycobacterium tuber
culosis,
Mycobacterium avium, HIV1, HBV, and HCV from various types of samples. 15,16,27
,28 Strand
Displacement Ampli cation (SDA) Strand displacement amplification (SDA) was origin
ally developed
and patented by Becton Dickinson, Inc. (Franklin Lakes, NJ) in 1991. 29,30,
31 One set of
primers incorporates a specific restriction enzyme site that is later attacked b
y an
endonuclease. The resulting nick created in only one strand by the restriction enz
yme allows
for dis placement of the amplified strands that then, in turn, serve as targets
for further
amplification and nick digestion. A modified deoxynucleotide (dATPS; one of the o
xygen molecules
in the triphosph te moiety h s been repl ced with sulfur) is used to synthe
size
double-str nded, hemiphosphorothio ted DNA recognition site for the restric
tion enzyme
cle v ge th t llows only single-str nd 1814_Ch11_166-185.qxd 7/10/09 2:55 PM
P ge 176 CHAPTER
11 Molecul r Di gnostic Techniques 177 nicking of the unmodified str nd inste d
of cutting
through both str nds. Becton Dickinson currently m rkets this methodology under
the l bel BD
ProbeTec nd is FDA- pproved for the detection of Legionell pneumophil
nd
combin tion kit
for Chl mydi tr chom tis nd Neisseri gonorrhoe e. 32 PROBE AMPLIFICATION R th
er th n directly
mplifying the t rget, there re sever l techniques th t mplify the detection m
olecule or probe
itself. The lig se ch in re ction is one ex mple of this technique. DNA lig se
mpli c tion, or
the lig se ch in re ction (LCR), p tented by Abbott L bor tories, represents one
m in type of
probe mpli c tion technique. 33 DNA lig se enzyme is
rep iring enzyme th t link
s preexisting
DNA str nds together by joining the 5 end of one str nd to the 3 end of nother st
r nd. 1 In
this c se, the enzyme is used to join two p irs of oligonucleotide probes only
fter they h ve
bound to the complement ry t rget sequence, which will hold them in precise
end-to-end
lignment. 1 E ch p ir of probes must hybridize to opposite ends (3 nd 5) of the
t rget DNA
molecule. Once the probes re in pl ce, the lig- se joins the two together. Aft
er this h s
occurred, e ch linked p ir of probes c n be m de single-str nded nd then ct s
templ te for
lig tion of ddition l probes (Fig. 119). The thermost ble lig se enzyme from T
qu ticus

rem ins ctive throughout the therm l cycling process. This technique h s been u
sed for detection
of Borreli burgdorferi, Mycob cteri species, nd Neisseri gonorrhoe e. 15 Lig
tion re ctions
re very speci c, bec use single b se p ir mism tch t the junction of the oligo
nucleotide
probe keeps lig tion from occurring. 2 Dis dv nt ges, however, include the possi
bility of
blunt-end lig tion of probes with- out the t rget being present. Thus, the ex ct
sequence of the
region to be mpli ed must be known to help prevent f lse-positive results from oc
curring. SIGNAL
AMPLIFICATION The l st c tegory of mpli c tion is b sed upon mpli c tion of the si
gn l
gener ted by the probe hybridizing to speci c t rget RNA or DNA. This technique
uses reporter
group (the l beled t g) being tt ched in gre ter numbers to the probe molecule
or incre sing the
sign l intensity gener ted by e ch l beled t g. 15 Bec use the p tient nucleic
cid itself is not
replic ted or mpli ed, this type of technique is less prone to cont min tion prob
lems. However,
the sensitivity m y be lower th n for enzyme mpli c tion methods, so it m y h ve
more limited
use in c ses of low numbers of t rget copies. 15 Reverse tr nscript se Primer T7
promoter site
RNA se T7 RNA polymer se T rget RNA sequence cDNA cDNA 2x cDNA 2 nd primer Multi
ple RNA t rget
templ tes to feed b ck into first step A B C D E FIGURE 118. Tr nscription-b sed
mpli c tion
systems. Non-PCR method of mplifying RNA t rgets. NASBA nd TMA re b sed upon
this methodology;
NASBA uses three enzymes (reverse tr nscript se, RN se H, nd T7 DNA-dependent R
NA polymer se)
while TMA uses only RNA polymer se nd
reverse tr nscript se with inherent RN
se H ctivity.
Initi l steps in the procedure involve gener tion of cDNAs from the t rget RNA u
sing primers
(which incorpor te T7 RNA polymer se binding site; A nd B). RN se H then dest
roys the initi l
str nds of t rget RNA from the RNA cDNA hybrids (C). The cDNA then serves s tem
pl te for the
gener- tion of double-str nded cDNA, with one str nd (cont ining the T7 binding
site) serving s
the templ te for reverse tr nscription nd synthesis of multiple copies of RNA u
sing the T7 RNA
polymer se (D). The RNAs then serve s templ tes for more cDNA templ tes to be m
de, nd the
cycles continue (E). 1814_Ch11_166-185.qxd 7/10/09 2:55 PM P ge 177 178 SECTI
ON 2 B sic
Immunologic l Procedures Perh ps the best known of the sign l mpli c tion systems
is the
br nched DNA (bDNA) sign l mpli c tion system origin lly developed by Chiron Corp
or tion nd now
sold through Siemens He lthc re Di gnostics (T rrytown, NJ). This technique empl
oys multiple
probes nd sever l simult - neous hybridiz tion steps. It h s been comp red to d
ecor ting
Christm s tree nd involves sever l s ndwich hybridiz tions. In the rst step, t r
get-speci c

oligonucleotide probes c p- ture the t rget sequence to solid support. Then

second set of
t rget-speci c probes c lled extenders hybridize to djoining sequences nd ct s
binding sites
for l rge piece c lled the br nched mpli c tion multimer. E ch br nch of the m
pli c - tion
multimer h s multiple side br nches c p ble of binding numerous (up to 10,000) e
nzyme l beled
oligonucleotides onto e ch t rget molecule .4,15 The most recent bDNA system h s
high speci city
nd c n provide qu ntit tive detection over
r nge of sever l orders of m gnitu
de (10 2 to 10 5
copies/mL; Fig. 1110). T rget DNA Den ture Anne l 1 2 1 2 4 3 4 3 4 3 4 3 1 2 1 2
1 2 4 3 Lig te
2 Copies fter one cycle 4 Copies fter two cycles 3 5 3 1 2 3 4 Add primers
5 + + FIGURE
119. LCR. Two p irs of probes re used to hybridize to the t rget DNA molecule. T
hey re joined
together by lig se enzyme. Once bound, the lig ted probes c n ct s templ t
e for ddition l
DNA replic tion. C pture on solid ph se Amplifier #1 Amplifier #2 L beled mplif
ier #3 T rget DNA
Den ture nd hybridize + + * * * * * FIGURE 1110. Br nched ch in mpli c tion. Se
ver l
different probes re used to mplify the sign l r ther th n the t rget DNA itsel
f. The rst probe
c ptures the t rget. Ampli er 1 binds in different pl ce nd forms the b se for
mpli er 2, the
br nched ch in. Ampli er 3 cont ins the sign l. 1814_Ch11_166-185.qxd 7/10/09 2:
55 PM P ge 178
CHAPTER 11 Molecul r Di gnostic Techniques 179 Br nched DNA systems re well
suited to
detecting nucleic cid t rget with sequence heterogeneity, such s hep titis C
nd HIV, bec use
if one or two of the c pture or extender probes f il to hybridize, the sign l-ge
ner ting c p city
is not lost s result of the presence of sever l rem ining probe complexes. Ad
dition lly, the
need for sev- er l independent probe-t rget hybridiz tion events provides
gre
t de l of
speci city. The d nger of f lse-positive results c used by c rryover of t rget m t
eri l is
decre sed, s the t rget itself is not mpli ed. However, nonspeci c loc liz - tion
of re gents
c n result in b ckground mplific tion. 2 Ass ys for hep titis B nd C, HIV-1,
nd CMV h ve been
developed using this method. 15 This system is lso used to qu ntit te HIV-1
with
lower
limit of detection t 75 copies/mL (T ble 111). 32 It h s been v lid ted for t
he qu ntit tion
of vir l lo d testing of ll the subtypes. Therefore, this technique c n
be used for
qu ntit tion nd to help monitor the ther peutic response. DRAWBACKS OF AMPLIFIC
ATION SYSTEMS
Ampli c tion systems in gener l re subject to sever l poten- ti l problems. The g
re test of
these is the potenti l for f lse-positive results c used by cont min ting nuclei
c cids. In the
c se of PCR, LCR, nd SDA, DNA products re the m in source of cont min tion. Fo
r NASBA nd TMA,

RNA products re the possible cont min nts. Therefore, it is nec- ess ry to use
some form of
product in ctiv tion s p rt of
qu lity-control progr m. 15 Sep r ting s mple
prep r tion re s
from mplific tion re s nd using in ctiv tion sys- tems, such s UV light nd
dUTP-UNG chemic l
system in PCR, help to llevi te cont min tion. Recent dv nces in utom ted mp
lific tion
systems employ closed systems where mpli c tion nd detection occur in the s me t
ube, resulting
in much less potenti l for cont min tion nd reducing turn round times nd
l bor costs. The
exquisite sensitivity of these techniques c n present problem in th t very sm
ll numbers of
p thogens present m y not be signi c nt. Ampli c tion procedures c n deter- mine if
RNA or
DNA from
p rticul r org nism
re present but reve ls nothing bout the vi
bility of the
org n- ism. There is lso the possibility th t inhibiting subst nces in clinic l
specimens m y
ffect enzym tic mplific tion methods nd c use f lse-neg tive re ctions. FUTUR
E OF MOLECULAR
DIAGNOSTIC TECHNIQUES When molecul r testing rst bec me v il ble for clinic l l
bor tory
testing in the l te 1980s, it w s restricted to rel tively few high-volume l b
or tories in the
re s of infec- tious dise se testing, hum n genetics, nd histocomp tibility. T
hose e rly ss ys
were speci lty tests nd did not repl ce convention l l bor tory ss ys. They te
nded to be costprohibitive nd very complex. However, t the beginning of the 21st century,
more
nd more
l bor tories beg n performing molecul r di gnostic testing; in some c ses, it i
s now considered
m instre m. This virtu l explosion of molecul r testing in clinic l l bs is due
to sever l
f ctors, including more re dily v il ble re gents, both FDA- pproved nd
n lyte-specific
re gents (ASRs), the trend tow rd re l-time mpli c tion technologies, more choice
s in utom ted
specimen processing (nucleic cid extr ction), nd now the f ct th t molecul r p
rocedures re the
gold st nd rd for di gnosis of m ny dise ses nd infections. 4 For ex mple, in m n
y l bs
testing for Chl mydi tr chom tis nd Neisseri gonorrhoe e, nucleic cidb sed te
sting is
performed inste d of convention l ss ys. Molecul r-b sed ss ys for these gent
s re
competitively priced, provide superior sen- sitivity, nd re rel tively e sy to
perform.
Molecul r testing c n be cost-effective in other w ys s well. For ex mple, r pi
d identific tion
of mycob cteri c n reduce p tient/ hospit l costs by elimin ting the need
for unnecess ry
respir tory isol tion for p tients suspected of h ving tuber- culosis. 15 Detect
ion of
multidrug-resist nt str ins of Mycob cterium tuberculosis m y le d to more tim
ely public he lth
control me sures. Identi c tion of genes for ntimi- crobi l resist nce in othe
r org nisms

will help in more effective tre tment for severe infections. 16 HCV genotyping nd
pretre tment vir l lo d testing in newly di gnosed infections provides the physi
ci n with
v lu ble inform tion to determine ther py regimens. 33 HIV vir l lo d testing c
n pl y role in
di gnosis of HIV in neon tes born to HIV- infected mothers nd in the di gn
osis of cute
HIV infections, which re de ned s the period fter exposure to the virus but bef
ore
seroconversion. 34 In ddition to direct detection of infectious microorg n- ism
s, there re
numerous pplic tions in immunology nd hem tology. Molecul r HLA typing h s rev
olutionized histocomp tibility testing. 4 The imp ct on cl ss II typing h s resulted in signi c
nt decre se in
morbidity nd mort lity in bone m rrow tr nspl nts. Hem tologic dise ses c n ls
o be followed
more e sily now, nd molecul r technology likely will influence tre tment choice
s in cute
leukemi c ses. 4 The problem of cont min tion is beginning to be ddress
ed in numerous
w ys. Multiple closed systems h ve been developed so th t both mplific tion nd
detection t ke
pl ce in the s me test tube. These newer techniques re lso more suit ble to u
tom tion, which
h s brought down l bor costs nd should continue to do so in the future. As di g
nostic kits nd
re gents become more widely v il- ble, use of these techniques will rise. The
incre sed
speci city nd sensitivity of molecul r test- ing will become the st nd rd of pr c
tice in
immunology nd microbiology. R pid turn round time plus incre sing utom ti
on will bring
down the cost nd will llow such techniques to be used in sm ller hospit ls nd
in l rger medic l centers. Molecul r techniques will not repl ce culture for those org nisms
th t re e sily
grown nd identi ed in the l bor tory, nor will it repl ce hem tologic procedures
th t re
st nd rdized nd efficient. However, for esoteric org nisms nd dise ses, molecu
l r techniques
will most 1814_Ch11_166-185.qxd 7/10/09 2:55 PM P ge 179 likely repl ce serol
ogic l techniques
th t re subject to cross- re ctivity nd the unpredict bility of the ntibody r
esponse in
individu l p tients. SUMMARY The unique ch r cteristics of DNA nd RNA m ke them
ide l tools to
use s di gnostic indic tors of dise se. Both re m de up of nucleotides th
t occur in
very specific sequences in different cells nd microorg nisms. They c n be used
to determine the
presence of microorg nisms, p r- ticul r genes th t would indic te c ncerous or
prec ncerous
st tes, nd much more. DNA exists s double-str nded molecule with very speci c
b se p ir
link ges: Adenine p irs with thymine, nd gu nine p irs with cytosine. When the
DNA molecule is
replic ted, the str nds re sep r ted, nd new str nd is cre ted th t is compl
ement ry to the

p rent str nd. This ex ct b se p iring forms the b sis for identi - c tion of unkn
own sequences
through the use of known short nucleic cid sequences c lled probes. Hybridiz ti
on is the p iring
of probe with complement ry DNA str nd. The probe is l beled with m rker f
or detection.
Dot-blots nd s ndwich hybridiz tion techniques re used to directly identify sp
eci c sequences
in p tient s mples. Det iled ch r cteriz tion of DNA c n be ccomplished through
the use of
enzymes c lled restriction endonucle- ses th t cle ve DNA t specific recogniti
on sites. Then
when the DNA is electrophoresed, the resulting fr gments c n be sep r ted on the
b sis of size.
Individu ls or speci c microorg nisms c n be identi ed on the b sis of unique p t- t
erns
obt ined, c lled RFLPs. If the resulting electrophoretic p ttern is tr nsferred
to membr ne for
use in hybridiz - tion, this is known s
Southern blot. Northern blot
s involve
sep r tion of RNA in simil r m nner. Individu l fr gments c n lso be isol ted
nd sequenced to
identify sin- gle nucleotide polymorphisms (SNPs), lso import nt for identi c tio
n purposes.
Hybridiz tion of probes with t rget nucleic cids c n lso t ke pl ce in solutio
n or in int ct
cells. The hybridiz tion protection ss y is n ex mple of the former. The l bel
on the probe is
only protected from degr d tion if it is tt ched to the t rget nucleic cid. If
int ct cells or
tissue is used, this is c lled in situ hybridiz tion. 180 SECTION 2 B sic Immuno
logic l
Procedures DNA/RNA chips represent
microenvironment in which thous nds of hybr
idiz tion
re ctions re possible. Chips re used to identify p rticul r str ins of viruses
such s HIV nd
help determine resist nce to p rticul r ther peutic drugs. Chip technology is l
so being used
more extensively to iden- tify lter tions of genes in v rious c ncers. If there
is not enough
nucleic cid in
specimen for detection, n lysis
mplific tion methods c n
be used to
incre se either the t rget or the detection system. Polymer se ch in re c
tion (PCR) is the
most widely used method of t rget mplific tion. Specific primers coupled with
thermost ble DNA
polymer se re used to m ke copies of the origin l DNA str nds. E ch cycle resul
ts in doubling
of specific t rget sequence, nd pproxim tely 30 cycles produce millions of c
opies of the
origin l mole- cules. This technique is used clinic lly to identify viruses such
s hep titis B,
hep titis C, nd HIV, nd to perform HLA typing nd me sure cytokine ctivity. T
MA, NASBA, nd
SDA re t rget mpli c tion methods b sed on the use of DNA templ te to m ke num
erous RNA
copies of the t rget sequence. Chl mydi tr chom tis, Mycob cterium tuberculosis
, Mycob cterium
vium, nd HIV re some ex mples of microorg nisms identified by this method. Pr
obes r ther th n

t rget nucleic cid c n be mpli ed by the lig se ch in re ction (LCR). LCR ctu l
ly splices two
DNA probes together to m ke
single-str nd copy of the t rget nucleic cid sequ
ences. Sign l
mpli c tion is nother me ns of detecting sm ll mounts of t rget nucleic cid. T
he br nched DNA
method uses
series of hybridiz tion steps, including
c pture probe,
n extender
probe, nd multimer probe to enh nce the sign l to
detect ble level. All mp
li c tion
techniques re subject to f lse-positive re ctions resulting from either cont mi
n tion with previously mplified products or b ckground re ctivity. In ddition, qu litycontrol
procedures nd l bor tory st n- d rdiz tion need to be developed. However,
molecul r
techniques re exceedingly speci c nd r pid nd will even- tu lly repl ce serolog
ic l techniques
for identific tion of org nisms th t re dif cult to culture nd ntigens such s
HLA, in which
sm ll differences must be distinguished. 1814_Ch11_166-185.qxd 7/10/09 2:55 PM
P ge 180
CHAPTER 11 Molecul r Di gnostic Techniques 181 A 23-ye r-old m le presented hims
elf to n urgent
c re center with urethr l disch rge nd
red r sh on his feet nd fore rms. He
st ted th t he
h d h d unprotected sex with prostitute within the p st 2 weeks. He w s tre te
d empiric lly for
chl mydi , gonorrhe (GC), nd Rocky Mount in spotted fever (RMSF). The followin
g l bor - tory
tests were ordered: Chl mydi /GC PCR; RPR; serology testing for RMSF; hep t
itis B, C; nd
HIV. All test results were neg tive except for Chl mydi PCR. Fourteen d ys l te
r, the p tient
presents to n infec- tious dise se clinic with he d che, fever of 38.1C, n u
se
nd
vomiting, my lgi , 2 weeks of f tigue
nd mouth ulcers,
nd
10 lb
weight loss
within the p st 3 weeks. L bor tory tests t this time reve l the p tient is l
ymphocytopenic,
nd his liver function enzymes re slightly elev ted. Questions . Wh t is
the most likely
di gnosis of this p tient: Chl mydi , HIV, HSV, syphilis, hep titis B, or
hep titis C? b.
Wh t l bor tory test would you like repe ted on the second visit, nd why? c. Wh
t molecul r test
could h ve been ordered on the p tients rst visit th t m y h ve likely been ble t
o provide
di gnosis for him? CASE STUDY 1814_Ch11_166-185.qxd 7/10/09 2:55 PM P ge 181
182 SECTION 2
B sic Immunologic l Procedures GEL ELECTROPHORESIS OF DNA Principle This experim
ent will
demonstr te the use of restriction enzymes to cle ve DNA from the b cterioph ge
l mbd (48,502
b se p irs in length). The resulting fr gments will be sep r ted out using gel e
lectrophoresis.
The three restric- tion endonucle ses used re B mHI, EcoRI, nd HindIII. B mHI
nd HindIII re
from H emophilus influenz e, nd EcoRI is from Escherichi coli. Three s mple tu
bes of l mbd DNA

re prep red nd incub ted t 37C with one of the enzymes in e ch tube. A fourth
tube, the
neg tive control, is incub ted without n endonucle se. The DNA s mples re then
lo ded into
wells of n g rose gel nd re electrophoresed. An electric l eld pplied cross
the gel c uses
the DNA fr gments in the s mples to migr te through the gel tow rd the positive
electrode. The
sm ller DNA fr gments migr te f ster th n the l rger ones. E ch enzyme will prod
uce different
set of b nds, representing different-sized fr gments. Only one b nd should be pr
oduced from the
control tube, in which no enzym tic digestion took pl ce. The resulting p ttern
for e ch is
c lled DNA nger- print. After electrophoresis, compound th t binds to DNA is
used to m ke
the b nds visible. A Pol roid c mer c n be used to m ke perm nent record of t
he results.
S mple Prep r tion Dehydr ted DNA is reconstituted by dding distilled or deioni
zed w ter. The
dehydr ted DNA nd enzymes used in the kit c n be stored t room temper
ture for up to
2 ye rs. DNA nd the enzymes re not of hum n origin, but they should be h ndled
using st nd rd
s fety prec utions. Re gents, M teri ls, nd Equipment Kit from C rolin Biologi
c l Supply
Comp ny, which con- t ins the following: Vi ls of l mbd DNA 6 vi ls B mHI 6 vi
ls EcoRI 6 vi ls
HindIII 6 vi ls lo ding dye 6 control re ction tubes Tris-bor te-ethylenedi mine
tetr cetic cid
(TBE) buffer concentr te 6 st ining tr ys 15 1.5 mL tubes Ag rose (3.2 gr ms for
400 mL) Flo ting
tube r ck Addition l equipment needed: Micropipettors (010 L or 020 L) Micropipett
e tips
Gel electrophoresis ch mbers nd power supplies Boiling w ter b th or microw ve
oven W ter b th
t 60C W ter b th t 37C White light box/UV light box with mid or long w velength
for viewing
st ined gels 0.25 N HCl nd 0.25 N N OH for decont min ting ethidium bromide st
ining solution
Option l equipment: Pol roid gun c mer or other c mer for recording results CA
UTION: Ethidium
bromide is mut gen nd sus- pected c rcinogen. If it is used for st i
ning, gloves
should be worn t ll times, nd st ining should be con- ned to
sm ll re of t
he l bor tory.
After st ining,
funnel c n be used to dec nt s much ethidium bro- mide
s p
ossible from
the st ining tr y b ck into the stor ge cont iner. The st in m y be reused t
o st in 15 or
more gels. Used st ining solutions nd gels need to be dis bled rst before disc r
ding. The steps
for doing so re listed in the procedure. Procedure* A. Restriction Digest 1. To
reconstitute the
DNA, dd 280 L of distilled or deionized w ter to e ch of two tubes of DN
A provided.
Allow to sit for 5 minutes. 2. Hold e ch closed tube rmly t the top, nd ick the
side of the
tube repe tedly to mix the contents. Do this for 1 full minute. 3. Allow tubes t
o st nd for n

ddition l 5 minutes. The DNA solution should look slightly op que. It is very i
mport nt th t the
DNA be tot lly dissolved. 4. Aliquot 90 L of DNA into e ch of six 1.5 mL tubes to
be used by
e ch l bor tory group. 5. Select four 0.2 mL tubes, e ch of which cont ins dif
ferent enzyme.
The blue tube cont ins B mHI, the pink tube cont ins EcoRI, the green tube cont
ins HindIII, nd
the yellow tube h s no enzyme t ll. *Procedure is t ken from the Te chers M nu
l included with
the kit c lled Restriction Enzyme nd DNA Kit from C rolin Biologic l Supply Co
mp ny,
Burlington, NC. EXERCISE 1814_Ch11_166-185.qxd 7/10/09 2:55 PM P ge 182 CHAPT
ER 11 Molecul r
Di gnostic Techniques 183 6. Add 20 L of DNA into e ch of the colored tubes. Mix
the DNA nd
enzymes by pipetting up nd down sever l times. There should be no concentr tion
of the blue
color in the bottom of the tube if DNA nd enzymes re mixed sufficiently. Use
fresh pipette
tip when
dding DNA into e ch re ction tube to prevent cross-cont min ti
on. 7. Incub te
ll re ction tubes for minimum of 20 minutes t 37C. B. Prep r tion of C st Ag
rose Gel 1.
Prep re the TBE buffer solution by pouring the con- tents of the 20X TBE concent
r te into 3 L
sk or c rboy. Add 2850 mL of distilled or deionized w ter for
n l volume of 3 L
. Stir for 1
to 2 minutes. 2. Prep re 0.8 percent g rose solution by dding 3.2 gr ms
of g rose to 400
mL of TBE buffer in cle n 1 L
sk. Cover with luminum foil nd he t in boili
ng w ter b th
for 10 to 20 minutes. Swirl to ensure th t no undissolved g rose rem ins.
Altern tively,
prep re four individu l
sks of g rose by weighing out 0.8 gr ms of g rose for
e ch sk nd
dding it to 100 mL of buffer in 250 mL
sk. Cover sks with p r lm, nd he t one
individu lly in
microw ve until the solution becomes cle r. 3. Pl ce sks in
60C w ter b th
to cool the g rose to 60C before pouring. 4. Se l ends of gel-c sting tr y with
t pe, or use
self- se ling tr y. Insert the well-forming comb. 5. C refully pour enough g ro
se solution into
the c st- ing tr y to ll it to depth of bout 5 mm. The gel should cover only
bout
one-third the height of comb teeth. While the g rose is still liquid, pipe
tte tip or
toothpick c n be used to move l rge bubbles or solid debris to the sides or end
of tr y. 6. Let
gel solidify without disturbing for bout 20 min- utes. Gel will become cloudy
s it solidi es.
7. Unse l ends of c sting tr y, nd pl ce the gel in the gel box for electrophor
esis with the
comb t the neg tive (bl ck) end. 8. Fill gel box with 1X TBE buffer to
level
th t just covers
the entire surf ce of the gel. 9. Gently remove comb by pulling str ight up, bei
ng c reful not to
rip the wells. 10. M ke sure th t the wells re completely covered with buffer.
If there re ny
dimples, slowly dd buffer until the dimples dis ppe r. C. Electrophoresis 1. Ad

d 2 L of lo ding
dye to e ch re ction tube, nd mix dye with digested DNA by t pping the tube on
the l b bench, or
pulse with microcentrifuge. 2. Use micropipette to lo d contents of e ch re
ction tube into
sep r te well in the gel. Lo d wells by ste dying pipette with two h nds. Expel
ny ir in the
micropipette tip end before lo ding gel. Pl ce pipette tip through surf ce of bu
ffer, nd
position the tip over well. Slowly expel the mixture. The lo ding dye should c
use the s mple
to sink to the bottom of the well. 3. Close the top of the electrophoresis ch mb
er, nd connect
electric l le ds to power supply, node to node nd c thode to c thode. 4. Tu
rn on power
supply, nd set volt ge for pproxi- m tely 125 volts. Note th t this volt ge wi
ll llow for
dequ te sep r tion on mini-gels in pproxi- m tely 1 hour. 5. Allow the DNA to
migr te until the
bromphenol blue b nd from the lo ding dye is ne r the end of the gel. 6. Turn of
f power supply,
disconnect le ds from the inputs, nd remove the top of the electrophoresis ch m
ber. 7. C refully
remove c sting tr y, nd slide gel into st ining tr y. 8. Flood gel with
ethidium bromide
solution, nd llow to st in for 10 to 15 minutes. Dec nt st in b ck into the co
nt iner, using
funnel. 9. Rinse gel nd tr y under running w ter to remove excess ethidium brom
ide solution. 10.
Pl ce on UV light box for viewing nd photogr phy. NOTE: Another version of this
kit comes with
C rolin BLU dye, which voids the h z rds of working with ethidium bromide nd
which c n be
viewed with reg- ul r light box inste d of one with UV light source. Dis bli
ng the Gels nd
Used St ining Solution 1. Add one volume of 0.05 M KMnO 4 , nd mix c refully. 2
. Add one
volume of 0.25 N HCl,
nd mix c refully. Let st nd t room temper ture fo
r sever l hours.
3. Add one volume of 0.25 N N OH, nd mix c refully. 4. Disc rd dis bled solutio
n down sink.
Disc rd gels in the regul r tr sh. Interpret tion of Results Digestion with B
mHI should
produce five fr gments, EcoRI should produce ve fr gments, nd HindIII should p
roduce six
fr gments. Bec use e ch enzyme recognizes
distinctive b se p ir sequence,
di
fferent p ttern
will be obt ined with e ch enzyme, nd no sep r tion will t ke pl ce in the cont
rol tube. This
s me technique is used with hum n DNA to obt in
DNA fingerprint or to look for
p rticul r
mut tions th t result in differences in numbers of b nds. 1814_Ch11_166-185.qxd
7/10/09 2:55 PM
P ge 183 1. Which technique is b sed on probe mpli c tion r ther th n mpli c tion
of the
t rget in question? . PCR b. TMA c. Dot-blot d. Br nched ch in mpli c tion 2. Ho
w re DNA nd
RNA different? . Only RNA cont ins ur cil. b. Only DNA cont ins cytosine. c. DN
A is less st ble
th n RNA. d. RNA is usu lly double-str nded. 3. All of the following re true of

nucleic cid
probe except . it is short nucleic cid ch in. b. it c n be m de up of either
DNA or RNA. c.
it is l beled with m rker for detection. d. it tt ches to double-str nded DNA
. 4. Which of
the following techniques uses restriction enzymes, electrophoresis, nd then
tr nsfer of DNA
fr g- ments onto solid m trix, followed by probing with l beled probes? . Dot
-blot b. Southern
blot c. Hybridiz tion protection ss y d. LCR 5. All of the following would be
dv nt ges of
nucleic cid mpli c tion techniques except . detection of nonvi ble org nisms. b
. extreme
sensitivity. c. e rly detection of dise se. d. low cost. 6. Which best describ
es the
principle of DNA chip technology? . Chips cont in multiple probes on their s
urf ce. b. Chips
cont in multiple copies of one unique probe. c. The s mple is l beled with
uore
scent t g. d.
Hybridiz tion is detected by the presence of r dio ctivity. 7. A hybridiz tion r
e ction involves
which of the following? . Binding of two complement ry DNA str nds b. Cle ving
of DNA into
sm ller segments c. Sep r ting DNA str nds by he ting d. Incre sing the number o
f DNA copies 8.
Which best describes the PCR? . Two probes re joined by lig ting enzyme. b.
RNA copies of the
origin l DNA re m de. c. Extender probes re used to detect positive re ction
. d. Primers re
used to m ke multiple DNA copies. 9. During PCR, wh t h ppens in the nne ling s
tep? . The
primers bind to the t rget DNA. b. Str nds re sep r ted by he ting. c. An RNA c
opy is m de. d.
Protein is m de from the DNA str nds. 10. Wh t is the function of restriction en
donucle ses? .
They splice short DNA pieces together. b. They cle ve DNA t speci c sites. c. The
y m ke RNA
copies of DNA. d. They m ke DNA copies from RNA. 11. To wh t does in situ hybrid
iz tion refer? .
Nucleic cid probes re ct with int ct cells within tissues. b. Probes re protec
ted from
degr d tion if hybridized. c. RNA polymer se copies messenger RNA. d. Hybridiz t
ion t kes pl ce
in solution. 12. Wh t is PCR used for in clinic l settings? . Detection of e rl
y HIV infection
b. Determin tion of speci c HLA ntigens c. Me surement of cytokines d. All of the
bove REVIEW
QUESTIONS 184 SECTION 2 B sic Immunologic l Procedures 1814_Ch11_166-185.qxd 7/
10/09 2:55 PM
P ge 184 CHAPTER 11 Molecul r Di gnostic Techniques 185 References 1. P rslow, T
G. Molecul r
genetic techniques for clinic l n lysis of the immune system. In Stites, DP, Te
rr, AI, nd
P rslow, TG (eds): Medic l Immunology, ed. 9. Appleton & L nge, St mford,
CT, 1997, pp.
309318. 2. Lo, DYM, nd Chiu, RWK. Principles of molecul r biology. In Burtis,
CA, Ashwood,
ER, nd Bruns, DE (eds): Tietz Textbook of Clinic l Chemistry nd Molecul r
Di gnostics, ed.
4. Elsevier S unders, St. Louis, 2006, pp. 13931406. 3. Wisec rver, J. The ABCs o

f DNA. L b Med
28:48, 1997. 4. Podzorski, RP. Molecul r methods. In Detrick, B, H milton, RG,
nd Folds, JD, et
l. (eds): M nu l of Molecul r nd Clinic l L bor tory Immunology, ed. 7. Am
eric n Society
for Microbiology, W shington, DC, 2006, pp. 2652. 5. Tenover, FC, nd Unger, ER.
Nucleic cid
probes for detec- tion nd identi c tion of infectious gents. In Persing, DH, Smi
th, TF, nd
Tenover, FC, et l. (eds): Di gnostic Molecul r Microbiology: Principles nd App
lic tions.
Americ n Society for Microbiology, W shington, D.C., 1993, pp. 325. 6. Forbes, BA
, S hm, DF, nd
Weissfeld, AS. B iley nd Scotts Di gnostic Microbiology, ed. 10. Mosby, St. Loui
s, 1998, pp.
188207. 7. Whetsell, AJ, Drew, JB, nd Milm n, G, et l. Comp rison of three nonr
dioisotopic
polymer se ch in re ction-b sed meth- ods for detection of hum n immunodeficienc
y virus type 1. J
Clin Microbiol 30:845, 1992. 8. H nsen, CA. Clinic l pplic tions of molecul r b
iology in
di gnostic hem tology. L b Med 24:562, 1993. 9. Unger, ER, nd Piper, MA. Molecu
l r di gnostics:
B sic prin- ciples nd techniques. In Henry, JB (ed): Clinic l Di gnosis nd M
n gement by
L bor tory Methods, ed. 20. WB S unders, Phil delphi , 2001, pp. 12751295. 10
. Friedrich,
MJ. New chip on the block. L b Med 30:180, 1999. 11. Check, W. Clinic l microbio
logy eyes nucleic
cid-b sed tech- nologies. ASM News 64:84, 1998. 12. Schrenzel, J, Hibbs, JR, n
d Persing, DH.
Hybridiz tion rr y technologies. In Henry, JB (ed): Clinic l Di gnosis
nd M n gement by
L bor tory Methods, ed. 20. WB S unders, Phil delphi , 2001, pp. 12961302. 13. S
nger, F,
Nicklen, S, nd Coulson, AR. DNA sequencing with ch in termin ting inhibitors. P
roc N tl Ac d Sci
USA 74:5463, 1977. 14. Wittwer, CT, nd Kusuk w , N. Nucleic cid techniques. In
Bruns, DE,
Ashwood, ER, nd Buris, CA (eds): Fund ment ls of Molecul r Di gnostics. S under
s Elsevier, St.
Louis, 2007, pp. 4679. 15. Persing, DH. In vitro nucleic cid mpli c tion techniqu
es. In
Persing, DH, Smith, TF,
nd Tenover, FC, et
l. (eds): Di gnostic Molec
ul r
Microbiology: Principles nd Applic tions. Americ n Society for Microbiology, W
shington, DC,
1993, pp. 5187. 16. Mitchell, PS, nd Persing, DH. Current trends in molecul r mi
crobiology. L b
Med 30:263, 1999. 17. Const ntine, N, nd Zh o, R. Molecul r b sed l bor tory te
st- ing nd
monitoring for hum n immunodeficiency virus infections. Clin L b Sci 18(4):2
63270, 2005. 18.
Weikersheimer, PB. Vir l lo d testing for HIV: Beyond the CD4 count. L b Med 30:
102, 1999. 19.
Eisenbrey, AB. Choosing
ppropri te methodologies on the clinic l environme
nt: Immunologic l
techniques versus molecul r methods. In Rose, NR, De M cArio, EC, nd Folds, JD,
et l. (eds):
M nu l of Clinic l L bor tory Immunology, ed. 5. Americ n Society for Microbiolo

gy, W shington,
DC, 1997, pp. 108115. 20. Perkins, HA. HLA typing for tr nspl nt tion of stem cel
ls form
unrel ted donors. L b Med 28:451, 1997. 21. Schmitz, JL. HLA typing using m
olecul r
methods. In Colem n, WB, nd Tsong lis, GJ (eds): Molecul r Di gnostics for the
Clinic l
L bor tori n, ed. 2. Hum n Press, Totow , NJ, 2006, pp. 485493. 22. P ng, J, Mod
lin, J, nd
Yolken, R. Use of modi ed nucleotides nd ur cil-DNA glycosyl se (UNG) for the con
trol of cont min tion in the PCR-b sed mpli c tion of RNA. Mol Cell Probes 6(3):251256, 1992.
23. Pruvost,
M, Gr nge, T, nd Geigl, EM. Minimizing DNA cont min tion by using UNG-coupled q
u ntit tive
re l-time PCR on degr ded DNA s mples: Applic tion to ncient DNA studies. Biote
chniques
38(4):569575, 2005. 24. Kwoh, DY, D vis, GR, Whit eld, KM, Ch pelle, LJ, DiMichele,
LJ, nd
Gi nge s, TR. Tr nscription-b sed mpli c tion sys- tem nd detection of mpli ed hu
m n
immunode ciency virus type 1 with be d-b sed s ndwich hybridiz tion form t. Proc
N tl Ac d Sci
USA 86:11731177, 1989. 25. Hill, CS. Molecul r di gnostics for infectious dise se
s. J Clin Lig
Ass y 19:43,1996. 26. New directions in molecul r di gnostic testing. Gen-Probe
Inc., S n Diego,
pp. 112. 27. http://www.gen-probe.com/prod_serv/clinic l. sp, ccessed November 1
, 2007. 28.
Nelson, NC. Molecul r tools for building nucleic cid IVDs. IVD Technology, M rc
h/April 1997. 29.
Little, MC, Andrews, J, Moore, R, Bustos, S, Jones, L, Embres, C, Durmo
wicz, G, et l.
Str nd displ cement mpli- c tion nd homogenous re l-time detection incorpor ted
in
second-gener tion DNA probe system. BDProbe TecET Clin Chem, 45:777784, 1999. 30.
W lker, GT,
Fr iser, ML, Schr m, JL, Little, MC, N dequ, JG, nd M linowski, DP. Str nd disp
l cement
mpli c tion n isotherm l in vitro DNA mplific tion technique. Nucl Acid Res 20:1
6911696,
1992. 31. W lker, GT, Little, MC, N de u, JG,
nd Sh nk, DD. Isotherm l
in vitro
mplific tion of DNA by restriciont enzyme/DNA polymer se system. Proc N tl Ac
d Sci USA,
89:392396, 1992. 32. http://www. mp.com/resources, ccessed November 30, 2007. 33
. Zein, NN.
Clinic l signi c nce of hep titis C virus genotypes. Clin Micro Rev 13:223235, 2000
. 34.
Ferreir -Gonz lez, A, Vers lovic, J, H becbu, S, nd C liendo, AM. Molecul r met
hods in di gnosis
nd monitoring of infec- tious dise ses. In Bruns, DE, Ashwood, ER, nd Burtis,
CA (eds):
Fund ment ls of Molecul r Di gnostics. S unders Elsevier, St. Louis, 2007, 1
71196. CHAPTER
11 Molecul r Di gnostic Techniques 185 1814_Ch11_166-185.qxd 7/10/09 2:55 PM
P ge 185 12 Flow
Cytometry nd L bor tory Autom tion Sus n M. Orton, PhD, MT (ASCP), D (ABMLI) LE
ARNING OBJECTIVES
After nishing this ch pter, the re der will be ble to: 1. List nd describe the

function of
e ch of the m jor components of
ow cytometer. 2. Discuss the difference between
intrinsic nd
extrinsic p r meters in ow cytometry. 3. List the dv nt ges nd dis dv nt ges of
utom ted
testing in clinic l immunology l b. 4. Discuss the principle of hydrodyn mic f
ocusing within
the ow cytometer. 5. De ne the concept of uorescence in ow cytometry. 6. Expl in the
difference between forw rd- ngle light sc tter (FSC) nd side sc tter (SSC). 7.
Discuss the
difference between n lyzing ow cytometry d t using single-p r meter histogr ms
nd
du l-p r meter dot plots. 8. List sever l clinic l pplic tions for ow cytometry.
9. Apply
knowledge of v rious T- nd B-cell surf ce ntigens to identify v rious cell pop
ul tions. 10.
Discuss dv nt ges nd dis dv nt ges of utom ted immuno ss y n lyzers. 11. Des
cribe the
difference between
r ndom ccess nd
b tch n lyzer. 12. De ne ccur cy, preci
sion,
report ble r nge, n lytic sensitivity, n lytic speci city, report ble r nge, ref
erence
interv l. KEY TERMS Accur cy An lytic sensitivity An lytic speci city Autom tic s
mpling B tch
n lyzer Du l-p r meter dot plot Extrinsic p r meter Flow cytometer Forw rd- ngl
e light sc tter
G te Immunophenotyping Intrinsic p r meter L ser light source Monoclon l ntibod
ies Precision
R ndom ccess n lyzer Reference interv l Report ble r nge Right- ngle light sc
tter
Single-p r meter histogr m 186 1814_Ch12_186-200.qxd 7/13/09 2:56 PM P ge 186
CHAPTER 12 Flow
Cytometry nd L bor tory Autom tion 187 CELL FLOW CYTOMETRY Principle The rst tru
e ow
cytometers were developed in the 1960s, prim rily for rese rch purposes. However
, in the e rly
1980s, physici ns st rted seeing p tients with new mysterious dise se ch r cte
rized by drop
in circul ting helper T cells, nd ow cytometry w s brought into the clinic l l b
or tory. Since
th t time, ow cytometry h s been routinely used to identify infection with HIV,
nd
immunophenotyping cellsidentifying their surf ce ntigen expressionh s become
m
jor component
of the worklo d in most clini- c l immunology l bor tories. Cell flow cytometry
is n utom ted
system in which single cells in
fluid suspension re n lyzed in term
s of their
intrinsic light-sc ttering ch r cteristics nd re simult neously ev lu ted for
extrinsic
properties (i.e., the presence of specific surf ce proteins) using fluores
cent l beled
ntibodies or probes. By using sever l different u- orochromes, cytometers c n
simult neously
me sure multiple cellul r properties. Another signi c nt dv nt ge of flow cyto
metry is th t
bec use the flow r te of cells within the cytometer is so r pid, thous nds
of cells c n be
n lyzed in seconds, llowing for the ccur te detection of very r re cells. The
m jor components

of
ow cytometer include the fluidics, the l ser light source, the optics
nd
photodetectors, nd
computer for d t
n lysis nd m n gement. 1 Instrument ti
on Fluidics For
cellul r p r meters to be ccur tely me sured in the ow cytometer, it is cruci l
th t cells p ss
through the l ser one t time in single file. Cells re processed into suspension, nd the
cytometer dr ws up the cell suspension nd injects the s mple inside c rrier s
tre m of isotonic
s line (she th uid) to form
l min r ow. The s mple stre m is constr ined by the
c rrier
stre m nd is thus hydron mi- c lly focused so th t the cells p ss single file t
hrough the
intersection of the l ser light source (Fig. 121). L ser Light Source E ch cell i
s interrog ted
by light source th t typic lly con- sists of one or more sm ll ir-cooled l se
rs. The
w velength of monochrom tic light emitted by the l ser in turn dict tes which uor
ochromes c n be
used. A uorochrome, or u- orescent molecule, is one th t
bsorbs light cro
ss
spectrum of w velengths nd emits light of lower energy cross spectrum of lon
ger w velengths.
E ch uorochrome h s distinctive spectr l p ttern of bsorption (excit tion) nd
emission. Not
ll fluorochromes c n be used with ll l sers, bec use e ch uorochrome h s distin
ct spectr l
ch r- cteristics. Therefore, the choice of fluorochromes to be used in n ss y
depends on the
light source used for exci- t tion (T ble 121). Most clinic l ow cytometers h ve
t le st one
l ser, typic lly rgon, th t emits t 488 nm. Newer cytometers lso h ve
secon
d l ser,
helium-neon (He-Ne), th t emits t 633 nm. This llows more fluorochromes to be
n lyzed in
single tube t one time. As
result of cell p ssing through the l ser, light
is sc ttered in
m ny direc- tions. The mount nd type of light sc tter (LS) c n provide v lu bl
e inform tion
bout cells physic l properties. Light t two specific ngles is me sured by th
e flow
cytometer: forw rd- ngle light sc tter (FSC), nd orthogon l right- ngle light
sc tter, or
90-degree- ngle light sc tter (SSC). CHAPTER OUTLINE CELL FLOW CYTOMETRY Princip
le
Instrument tion D t Acquisition nd An lysis S mple Prep r tion Clinic l Applic
tions
IMMUNOASSAY AUTOMATION V lid tion SUMMARY CASE STUDIES REVIEW QUESTIONS REFERENC
ES T ble 55-2
Comp rison of Tests Used EXCITATION FLUOROCHROME EMISSION WAVELENGTH OR DYE
WAVELENGTH 488
nm ( rgon Fluorescein 530 l ser) isothiocy n te (FITC) Phycoerythrin (PE) 580
Propidium iodide
(PI) 620 Peridinin chlorophyll 670 (PerCP) 633 nm (He-Ne Allophycocy nin 670
l ser) (APC) Cy-5
670 T ble 12-1. Fluorochromes Commonly Used in Clinic l Flow Cytometry 1814_Ch12
_186-200.qxd
7/13/09 2:56 PM P ge 187 FSC, or light sc ttered t less then 90 degrees, is c
onsidered n

indic tor of size, while the SSC sign l is indic tive of gr nul rity or intr cel
lul r complexity
of the cell. Thus, these two v lues, which re considered intrinsic p r me- ters
, c n be used to
ch r cterize different cell types. If one looks t s mple of whole blood on
o
w cytometer,
where ll the red blood cells h ve been lysed, the three m jor pop- ul tions of
white blood cells
(lymphocytes, monocytes, nd gr nulocytes) c n be roughly differenti ted from e
ch other b sed
solely on their intrinsic p r meters (FSC nd SSC; Fig. 122). Unlike FSC nd SSC,
which
represent light-sc ttering properties th t re intrinsic to the cell, extrinsic
p r me- ters
require the ddition of
fluorescent probe for their detection. Fluorescent l b
eled ntibodies
bound to the cell re interrog ted by the l ser. By using uorescent molecules wit
h v rious
emission w velengths, the l bor tori n c n simult neously ev lu te n individu l
cell for sever l
extrin- sic properties. The clinic l utility of such multicolor n lysis is enh
nced when the
uorescent d t re n lyzed in con- junction with FSC nd SSC. 2 188 SECTION 2 B
sic
Immunologic l Procedures 90-degree light sc tter detector Filter Filter Fluoresc
ence detector
Electric l sign ls Computer Printer Dichroic mirror Lens Forw rd ngle light sc
tter detector
Ch rged pl tes c n deflect drops to sep r te popul tions L ser Lens Sheth fluid
Cells in liquid
suspension Air pressure FIGURE 121. Flow cytometry. Components of
l ser-b sed ow
cytometer
include the uidics sys- tem for cell tr nsport tion, l ser for cell illumin tio
n, photodetectors for sign l detection, nd
computer-b sed m n gement sys- tem. Both forw r
d nd 90-degree
LS re me sured, indic ting cell size nd type. 1000 800 600 400 200 0 0 200 1.
Lymphocytes 2.
Monocytes 3. Gr nulocytes 400 600 FSC-Height S S C - H e i g h t 800 1000 1 2 3
FIGURE 122.
Peripher l blood leukocyte n lysis by simult neous ev lu tion of forw rd- ngle
light sc tter
(FCS) nd 90-degree LS (SSC). B sed on the intrinsic ch r cteristics of size (FS
C) nd
gr nul rity (SSC) only, the three m in popul tions of white cells (lymphocytes,
monocytes, nd
gr nulocytes) c n be discrimin ted into individu l popul tions. 1814_Ch12_186-20
0.qxd 7/13/09
2:56 PM P ge 188 CHAPTER 12 Flow Cytometry nd L bor tory Autom tion 189 Optics
The v rious
sign ls (light sc tter nd uorescence) gener- ted by the cells inter ction with t
he l ser re
detected by photomultiplier tubes nd detectors. The number of uo- rochromes c p
ble of being
me sured simult neously is limited by the number of photodetectors in the ow cyto
me- ter. The
speci city of e ch photodetector for given b nd length of w velengths is chieve
d by the
rr ngement of series of optic l lters th t re designed to m ximize collec- ti
on of light

derived from
specific fluorochrome while minimizing collection of light from o
ther
fluorochromes used to st in the cells. The newer ow cytometers ctu lly use ber-op
tic c bles to
direct light to the detectors. Most clinic l ow cytometers in use tod y re c p b
le of three- to
six-color detection using one to two l sers. When fluorescent light re ches
the
photomultiplier tubes, it cre tes n electric l current th t is converted into
volt ge pulse.
The volt ge pulse is then converted (using v rious methods depending on the m nu
f cturer) into
dig- it l sign l. The digit l sign ls re proportion l to the intensity
of light
detected. The intensity of these converted sign ls is me sured on rel tive sc
le th t is
gener lly set into 1 to 256 ch nnels, from lowest energy level or pulse to the h
ighest level.
D t Acquisition nd An lysis Once the intrinsic nd extrinsic cellul r properti
es of m ny cells
(typic lly 10,000 to 20,000 events re collected for e ch s mple) h ve been collec
ted nd the
d t digit lized, it is re dy for n lysis by the oper tor. E ch p r meter c n b
e n lyzed
independently or in ny combin tion. Gr phics of the d t c n be represented in
multiple w ys.
The first level of represent tion is the single-p r meter histogr m, which plots
chosen
p r meter (gener lly uorescence) on the x- xis versus the number of events on the
y- xis, so
only
single p r meter is n lyzed using this type of gr ph (Fig. 123).
The oper tor
c n then set m rker to differ- enti te between cells th t h ve low levels of f
luorescence
(neg tive) from cells th t h ve high levels of fluorescence (positive) for p r
ticul r
fluorochrome l beled ntibody. The computer will then c lcul te the percent ge o
f neg - tive
nd positive events from the tot l number of events collected. The next level of r
epresent tion
is the biv ri nt histogr m, or du l-p r meter dot plot, where e ch dot represent
s n individu l
cell or event. Two p r meters, one on e ch xis, re plotted g inst e ch other.
E ch p r meter
to be n - lyzed is determined by the oper tor. Using du l-p r meter dot plots,
the oper tor c n
then dr w g te round
pop- ul tion of interest nd n lyze v rious p r meters
(extrinsic
nd intrinsic) of the cells cont ined within the g ted region (Fig. 124). This l
lows the
oper tor to screen out debris nd isol te subpopul tions of cells of interest. W
hen n lyzing
popul tion of cells using
du l- p r meter dot plot, the oper tor chooses
which p r meters
to n lyze on both the x- nd y- xes. He or she then divides the dot plot into f
our qu dr nts,
sep r ting the positives from the neg tives in e ch xis (Fig. 125). Qu dr nt 1 c
on- sists of
cells th t re positive for uorescence on the y- xis nd neg tive for uorescence o
n the x- xis.

Qu dr nt 2 con- sists of cells th t re positive for uorescence on both the x- n

d y- xes.
Qu dr nt 3 consists of cells th t re neg tive for fluorescence on both the xnd y- xes. And
qu dr nt 4 consists of cells th t re positive for uorescence on the x- xis nd
neg tive for
fluorescence on the y- xis. The computer will then c lcul te the percent ge
of cells in e ch
qu dr nt b sed on the tot l number of events counted (typ- ic lly 10,000 to 20,0
00 events per
s mple). For ex mple, g te c n be dr wn round
popul tion of cells
b sed on their
FSC versus SSC ch r cteristics, nd the extrinsic 170 0 10 0 10 1 10 2 10 3 10 4
M rker defines %
+ cells Fluorescent intensity N u m b e r
o f
c e l l s
FIGURE 123. Ex mple of single p r meter ow histogr m. The y- xis consist
s of the number
of events. The x- xis is the p r meter to be n lyzed, which is chosen by the op
er tor, usu lly
n extrinsic p r meter, such s
uorescent l beled ntibody. The oper tor c n th
en set
m rker
to isol te the positive events. The computer will then c lcul te the percent pos
itive events
within the design ted m rkers. 1000 800 600 400 200 0 0 200 400 600 FSC-Heigh
t G te dr wn
round cells of interest S S C - H e i g h t
800 1000 FIGURE 124. A du l-p r meter dot plot. Both p r meters on the x
- nd y- xes re
chosen by the oper tor. In this c se, lysed whole blood is n lyzed on FSC (x- x
is) nd SSC
(y- xis). The oper tor then dr ws
g te or isol tes the popul tion of interest (e
.g., lymphocytes) for further n lysis. 1814_Ch12_186-200.qxd 7/13/09 2:56 PM P ge 189 1
90 SECTION 2
B sic Immunologic l Procedures ch r cteristics of the g ted popul tion c n be n
lyzed th t is,
lymphocytes c n be g ted, nd then the subpopul tions of T cells (CD3 nd CD4 or CD2 )
nd B
cells (CD2 , CD19 ) c n be
n lyzed (Fig. 126). The
bsolute count of
p r
ticul r
cell typefor inst nce, CD4 T lymphocytesis obt ined by multiplying the bsolute
cell count
of the popul tion of interest (e.g., lymphocytes) derived from hem tolog
y n lyzer by the
percent ge of the popul tion of interest in the s mple (CD3 nd CD4 lymphocytes). 1
,2 Det iled
phenotypic n lysis c n determine the line ge nd clon lity, s well s the degr
ee of
differenti tion nd ctiv tion of
cell popul tion. This is useful for differen
ti l di gnosis or
cl ri c tion of closely rel ted lymphoprolifer- tive disorders. S mple Prep r tio
n S mples
commonly used for n lysis include whole blood, bone m rrow, nd fluid spir tes
. Whole blood

should be collected into ethylenedi minetetr cetic cid (EDTA), the ntico gul
nt of choice
for s mples processed within 30 hours of collection. Hep rin c n lso be used
for whole blood
nd bone m rrow nd c n provide improved st bil- ity in s mples over 24 hours ol
d. Blood should
be stored t room temper ture (20C to 25C) prior to processing nd should be well
mixed before
being pipetted into st ining tubes. 2,3 Hemolyzed or clotted specimens should be
rejected.
Peripher l blood, bone m rrow, nd other s mples with l rge numbers of red cells
require
erythrocyte remov l to llow for ef cient n lysis of white cells. Historic lly, d
ensity gr dient
centrifug tion with Ficoll- Hyp que (Sigm , St. Louis, MO) w s used to gener te
cell suspension
enriched for lymphocytes or bl sts. However, this method results in selective lo
ss of some cell
popul - tions. 4 Altern tively, there re numerous erythrocyte lysis techniques
v il ble, both
commerci l nd noncommerci l. 4 Tissue specimens re best collected nd tr nspor
ted in tissue
culture medium (RPMI 1640) t either room temper- ture or 4C, if n lysis will b
e del yed. The
specimen is then dis ggreg ted to form single cell suspension, either by mech
nic l
dissoci tion or by enzym tic digestion. Mech nic l dis ggreg tion, or te sing
, is preferred
nd is ccomplished by the use of either
sc lpel nd forceps, needle nd syr
inge, or wire
mesh screen. 5 Antibodies re then
dded to the resulting cellul r prep r
tion nd
processed for n lysis. The ntibodies used re typic lly monoclon l, e ch with
different
uorescent t g. Clinic l Applic tions Routine pplic tions of flow cytometry
in the
clinic l l bor tory include immunophenotyping of peripher l blood lymphocyte
s, enumer tion of
CD34 stem cells in peripher l blood nd bone m rrow for use in stem cell tr ns- p
l nt tion, nd
immunophenotypic ch r cteriz tion of cute leukemi s, non-Hodgkins lymphom s, nd
other lymphoprolifer tive disorders. Immunophenotyping by ow cytometry h s become n import n
t component of
initi l ev lu tion nd subsequent post-ther peutic monitoring in leukemi nd ly
mphom
m n gement. Flow cytometric ndings h ve been incorpo- r ted into current leukemi
nd lymphom
cl ssi c tions, beginning with the Revised Europe n-Americ n Lymphom cl ssific ti
on in 1994 nd,
more recently, in the proposed World He lth Org niz tion (WHO) cl ssi c tions. 6,7
One of the
most import nt components of ow cytometric n ly- sis is the str tific tion of he
m topoietic
m lign ncies by 1 2 3 FL1 FL2 4 A B 10 4 10 3 10 2 10 1 10 0 10 2 CD3 FITC CD4
+ T cells C D 4
P E 10 3 10 4 10 1 10 0 FIGURE 125. Qu dr nt n lysis of
du l-p r meter
dot plot. The
oper tor chooses which p r meters to n - lyze on e ch xis. (A) On e ch xis th

ere re positive
( uorescence positive) nd neg tive ( uorescence neg tive) cells. (B) Ex mple of d
u l-p r meter
dot plot to identify CD4 T cells: CD3 on the x- xis nd CD4 on the y- xis. The cel
ls in qu dr nt
2 th t re both positive for CD3 nd CD4 re true CD4 T cells. 1814_Ch12_186-200.q
xd 7/13/09
2:56 PM P ge 190 CHAPTER 12 Flow Cytometry nd L bor tory Autom tion 191 their
line ge (i.e.,
B cell, T cell, or myeloid) nd the degree of differenti tion. Some of th
e more common
cell- differenti tion ntigens re listed in T ble 122 nd include CD2, CD3, CD4,
CD7, CD8 found
on T cells, CD19, CD20, CD22 found on B cells, CD11b, CD13, CD15, CD16
on myeloid
cells, nd CD11b, CD14, HLA-DR (MHC cl ss II) on monocytoid cells. 8,9 CD45
is
p n-leukocyte m rker present on ll white cells but with v rying levels of expre
ssion; this
results in v rying levels of uorescence. This v ri nce in expression is b sed on
the cells
m turity. Bl sts express lower levels of CD45 (low uorescence) but show n incre
se of CD45
expression s the cell m tures, so m ture white cells h ve much brighter fluores
cence comp red to
their e rlier progenitor st ges. This v rying level of CD45 expression is useful
in differenti ting v rious popul tions of white cells. Immunophenotyping of white blood cel
l popul tions is
essenti l when n immunodeficiency is suspected. Enumer tion of peripher l
blood CD4 T
cells in HIV- infected p tients rem ins the highest volume test performed in the
ow cytometry
l bor tory, bec use it is used in cl s- sifying st ges of HIV dise se nd determ
ining tre tment
options. 10,11 HIV type 1 infections c use r pid, profound decre se in CD4 T
cell numbers
nd n exp nsion of CD8 T-cell levels during the e rly course (12 to 18
months) of the
illness. 12,13 Some individu ls continue to r pidly lose CD4 T cells nd progress
to AIDS, while
oth- ers m int in rel tively st ble CD4 T-cell counts
nd rem in AIDS-free
for ye rs.
During this chronic ph se of HIV-1 dise se, the decline in CD4 T cells c n be slow
over m ny
ye rs due to m inten nce of homeost tic mech nisms. However, s these homeost ti
c mech nisms
st rt to f il, A 10 4 10 3 10 2 10 1 10 0 10 2 CD3 FITC C D 4
P E
10 3 10 4 10 1 10 0 1000 800 600 400 200 0 0 200 400 600 FSC-Height R
1 S S C - H e i g h t
800 1000 B 10 4 10 3 10 2 10 1 10 0 10 2 CD2 FITC C D 1 9
P E
sets in

10 3 10 4 10 1 10 0 FIGURE 126. G ting str tegy to n lyze lymphocyte sub

s mple of

whole blood. Whole blood is incub ted with uorescent-l beled ntibodies speci c for
CD2, CD3,
CD4, nd CD19. The s mple is w shed, RBCs re lysed, nd the s mple is n lyzed
on the ow
cytometer. To n lyze using g ting str tegies, the s mple is rst plotted on FSC v
ersus SSC. (A)
A g te, or region, is dr wn round the lymphocyte popul tion. (B) On the subsequ
ent plots of
uorescent m rkers, only the lymphocyte popul tion is n lyzed. The dot plot is di
vided into four
qu dr nts to isol te positive from neg tive popul tions. The computer c lcul tes
the percent
positive cells in e ch qu dr nt. Ex mple of
ow p ttern n lyzing two different
cell surf ce
m rkers. CD3 identi es the T-cell popul tion. CD4 identi es the percent ge of the Tcell
popul tion th t re T helper cells (CD4 ). Three distinct popul tions re identi ed:
CD3 CD4 in
the upper right qu dr nt; CD3 CD4 in the lower right qu dr nt; nd CD3 CD4 in the lo
wer left
qu dr nt. 1814_Ch12_186-200.qxd 7/13/09 2:56 PM P ge 191 192 SECTION 2 B sic
Immunologic l
Procedures there is further decline in CD4 T nd CD8 T cells, which eventu lly
le ds to
the development of AIDS. 14 CD4 T-cell levels re used to st ge HIV dise se pro
gres- sion, re
prognostic for the development of AIDS, nd re used to monitor response to nti
retrovir l
ther py. The Centers for Dise se Control nd Prevention (CDC) guide- lines st g
e HIV-1 dise se
by CD4 T-cell level into three groups: 500 CD4 cells/mm3, or 28 percent CD4 cel
ls within
lymphocytes; 200 to 500 CD4 cells/mm3, or 14 to 28 percent CD4 cells; nd 200 CD4
cells/mm3, or
14 percent CD4 cells. 11 Addition l ex mples of flow cytometry use include the de
termin tion of
DNA content, or ploidy st tus of tumor cells. This c n provide the physici n wit
h import nt prognostic inform tion. 15 Monitoring p tients who h ve been tre ted for leukemi or
lymphom for
minim l residu l dise se h s lso become nother import nt function of the ow cytom
eter, since
st tistic lly signi c nt r re cell events c n be e sily detected. In the c se of
fet lm tern l
hem- orrh ge, using flow cytometry to detect hemoglobin F positive cells i
s much more
sensitive th n the tr dition l Kleih uer-Betke method. 16,17 Flow cytometry is
lso being used in
hum n leukocyte ntigen typing nd cross-m tching for solid org n tr nspl nt tio
n. 1,15
Immunophenotyping by flow cytometry, in wh tever c p city th t it is used,
is not possible
without the use of uorescent-l beled monoclon l nd polyclon l ntibodies. Monocl
on l ntibodies
speci c for v rious surf ce ntigens re prefer ble to using polyclon l ntibodies
. The bility
to produce monoclon l ntibodies through hybridom nd recombin nt DNA technique
s h s contributed
gre tly to the ccur cy of flow cytometry nd h s widened its use. (See Ch pter
4 for

discussion of monoclon l ntibody production.) T ble 12-2. Surf ce M rkers on T

nd B Cells
ANTIGEN CELL TYPE FUNCTION CD2 Thymocytes, T cells, NK cells Involved in T-c
ell ctiv tion
CD3 Thymocytes, T cells Associ ted with T-cell ntigen receptor; role in TCR sig
n l tr nsduction
CD4 Helper T cells, monocytes, m croph ges Coreceptor for MHC cl ss II; receptor
for HIV CD5
M ture T cells, thymocytes, subset of B cells (B1) Positive or neg tive modul ti
on of T- nd
B-cell receptor sign ling CD8 Thymocyte subsets, cytotoxic T cells Coreceptor fo
r MHC cl ss I
CD10 B- nd T-cell precursors, bone m rrow Prote se; m rker for pre-B CALLA str
om l cells CD11b
Myeloid n NK cells M subunit of integrin CR3, binds complement component iC3b,
CD13
Myelomonocytic cells Zinc met lloprote se CD14 Monocytic cells Lipopolys cch rid
e receptor CD15
Neutrophils, eosinophils, monocytes Termin l tris cch ride expressed on glycolip
ids CD16
M croph ges, NK cells, neutrophils Low f nity FC receptor, medi tes ph gocytosis
nd ADCC CD19 B
cells, follicul r dendritic cells P rt of B-cell coreceptor, sign l tr nsduction
molecule th t
regul tes B-cell development nd ctiv tion CD21 B cells, follicul r dendritic c
ells, subset of
Receptor for complement component C3d; p rt of B-cell imm ture thymocytes corece
ptor with CD 19
CD23 B cells, monocytes, follicul r dendritic cells Regul tion of IgE synthesis;
triggers rele se
of Il-1, Il-6, nd GM-CSF from monocytes CD25 Activ ted T cells, B cells, monocy
tes Receptor for
IL-2 CD44 Most leukocytes Adhesion molecule medi ting homing to peripher l lymph
oid org ns CD45
All hem topoietic cells Tyrosine phosph t se, ugments sign ling CD45R Different
forms on ll
hem topoietic cells Essenti l in T- nd B-cell ntigen-stimul ted ctiv tion CD
56 NK cells,
subsets of T cells Not known CD 94 NK cells, subsets of T cells Subunit of NKG2A complex
involved in inhibition of NK cell cytotoxicity NK N tur l killer; TCR CD3- rcptor
complx;
MHC major histocompati
ility class; HIV human immuno cincy virus; CALLA common acu
t
lympho
lastic lukmia antign; FC Fragmnt crystallia
l; ADCC anti
oy-pnn
t cll
cytotoxicity; IgE immunoglo
ulin E; GM-CSF granulocyt-macrophag colony-stimulati
ng factor.
1814_Ch12_186-200.qx 7/13/09 2:56 PM Pag 192 CHAPTER 12 Flow Cytomtry an
La
oratory
Automation 193 IMMUNOASSAY AUTOMATION In aition to th us of ow cytomtry, av
ancs in automat tchnology ar
coming mor vint in clinical immunology la
s with th
avnt of
immunoassay analy- rs. Rlia
l immunoassay instrumntation, xcluing ra
ioimmunoassay,
was first availa
l in th arly 1990s. Using a soli support for sparating fr
 an
oun analyts, ths instrumnts hav ma it possi
l to automat htrognous immunoa
ssays vn for

low-lvl pptis, such as ppti hormons. 18 Currntly thr ar ovr two o
n iffrnt
typs of automat immunoassay analyrs that ar capa
l of prforming almost a
ll common
iagnos- tic immunoassays, 19 an thy hav largly rplac manual tsting, sp
cially in largr
la
oratoris. Th riving moti- vation for th vlopmnt of immunoassay analy
rs has
n th
n to crat an automat systm capa
l of rucing or liminating th many m
anual tasks
rquir to prform analytical procurs. Eliminating manual stps crass t
h liklihoo
of rror, sinc th potntial rror u to fatigu or rronous sampling is
ruc. 20
La
oratory profssionals ar also trying to stramlin tst prformanc to ruc
 turnaroun tim
an th cost pr tst. Automation, in som cass, is much mor accurat an prc
is compar to
manual mthos an, pning on th assay platform, may
 mor snsitiv as w
ll. Othr
potntial
n ts of immunoassay automation inclu th a
ility to provi mor s
rvic with lss
staff; saving on con- trols, uplicats, ilutions, an rpats; longr shlf li
f of ragnts
an lss isposal u to outating; an th potn- tial for automation of sampl
livry with

ar cos for
ttr sampl inti cation. 21 Du to th wi varity of automat
immunoassay
ana- lyrs availa
l, it can
 ifficult to trmin th
st instrumnt for
any givn
la
oratory. Thr ar many fac- tors to consir othr than th analytic quality
of assays that
ar availa
l. Ta
l 123 offrs a partial list of factors to consir in trmin
ing what typ
of analyr will ful ll a la
oratorys ns. It is important for all thos involv
in th
instrumnts slction to prioriti th proprtis of any analyr to mt th 
mans of th
la
oratory. Thr ar currntly two main typs of immunoassay ana- lyrs on th
markt:
atch
analyrs an ranom accss analyrs (Ta
l 124). Batch analyrs can xamin mu
l- tipl
sampls an provi accss to th tst sampls for th formation of su
squnt r
action mixturs.
Howvr, such
atch analyrs prmit only on typ of analysis at a tim. In som
 cass, this may

 consir a raw
ack; stat sam- pls cannot
 loa ranomly, an th
r cannot

multipl analyss on any on sampl. Partially for thos ra- sons, th nxt gn
ration of
analyrs was sign in a moular systm that coul
 con gur to masur mult
i- pl analyts
from multipl sampls. Ths typs of analyrs ar call ranom accss analy
rs, in which
multipl tst sampls can
 analy, an multipl tsting can
 pr- form o
n any tst
sampl. 22 Ta
l 55-2 Comparison of Tsts Us Ta
l 12-3. Factors for Consira
tion in Slcting
an Automat Immunoassay Instrumnt CATEGORY FACTOR Analytical Snsitivity Pr
cision

Accuracy/tst stanariation Linarity Intrfrncs Carryovr ffcts Economic

Purchas cost
Las options Maintnanc costs Ragnt costs Oprator tim an costs Disposa
l
costs Instrumnt
Maintnanc rquirmnts Automation compati
ility Spac rquirmnts Utility rq
uirmnts
Rlia
ility Clot rror tction Manufacturr Futur prouct plans Rputation T
chnical support
Mnu xpansion plans Oprational Tst mnu Throughput Ragnt capacity Ragnt s
ta
ility STAT
capa
ility R x tsting a
ility Ragnt kit si Training rquirmnts Oprating
complxity
Wast rquirmnts Ragnt storag rquirmnts Downtim plans Rprint from R
maly, AT, an
Hortin, GL. Protin analysis for iagnostic applica- tions. In Dtrick, B, Hamil
ton, RG, an
Fols, JD, t al. (s): Manual of Molcular an Clinical La
oratory Immunology,
. 7. Amrican
Socity for Micro
iology, Washington, DC, 2006, p. 15. 1814_Ch12_186-200.qx 7/
13/09 2:56 PM
Pag 193 194 SECTION 2 Basic Immunological Procurs Automation can an os oc
cur in all thr
stags of la
oratory tsting: th pranalytical, analytical, an postan- alytica
l stags. For th
purposs of this chaptr, iscussion is limit to automation within th analyti
cal stag of
tsting. Th tasks to
 consir uring th analytical stag inclu introuc
ing sampl,
aing ragnt, mixing ragnt an sampl, incu
ating, tcting, calculating, a
n rport- ing
raout or rsults. All or som of ths tasks may
 automat on various immun
oassay analyrs.
Automatic sampling can
 accomplish
y svral iffrnt mth- os: pristalt
ic pumps (olr
tchnology) an positiv-liqui isplacmnt piptts (nwr tchnology) ar two
xampls. In
most systms, sampls ar piptt using thin, stainlss- stl pro
s. Issus t
o consir with
pro
 tchnology ar th inclusion of clot tctors that will automatically rj
ct a sampl if a
clot is tct an th a
ility of th sampl pro
 to tct th amount of th
sampl liqui,
using a liq- ui lvl snsor. This lattr snsor must
 a
l to tct th lac
k of sampl in a
tu
, usually
caus of a short raw, an rjct such sampls. Anothr iss
u associat
with rusa
l piptt pro
s is carryovr or contamination of on sampl with ma
trial from th
prvious sampls. Various mthos hav
n vlop to ruc carryovr, inclu
- ing th us of
isposa
l piptt tips to initially transfr sampls. Ragnt us in automat
 immunoassay
analyrs rquirs consiration of th following factors: hanling, prparing a
n storing, an
ispnsing. Som ragnts com ray for us,
ut if not, th analyr must
 a

l to prop- rly
ilut ragnts
for thy can
 us. Most ragnts com with
ar cos that
ar ra
y th
analyr to ruc oprator rror, so if th wrong ragnt is loa into th an
alyr
y

mistak, th analyr will tct th rror an gnrat an rror mssag. Aftr
ragnts hav

n a to th sampls, th nxt concrn is propr mixing to o
tain rlia
l
rsults.
Analyrs us iffrnt mthos for mixing, incluing magntic stir- ring, rotat
ion pals,
forcful ispnsing, an vigorous latral shaking. Whichvr mtho us, it is
imprativ that
thr
 no splashing
twn sampl wlls to prvnt rronous rsults. Tim i
ncu
ation is thn
carri out at am
int tmpr- aturs, or analyrs n to hav
uilt-in incu
a
tors for
tmpratur-controll incu
ation. Hat mtal
locks ar wily us to incu
a
t ragnt wlls
or cuvts. Dtction of th nal analyt pns on th chmistry involv in th
 immunoassay.
In th past, colorimtric a
sorption spctroscopy has
n th principl ma
ns of
masurmnt, u to th a
ility to masur a wi varity of compouns. Othr m
thos of
tction inclu uorscnc Ta
l 12-4. Automat Immunoassay Analyrs MANUFACT
URER
INSTRUMENT OPERATIONAL TYPE ASSAY PRINCIPLE A

ott Diagnostics AxSym Continu
ous Ranom Accss
FPIA, MEIA Architct Batch, ranom accss, cont. Enhanc chmiluminscnc ra
nom accss
Bckman Coultr Accss Cont. ranom accss Chmiluminscnc BioMriux VIDAS Batc
h, ranom
accss Fluorscnc/EIA coat SPR Th Bining Sit, Inc. DSX automat systm B
atch EIA Bio-Ra
La
s, Clin. Diag. Group BioPlx Cont. ranom accss Ba ow cytomtric (multiplx
) PhD Systm
Batch EIA Diamix Corp. Mago Plus Automat Batch, ranom accss EIA EIA Proc
ssor Diasorin,
Inc. ETI-Max Batch, ranom accss EIA Hycor Biomical, Inc. HY-TEC Ranom
atch
s EIA Invrnss
Mical Profssional AIMS Batch EIA, multiplxing/
a Diagnostics Ortho-Clinic
al Diag., a J&J
Co. VITROS Cont. ranom accss Chmiluminscnc (nhanc) Phaia ImmunoCAP Con
t. ranom accss
FEIA Simns Mical Solutions Diag. ADVIA Cntaur Cont. ranom accss Chmilumi
nscnc IMMULITE
Cont. ranom accss Chmiluminscnc TOSOH Bioscinc, Inc. AIA Cont. ranom ac
css
Fluorscnc, EIA Sourc: CAP Toay, Jun 2007. EIA nym immunoassay; FEIA
uoro
nym
immunoassay; FPIA
uorscnt polariation immunoassay; MEIA microparticl nym i
mmunoassay;
SPR
soli phas rcptacl 1814_Ch12_186-200.qx 7/13/09 2:56 PM Pag 194 in
th analysis.
Analytic snsitivity is  n as th lowst masura
l amount of an analyt, whi
l analytic
spci- city is th assays a
ility to gnrat a ngativ rsult whn th analyt i
s not
prsnt. Th rporta
l rang is  n as th rang of valus that will gnrat
a positiv
rsult for th spcimns assay
y th tst procur. Not that this may not i
nclu th ntir
ynamic rang of th analytic instrumnt us to prouc th rsult. Finally, th
 rfr- nc

intrval is th rang of valus foun in halthy iniviuals who o not h

av th conition
that is tct
y th assay. This is us to  n th xpct valu of a ngativ tst.
Choosing an automat immunoassay analyr is com- plicat an pns grat
ly on th main
tasks to
 prform in a particular la
oratory. Batch analyrs may work

st if only on
typ of tsting is prform on a larg scal. Ranom accss analyrs allow for
mor
flxi
ility, inclu rapi procssing of stat sampls. In ithr cas, any nw i
nstrumnt
rquirs xtnsiv valiation
for patint rsults can
 rport with confi
nc.
Instrumntation can incras turnaroun tim for tsting, rmov th pos- si
ili
ty of manual
rrors, an allow for gratr snsitivity in trmining th prsnc of low-lv
l analyts.
SUMMARY Flow cytomtry is a powrful tool to intify an numr- at various
cll
populations. It was first us in clinical la
oratoris to prform CD4 T
-cll counts
in HIV- infct iniviuals. A flow cytomtr masurs multipl proprtis of
clls suspn
in a moving ui mium. As ach cll or particl passs singl l through a lasr
light sourc,
it proucs a charactristic light pattrn that is mas- ur
y multipl tct
ors for scattr
light (forwar an 90 grs) an uorscnt missions (if th cll is stain wi
th a
uorochrom). Forwar scattr is a masur of cll si, whil si scattr trm
ins a clls
intrnal complx- ity, or granularity. Dtrmining an iniviuals lymphocyt popu
lation is
ssntial in iagnosing such conitions as lymphomas, immunoficincy i
sass,
unxplain infctions, or acquir immun isass such as AIDS. Lymphocyts
ar inti 
using monoclonal anti
ois irct against sp- ci c surfac antigns. Ractions
can

inti  manually
y mploying a uorscnc microscop or
y immunon- ym staini
ng mthos.
Howvr, flow cytomtry is th most commonly us mtho for immunophnotyping o
f lymphocyt
populations. Othr instrumntation in th clinical immunology la
o- ratory inclu
s a varity of
automat immunoassay analyrs that ar currntly availa
l. Thy hav rplac
manual
immunoassay procurs in many la
oratoris. Ths auto- mat systms can gnr
at mor
accurat, prcis, an snsitiv analysis of many analyts compar to ma
nual
immunoassays. Thr ar many factors to consir in tr- mining which analy
r will fill
th ns of a particular CHAPTER 12 Flow Cytomtry an La
oratory Automatio
n 195 an
chmiluminscnc,
oth of which rquir uorscnc tctors. Valiation Rgarl
ss of th
instrumntation consir, propr vali- ation of nw instrumntation or mtho
ology must always


 prform. Th la
oratory ns to trmin how it will mt Clinical La
ora
tory Improvmnt
Amnmnt (CLIA) rgulations for vrifying th manufacturrs pr- formanc spci
fications. Th
rgulations apply to ach nonwaiv tst or tst systm
rought into th la

oratory for th
first tim. Valiation of th nw instrumnt or mtho must
 complt

for patint
rsults using that instrumnt can
 rport. Thr ar multipl rsourcs avai
la
l on th
topic of mtho valiation. Th Cntrs for Micar an Micai Srvics has a
n ovrviw of th
CLIA (availa
l at http://www.cms.hhs.gov/CLIA). Th spci c rquirmnts for
mtho
valiation for non- waiv an moifi tsts (Su
part K) can
 foun
at
http://www.cms.hhs.gov/CLIA/ownloas/apcsu
k1.pf. Ta
l 125 lists othr govrnm
ntal w
sits
with infor- mation rgaring mtho/instrumnt valiation. 23 As signat
y C
LIA, th rquir
vri cations to
 trmin for a nw mtho ar accuracy, prcision, ana- lytic
snsitivity,
analytic spcificity to inclu intrfring su
stancs, rporta
l rang, an
rfrnc
intrvals. Accuracy rfrs to th tsts a
ility to actually masur what it claim
s to masur.
For xampl, th assay may
 tst using prviously known positivs or ngativ
s as provi
y
proficincy tsting or intrla
oratory xchang. Also, paralll tsting with an
altrnativ
mtho or tchnology is a form of accuracy tsting. Prcision rfrs to th a
il
ity to
consistntly rprouc th sam rsult on rpat tsting of th sam sampl. C
LIA 88 spcifis
that th stanar viation an cof cint of variation shoul
 calculat from
10 to 20
ay-to-ay quality-control rsults. 24 At last on normal an on a
normal cont
rol shoul

inclu Ta
l 55-2 Comparison of Tsts Us AGENCY WEBSITE Cntrs for Disas

www.phppo.cc.gov/clia/fault.aspx Control an Prvntion Foo an Drug
www.fa.gov/crh/CLIA/inx/html Aministration Th Collg of Amrican www.cap.
org/apps/ocs/
Pathologists La
oratory la
oratory_accriation/chcklists/ Accritation Prog
ram
chmistry_an_toxicology_ Inspction Chcklist for april2006.pf Chmistry Clini
cal La
oratory
www.clsi.org Stanars Institut Evaluation Protocol Ta
l 12-5. CLIA-Rlat Go
vrnmntal
W
sits 1814_Ch12_186-200.qx 7/13/09 2:56 PM Pag 195 196 SECTION 2 Basic I
mmunological
Procurs la
oratory, incluing ciing whthr a
atch or a ranom accss ana
lyr can
st
srv tsting ns. Automation is incorporat in all stags of la
oratory tst
ing:
pranalytical, analytical, an postanalytical. Onc an analyr has
n purchas
, a thorough
valiation of all assays to
 prform must
 on to nsur quality. Th val
iation shoul
inclu at last a trmination of accu- racy, prcision, rporta
l rang, rf

rnc rang,
analytic snsitivity, an analytic spci city. Automat analyrs ar costly
ut
can ruc
turnaroun tim an hans-on tim
y th tchnical staff an can provi snsiti
v an prcis
rsults. 1814_Ch12_186-200.qx 7/13/09 2:56 PM Pag 196 CHAPTER 12 Flow Cytom
try an
La
oratory Automation 197 1. A la
oratory has just purchas a nw immunoassay a
nalyr, an
valiation is
ing on
for patint rsults can
 rport out. Twnty ran
om patint
sampls ar run
y
oth th ol an nw mthoology. Accoring to th nwr inst
rumntation,
thr sampls that wr ngativ
y th ol mtho ar positiv
y th nw instr
umnt. Qustions
a. What sort of possi
l rrorthat is, snsitivity, spci- city, accuracy, or prc
isionos
this rprsnt?
. What stps shoul
 takn to rsolv this iscrpancy? 2. A
3-yar-ol fmal
is snt for immunologic tsting
caus of rcurring rspiratory infctions, inc
luing svral

outs of pnumonia. Th rsults show cras immunoglo
ulin lvls, spcially
of IgG. Although
hr whit
loo cll count was within th normal rang, th lymphocyt count w
as low. Flow
cytomtry was prform to trmin if a particular su
st of lym- phocyts wa
s low or missing.
Figur 127 shows th ow cytomtry rsults o
tain. Qustions a. What o th ow cyt
omtry
pattrns inicat a
out th population of lymphocyts affct?
. How can thi
s account for
th chils rcurring infctions? c. What furthr typ of tsting might
 inic
at? CASE
STUDIES C D 3 CD19 A 10 4 10 3 10 2 10 1 10 0 10 2 CD3-FITC C D 4 - A P C 10 3 1
0 4 10 1 10 0 B
10 4 10 3 10 2 10 1 10 0 10 2 CD3-FITC Patint 23% 40% Normal onor C D 4 - A P
C 10 3 10 4 10 1
10 0 FIGURE 127. Flow cytomtry pattrns for cas stuy. (A) Plot of CD3 vrsus C
D19. (B) Plot
of CD3 vrsus CD4. 1814_Ch12_186-200.qx 7/13/09 2:56 PM Pag 197 198 SECTION
2 Basic
Immunological Procurs 1. Flow cytomtry sparats clls on th
asis of which
of th
following? a. Forwar an 90-gr si scattr of an intr- rupt
am of lig
ht
. Front-angl
scattr only of an intrrupt light
am c. A
sor
anc of light
y iffrnt ty
ps of clls .
Transmittanc of light
y clls in solution 2. Forwar-angl light scattr is an
inicator of
cll a. granularity.
. nsity. c. si. . num
r. 3. What is th singl most
important
rquirmnt for sam- pls to
 analy on a ow cytomtr? a. Whol
loo is col
lct into a
srum-sparator tu
.
. Clls must
 in a singl-cll suspnsion. c. Sampls m
ust
 x in
formalhy prior to procssing. . Bloo must
 kpt rfrigrat whil proc
ssing. 4. Which
rprsnts th
st xplanation for a ow cytom- trs a
ility to tct svral c
ll surfac
markrs at th sam tim? a. Th forwar scattr can sparat out clls on th

asis of
complxity.
. On tctor can
 us to tct many iffrnt wavlngths. c.
For ach markr,
a spci c uorochromanti
oy com
ination is us. . Intrinsic paramtrs ar spara
t out on
th
asis of amount of si scattr. 5. Which of th following cll surfac mark
rs woul

prsnt on a population of T hlpr clls? a. CD3 an CD4
. CD3 an CD8 c. CD3
only . CD4 only
6. Which cll surfac markr is trm th common acut lympho
lastic lukmia m
arkr? a. CD19
.
CD10 c. CD23 . CD21 7. All of th following ar clinical applications for flow
cytomtry xcpt
a. ftal hmoglo
in.
. immunophnotyping of lymphocyt su
populations. c. HIV v
iral loa
analysis. . numration of stm clls in a priphral
loo mononuclar cll pr
ouct. 8. Which
typ of analyr allows on to masur multi- pl analyts from multipl sampls
, loa at any
tim? a. Batch analyr
. Ranom accss analyrs c. Front-n loa analyrs
. Squntial
accss analyrs 9. All of th following ar
n ts of automation xcpt a. grat
r accuracy.
.
incras turnaroun tim. c. savings on controls. . lss isposal of outat
ragnts. 10. If
an analyr gts iffrnt rsults ach tim th sam sampl is tst, w
hat typ of
pro
lm os this rprsnt? a. Snsitivity
. Spci city c. Accuracy . Prcision
REVIEW
QUESTIONS 1814_Ch12_186-200.qx 7/13/09 2:56 PM Pag 198 CHAPTER 12 Flow Cyto
mtry an
La
oratory Automation 199 Rfrncs 1. Marti, GE, Stlr-Stvnson, M, Blsing
, JJH, an
Flishr, TA. Introuction to flow cytomtry. Sminars in Hm 38(2):93, 2
001. 2. National
Committ for Clinical La
oratory Stanars. Clinical applications of flo
w cytomtry
quality assuranc an immunophnotyping of priphral
loo lymphocyts. H42-A.
National
Committ for Clinical La
oratory Stanars, Wayn, PA, 1998. 3. Rni, P, an G
inns, LC.
Analysis of T cll su
sts in normal aults. Comparison of whol
loo lysis tc
hniqu to FicollHypaqu sparation
y flow cytomtry. Immunol Mthos 98:5356, 1987. 4. Cartr, P
H, Rsto-Rui,
S, Washington, GC, Ethrig, S, Palini, A, Vogt, R, Waxal, M, Flishr, T, Nogu
chi, PD, an
Marti, GE. Flow cytomtric analysis of whol
loo lysis, thr anticoagulants,
an v cll
prparations. Cytomtry 13:6874, 1992. 5. Braylon, RC, an Bnson, NA. Flow cytom
tric analysis
of lymphomas. Arch Pathol La
M 113:627633, 1989. 6. Harris, HL, Jaff, ES,
Stin, H,
Banks, PM, Chan, JKC, Clary, ML, DElsol, G, t al. A rvis Europan-Amri
can
classification of lymphoi noplasms: A proposal from th Intrnational Lymphoma
Stuy Group.
Bloo 84:13611392, 1994. 7. Harris, NLE, Jaff, ES, Di
ol, J, Flanrin,
G, MullrHrmlink, HK, Variman, J, Listr, TA, an Bloom l, CD. Th Worl Halth Organi

ation
classification of noplastic isass of th hmatopoitic an lymphoi tissus.
Rport of th
clinical Avisory Committ mting, Airli Hous, Virginia. Ann Oncol 10:1
4191432, 1999.
8. Woo, BL. Immunophnotyping of lukmia an lymphoma
y ow cytomtry. In Dtri
ck, B,
Hamilton, RG, an Fols, JD, t al. (s): Manual of Molcular an Clinical La
o
ratory
Immunology, . 7. Amrican Socity for Micro
iology, Washington, DC, 2006
, pp. 171186. 9.
Blsing, JJH, an Flishr, TA. Immunophnotyping. Sminars in Hm 38(2):1
00, 2001. 10.
Cntrs for Disas Control an Prvntion. 1997 rvis guilins for prformi
ng CD4 T cll
trminations in pr- sons infct with human immunoficincy virus (HIV). Mo
r
Mortal Wkly
Rp 46:129, 1997. 11. Cntrs for Disas Control. 1993 rvis classi cation systm for HIV
infctions an xpan cas  nition for AIDS among aolscnts an aults. Mor

Mort Wkly Rp
41:119, 1992. 12. Giorgi, JVA, Ksson, M, an Chou, CC. Immuno cincy an infcti
ous isass.
In Ros, NL, Macario, CE, Fahy, JL, Friman, H, an Pnn, GM (s): Manual o
f Clinical
La
oratory Immunology, . 4. Amrican Socity for Micro
iology, Washingto
n, D.C., 1992,
pp. 174181. 13. Zaunrs, J, Carr, A, McNally, L, Pnny, R, an Coopr, DA. Effc
ts of primary
HIV-1 infctions on su
sts of CD4 an CD8 T lymphocyts. AIDS 9:561566, 1995. 14. M
argolick,
JB, Muno, A, Donnn
rg, A, Park, LP, Galai, N, Giorgi, JV, OGorman, MRG, an F
r
as, J.
Failur of T- cll homostasis prcing AIDS in HIV infction. Nat M 1:674680,
1995. 15.
Diguispp, JA. Flow cytomtry. In Colman, WB, an Tsongalis, GJ (s):
Molcular
Diagnostics: For th Clinical La
oratorian, . 2. Humana Prss, Inc., Totowa, N
J, 2006, pp.
163172. 16. Chn, JC, Davis, BH, Woo, B, an Warynski, MJ. Multicntr
clinical
xprinc with ow cytomtric mtho for ftomatrnal hmorrhag tction. Ctyom
try
50:285290, 2002. 17. Frnans, BJ, vonDalsn, P, Faal, I, Bansil, N, an Rya
n, G: Flow
cytomtric assssmnt of fto-matrnal hmorrhag; a comparison with Btk-Kl
ihaur. Prnat
Diagn 27(7): 641643, 2007. 18. Rmaly, AT, an Hortin, GL. Protin analysis for
iagnos- tic
applications. In Dtrick, B, Hamilton, RG, an Fols, JD, t al. (s). Manual o
f Molcular an
Clinical La
oratory Immunology, . 7. Amrican Socity for Micro
iology,
Washington, DC,
2006, pp. 721. 19. Whlr, MJ. Automat immunoassay analysrs. Ann Clin Biochm
38:217, 2001.
20. Sunhimr, RL, an Thratt, G. Analysis: Clinical la
ora- tory automation.
In McPhrson, RA,
an Pincus, MR (s): Hnrys Clinical Diagnosis an Managmnt
y La
oratory Mth
os, . 21.
Saunrs Elsvir, Philalphia, 2007, pp. 5663. 21. Turgon, ML. Automat pr

ocurs. In
Immunology an Srology in La
oratory Micin, . 3. Mos
y, St. Louis, 2003, p
p.157164. 22.
http://www.patntstorm.us/patnts/5358691-scription.html, last accss March
2, 2008. 23.
Moon, TC, an Lgrys, VA. Taching mtho valiation in th clinical la
oratory
scinc
curriculum. Clin La
Sci21(1): 1924, 2008. 24. Association for Molcular Patho
logy.
Association for Molcular Pathology Statmnt: Rcommnations for in-hous
vlopmnt an
opration of molcular iagnostic tsts. Am J Clin Path 111:449463, 1999. 1814_Ch
12_186-200.qx
7/13/09 2:56 PM Pag 199 1814_Ch12_186-200.qx 7/13/09 2:56 PM Pag 200 III
SECTION Immun
Disorrs 201 1814_Ch13_201-223.qx 7/10/09 2:56 PM Pag 201 13 Hyprsnsitiv
ity LEARNING
OBJECTIVES Aftr nishing this chaptr, th rar will
 a
l to: 1. D n hyprs
nsitivity,
atopy, an allrgn. 2. Discuss th ky immunologic ractant involv in immia
t
hyprsnsitivity. 3. Dscri
 th changs that tak plac in IgE-coat mast cl
ls an
asophils
whn
ining with a spci c antign occurs. 4. Rlat clinical manifstations of i
mmiat
hyprsnsitivity to spci c miators rlas from mast clls an
asophils. 5. D
scri

anaphylaxis. 6. Discuss th avantags an isavantags of skin tsting for imm
iat
hyprsnsitivity. 7. Compar total IgE an allrgn-spci c IgE tsting as to sns
itivity an th
signi canc of th iagnostic nings. 8. Discuss how cllular amag occurs in typ
II
hyprsnsitivity. 9. Giv xampls of typ II hyprsnsitivity ractions. 10. Ex
plain th
signi canc of a positiv irct antiglo
ulin tst. 11. Distinguish
twn typ I
I an typ III
hyprsnsitivity ractions on th
asis of th natur of th antign involv an
 th mchanisms
of cllular injury. 12. Discuss th Arthus raction an srum sicknss as xampl
s of typ III
hyprsnsitivity ractions. 13. Rlat how typ IV snsitivity iffrs from th
othr thr typs
of hyprsnsitivity ractions. 14. Explain th natur of th immunologic ractio
n in contact
rmatitis an hyprsnsitivity pnumonitis. KEY TERMS Anaphylaxis Arthus racti
on Atopy Col
autoagglutinins Contact rmatitis Dlay hyprsnsitivity Eosinophil chmotact
ic factor of
anaphylaxis (ECF-A) Hmolytic isas of th nw
orn (HDN) Histamin Hyprsnsit
ivity
Hyposnsitiation Immiat hyprsnsi- tivity Isohmagglutinin Nutrophil chmo
tactic factor
Passiv cutanous anaphylaxis Raioallrgosor
nt tst (RAST) Raioimmunosor
nt
tst (RIST)
Srum sicknss Warm autoimmun hmolytic anmia 202 1814_Ch13_201-223.qx 7/10/
09 2:56 PM Pag
202 CHAPTER 13 Hyprsnsitivity 203 In prvious chaptrs, th immun rsp
ons has
n
scri
 as a fns mchanism
y which th
oy ris itslf of potntially ha

rmful antigns.
In som cass, how- vr, this procss can n up causing amag to th host. Wh
n this typ of
raction occurs, it is trm hyprsn- sitivity, which can
 fin as a
hightn
stat of immun rsponsivnss. Typically, it is an xaggrat rspons
to a harmlss
antign that rsults in injury to th tissu, isas, or vn ath. Briti
sh immunologists
P. G. H. Gll an R. R. A. Coom
s vis a classi cation systm for such raction
s
as on four
iffrnt catgoris. In typ I ractions, cll-
oun anti
oy racts with antig
n to rlas
physiologically activ su
stancs. Typ II rac- tions ar thos in which fr a
nti
oy racts
with antign associat with cll surfacs. In typ III hyprsnsitivity, anti
o
y racts with
solu
l antign to form complxs that prcipitat in th tissus. In ths latt
r two typs,
compl- mnt plays a major rol in proucing tissu amag. Typ IV hyprsnsiti
vity iffrs
from th othr thr,
caus snsiti T clls rathr than anti
oy ar r
sponsi
l for th
symptoms that vlop. Figur 131 givs an illustra- tiv comparison of th four
main typs of
hyprsnsitivity. CHAPTER OUTLINE TYPE I HYPERSENSITIVITY Triggring of Typ I R
actions
y IgE
Rol of Mast Clls an Basophils Miators Rlas from Granuls Clinical Manif
stations of
Immiat Hyprsnsitivity Tratmnt of Immiat Hyprsnsitivity Tsting for I
mmiat
Hyprsnsitivity TYPE II HYPERSENSITIVITY Transfusion Ractions Hmolytic Disas
 of th Nw
orn
Autoimmun Hmolytic Anmia Typ II Ractions Involving Tissu Antigns Tsting
for Typ II
Hyprsnsitivity TYPE III HYPERSENSITIVITY Arthus Raction Srum Sicknss Autoim
mun Disass
Tsting for Typ III Hyprsnsitivity TYPE IV HYPERSENSITIVITY Contact Drmatiti
s
Hyprsnsitivity Pnumonitis Tu
rculin-Typ Hyprsnsitivity Tsting for Dlay

Hyprsnsitivity SUMMARY CASE STUDIES EXERCISE: DIRECT ANTIGLOBULIN TEST REVIEW
QUESTIONS
REFERENCES IgE-coat mast cll Rlas of miators Dgranulation Typ I Antig
n IgG- or
IgM-coat r clls Cllular struction Bining to macrophags or activation o
f complmnt Typ
II Complmnt
Macrophag Antignanti
oy complxs Tissu struction Dposit
ion of
complxs in tissu Typ III Nutrophil T cll Inflammation Typ IV Snsiti T
cll rlass
cytokins Antign + T
T FIGURE 131. Comparison of hyprsnsitivity ractions
for
typs I to IV. (Typ I) Antign-spci c IgE is
oun to mast clls. Antign
rig
s ajacnt
anti
oy molculs, causing isruption of th cll mm
ran with rlas of mi
ators. (Typ II)
Anti
oy is irct against cllular antigns. Complmnt is activat, an tar
gt clls ar
stroy. (Typ III) Solu
l antigns com
in with anti
oy to form immun comp

lxs. Ths ar

posit in tissus. Complmnt activation an xocytosis caus tissu amag.
(Typ IV) T clls

com snsiti to antign an rlas lymphokins on scon xposur to that
antign. A
locali in ammatory raction occurs. 1814_Ch13_201-223.qx 7/10/09 2:56 PM Pa
g 203 204
SECTION 3 Immun Disorrs Although som isas manifstations may ovrlap
among ths
catgoris, knowlg of th gnral charac- tristics of ach will hlp in un
rstaning th
immun procsss that triggr such tissu amag. Typs I through III hav prvi
ously
n
rfrr to as immiat hypr- snsitivity,
caus symptoms vlop within a f
w minuts to a
fw hours. Typ IV hyprsnsitivity has
n call lay hyprsnsitivity,

caus its
manifstations ar not sn until 24 to 48 hours aftr contact with antign. For
ach of th
four main raction typs, th natur of th immun ractants is iscuss
, xampls ar
givn, an rl- vant tsting is rviw. Rfr to Ta
l 131 for a summa
ry of th main
charactristics that istinguish ach class. TYPE I HYPERSENSITIVITY Th isting
uishing fatur
of typ I hyprsnsitivity is th short tim lag, usually scons to minuts,

twn xpo- sur
to antign an th onst of clinical symptoms. Th ky ractant prsnt in typ
I, or immiat
snsitivity ractions, is IgE. Antigns that triggr formation of IgE ar call
atopic antigns,
or allrgns. Atopy rfrs to an inhrit tn- ncy to rspon to naturally oc
curring inhal
an ingst allrgns with continu prouction of IgE. 1 Th prva- lnc of a
llrgic isass
has incras gratly in vlop countris in th last 20 to 30 yars, an it
is stimat that
as much as 30 prcnt of th U.S. population hav som typ of allrgic symptoms
. 2,3 Typically,
patints who xhi
it allrgic or immiat hyprsnsitivity ractions prouc a
larg amount of
IgE in rspons to a small concntration of antign. IgE lvls appar to pn
on th
intraction of
oth gntic an nvironmntal factors. Carl Wilhlm Prausnit an
 Hin Kstnr
wr th rst rsarchrs to show that a srum factor was rsponsi
l for typ I r
actions. Srum
from Kstnr, who was allrgic to sh, was injct into Prausnit. A latr xposur
to sh antign at th sam sit rsult in an allrgic skin raction. 1 This typ of ract
ion is known as
passiv cutanous ana- phylaxis. It occurs whn srum is transfrr from an all
rgic iniviual
to a nonallrgic iniviual, an thn th scon iniviual is challng with s
pci c antign.
Although this xprimnt was conuct in 1921, it was not until 1967 that th s
rum factor
rsponsi
l, namly IgE, was intifi. Charactristics of IgE ar scri
 in
Chaptr 4.
Triggring of Typ I Ractions
y IgE IgE is primarily synthsi in th lympho

i tissu of th
rspiratory an gastrointstinal tracts. Normal lvls ar in th rang of appro
ximatly 150
ng/mL. 1,4 Th rgulation of IgE prouction appars to
 a function of a su
st
of T clls
call typ 2 hlpr clls (Th2). 1,5 Th normal immun rspons to microorgan
isms an
possi
l allrgns is a function of typ 1 hlpr clls (Th1), which prouc
intrfron- gamma
(IFN- ). IFN- , along with intrlukin-12 an intrlukin-18, which ar prouc

y
macrophags, may actually supprss prouction of IgE typ anti
ois. 4 Ho
wvr, in popl
with allrgis, Th2 clls rspon insta an prouc intrlukin-3 (IL-3),
intrlukin-4
(IL-4), intrlukin-5 (IL-5), intrlukin-9 (IL-9), an intrlukin-13 (IL-13).
4 S Chaptr 5
for a iscussion of cytokins. IL-4 an IL-13 ar rsponsi
l for th nal iffr
ntiation that
occurs in B clls, initiating th transcription of th gn that cos for th 
psilon-havy
chain of immunoglo
ulin molculs
long- ing to th IgE class. 1,4,5 IL-5 an I
L-9 ar involv
in th vlopmnt of osinophils, whil IL-4 an IL-9 promot vlopmnt of ma
st clls. IL-4,
IL-9, an IL-13 all act to stim- ulat ovrprouction of mucus, a charactristic
of most allrgic
ractions. This propnsity to scrt cytokins that promot pro- uction of IgE
is link to a
gn locus on chromosom 5 that ncos cytokins IL-3, IL-4, IL-5, IL-9, IL-13,
an
granulocyt-monocyt colony stimulating factor (GM- CSF). 6 All of ths cyt
okins ar ky to
a switch to a Th2 rspons. Il-4 an IL-13 activat transcription of th psilon
gn in B clls
whn thy
in to spci c rcptors. 4,7 Ta
l 13-1. Comparison of Hyprsnsitivit
y Ractions
TYPE I TYPE II TYPE III TYPE IV Immun Miator IgE IgG IgG or IgM T clls Antig
n Htrologous
Autologous or Autologous or Autologous or htrologous htrologous htrologo
us Complmnt
Involvmnt No Ys Ys No Immun Mchanism Rlas of miators Cytolysis u t
o anti
oy
Dposits of antign Rlas of cytokins from mast clls an an complmnt anti

oy complxs

asophils Exampls Anaphylaxis, hay fvr, Transfusion ractions, Srum sickns
s, Arthus Contact
rmatitis, foo allrgis, asthma autoimmun hmolytic raction, lupus tu
rcu
lin tst, anmia,
HDN rythmatosus pnumonitis HDN
hmolytic isas of th nw
orn 1814_Ch13_201
-223.qx
7/10/09 2:56 PM Pag 204 CHAPTER 13 Hyprsnsitivity 205 Although actual ant
i
oy synthsis
is rgulat
y th action of cytokins, th tnncy to rspon to spcific all
rgns appars to

 link to inhritanc of crtain major histocompati
ility complx (MHC) gns
. Various human
lukocyt antign (HLA) class II molculs, sp- cially HLA-DR2, DR4, an DR7,
sm to

associat with a high rspons to iniviual allrgns. 7 HLA-D mol- culs ar

known to play a
rol in antign prsntation (s Chaptr 3), an thus iniviuals who possss p
artic- ular HLA
molculs ar mor likly to rspon to crtain allrgns. 6 In aition, ini
viuals who ar
pron to allrgis xhi
it crtain variations in th gn foun on chromo- so
m 11q that cos
for rcptors for IgE foun on svral typs of clls. 57 It is thought that poly
morphisms in
th
ta chain of ths rcptors ar link to atopy. Ths high-affinity rcp
tors, nam
FC-RI rcptors,
in th fragmnt crystallia
l (FC) rgion of th psilon-hav
y chain an ar
foun on
asophils an mast clls. A singl cll may hav as many as 200,000 suc
h rcptors. 4,6
Othr clls, such as monocyts, osinophils, Langrhans clls, an nritic cl
ls, also hav
rcptors for IgE,
ut thir concntration is low. Howvr, Langrhans an nr
itic clls
intrnali an procss allrgns from th nviron- mnt an transport th allr
gn-MHC class II
complx to local lymphoi tissu, whr synthsis of IgE occurs. 4 Bining of
IgE to cll
mm
rans incrass th half- lif of IgE from 2 or 3 ays up to at last 10
ays. Onc
oun,
IgE srvs as an antign rcptor on mast clls an
asophils. Cross-linking of
at last two
anti
oy mol- culs
y antign triggrs rlas of miators from ths clls.
Whn
cross-linking occurs, intracllular signaling vnts u to multipl phosphoryla
tion ractions
caus an incras in calcium. Th influx of calcium promots syn- thsis of arac
hionic aci from
mm
ran lipis. It also promots cytokin synthsis an rlas of prform m
iators u to
granulation. 1 Figur 132 picts this raction. Rol of Mast Clls an Basophi
ls Mast clls
ar th principl ffctor clls of immiat hyprsnsitivity, an thy ar r
iv from
prcursors in th
on marrow that migrat to spcific tissu sits to matur. A
lthough thy ar
foun throughout th
oy, thy ar most prominnt in connctiv tissu, th ski
n, th uppr an
lowr rspiratory tract, an th gastroin- tstinal tract. 1 In most organs,
mast clls
tn to
 concntrat aroun th small
loo vssls, th lymphat- ics, th
nrvs, an
th glanular tissu. 7 Ths clls contain numrous cytoplasmic granuls tha
t ar nclos
y
a
ilayr mm
ran. Histamin, which compriss approximatly 10 prcnt of th
total wight of
granular constitunts, is foun in 10 tims gratr supply pr cll than in
aso
phils. 6 Mast
cll populations in iffrnt sits iffr in th amounts of allrgic m
iators that thy
contain an in thir snsitivity to activating stimuli an cytokins. All typs
of clls ar
triggr in th sam mannr, howvr. Thy rlas a varity of cytok
ins an othr
miators that nhanc th allrgic rspons. 1 Basophils rprsnt approximat

ly 1 prcnt of
th whit
loo clls in priphral
loo. Thy hav a half-lif of a
out 3 ays
. Thy contain
histamin-rich granuls an high-affinity rcptors for IgE, just as in mast cl
ls. Thy rspon
to chmotactic stimulation an tn to accumulat in th tissus uring an in amma
tory raction.
In th prs- nc of IgE, th num
r of rcptors has
n foun to incras, in
icating a
possi
l mchanism of uprgulation uring an allrgic raction. 4,6
Allrgn CD4
T cll APC
procsss allrgn APC
B cll T cll provis hlp to B cll Allrgn-spcifi
c IgE A
Anti
oy-coat mast cll Allrgn Rlas of histamin + othr miators
Binin
g an
granulation of mast cll Anaphylaxis asthma hivs rhinitis B + FIGURE 132. Typ
I
hyprsnsi- tivity. (A) Snsitiation. Formation of antign-spci c IgE that attac
hs to mast
clls. (B) Activation. Rxposur to sp- ci c antign with su
squnt granulati
on of mast
clls an rlas of miators. (APC
antign-procssing cll.) 1814_Ch13_201-223
.qx 7/10/09
2:56 PM Pag 205 206 SECTION 3 Immun Disorrs Miators Rlas from Granul
s Prform
Miators Cross-linking of surfac-
oun IgE on
asophils an mast clls
y a sp
ci c allrgn
causs changs in th cll mm-
ran that rsult in th rlas of mia
tors from th
cytoplasmic granuls. Ths prform or primary mia- tors inclu histamin,
hparin,
osinophil chmotactic factor of anaphylaxis (ECF-A), nutrophil chmotactic fac
tor, an
protass. 4 Rlas of ths su
stancs is rspon- si
l for th arly phas sy
mptoms sn in
allrgic ractions, which occur within 30 to 60 minuts aftr xposur to th al
lrgn. Th
ffct of ach of ths miators is iscuss an a summary is prsnt in Ta

l 132.
Histamin, a major componnt of mast cll granuls, is a low-molcular-wight va
soactiv amin.
Its ffcts, which appar within 30 to 60 scons aftr rlas, ar pnnt o
n activation of
four iffrnt typs of rcptors foun on clls in various typs of tissu. 8 A
ctivation of H 1
rcptors rsults in contraction of smooth muscl in
ronchiols,
loo vssls
, an th
intstins, an gnrally inucs proin ammatory activity. 6,8 In aition, th
r is incras
capillary prma
ility, altr cariac contractility, an incras mucous
glan scrtion
in th uppr-rspiratory tract. Bining to H 2 rcptors incrass gastric aci
scr- tion,
airway mucus prouction, an prma
ility of capillaris an vnuls. 6 H 3 rc
ptors ar foun
on cntral an priph- ral nural tissu. Bon marrow clls, mast clls,
an priphral

loo clls such as osinophils, nutrophils, an
asophils all hav H 4 rcpto
rs. 6,8 H 4
rcptors appar to
 involv in immun rgulation, incluing chmotaxis of ma

st clls;
rcruitmnt of osinophils, nutrophils, an
asophils; an cytokin scrti
on. 8 In th
skin, histamin is rsponsi
l for local rythma or rnss an whal an flar
formation.
Contraction of th smooth muscl in th
ronchiols may rsult in air ow o
structi
on. Incras
vascular prma
ility may caus hypotnsion or shock. Dpning on th rout

y which an
iniviual is xpos to th triggring allrgn, on or mor of ths ffcts m
ay
 sn.
Eosinophil chmotactic factor of anaphylaxis (ECF-A) is anothr prform factor
rlas from
granuls. This attracts osinophils to th ara an inucs xprssion of osino
phil rcptors
for C3
. 7 Eosinophils hav granuls that contain major
asic protin, osinophi
l cationic protin, osinophil proxias, osinophil riv nurotoxins, histaminass, an p
hospholipas D. 4
All of ths proucts ar toxic to
ronchial pithlial clls an to hlminth pa
ra- sits.
Killing of parasits is thought to
 th rason that IgE prouction volv. On
 of th
protolytic nyms rlas is tryptas. Tryptas clavs kininogn to gn
rat
raykinin,
which inucs prolong smooth muscl contraction an incrass vascular prma

ility an
scrtory activity. 1 Complmnt split proucts ar also rlas whn C3 is con
vrt to C3a an
C3
. Nwly Synthsi Miators In aition to immiat rlas of prform m
iators, mast
clls an
asophils ar triggr to synthsi crtain Ta
l 13-2. Miators of
Immiat
Hyprsnsitivity MEDIATOR STRUCTURE ACTIONS Prform Histamin MW 111 Smooth mu
scl contraction,
vasoilation, incras vascular prma
ility ECF-A MW 3802000 Chmotactic for o
sinophils
Nutrophil MW 600,000 Chmotactic for nutrophils chmotactic factor Protass
MW 130,000
Convrt C3 to C3
, mucus prouction, activation of cytokins Nwly Synthsi P
GD 2 Arachionic
aci rivativ Vasoilation, incras vascular prma
ility LTB 4 Arachionic
aci rivativ
Chmotactic for nutrophils, osinophils LTC 4 , LTD 4 , LTE 4 Arachionic aci
rivativs
Incras vascular prma
ility,
ronchoconstriction, mucous scrtion Platlt
activating MW
300500 Platlt aggrgation factor Cytokins IL-1, TNF-a, Small MW protins Incr
as
in ammatory clls in ara, an incras IL-3, IL-4, IL-5, IL-6, IgE prouction IL
-9, IL-13,
IL-14, IL-16, GM-CSF MW
molcular wight; ECF-A osinophil chmotactic factor of
anaphylaxis;
PGD
prostaglanin; LT
lukotrin 1814_Ch13_201-223.qx 7/10/09 2:56 PM Pag
206 CHAPTER
13 Hyprsnsitivity 207 othr ractants from th
rakown of phospholipis in
th cll
mm
ran. Ths proucts ar rsponsi
l for a lat- phas allrgic raction s
n within 6 to 8
hours aftr xposur to antign. Nwly form miators inclu platlt-

activating factor
(PAF); prostaglanin (PG) D 2 ; lukotrins (LT) B 4 , C 4 , D 4 , an E 4 ); a
n cytokins.
1,4,5,7 Prostaglanins an lukotrins ar riv from arachionic aci, a mm
-
ran lipi,
y
two sparat mta
olic pathways. In on pathway, th nym 5-lipoxygnas clav
s arachionic
aci to gnrat lukotrins. Th othr pathway uss cyclooxy- gnas, which r
sults in
prostaglanin prouction. PGD 2 is th major prouct of th cyclooxygnas pathway. Whn
rlas
y mast clls, it mimics th ffcts of histamin, causing
ronchial co
nstriction an
vasoilation. 1,6 It is much mor potnt than histamin,
ut it is rlas in s
mallr
quantitis. In skin ractions, PGD 2 triggrs whal an ar formation. Thus, th
ovrall ffct
is to nhanc an potntiat th action of histamin. Lukotrins, rsulting fr
om th
5-lipoxygnas path- way of arachionic aci mta
olism, ar also rsponsi
l fo
r lat-phas
symptoms of immiat snsitivity. Lukotrins C 4 , D 4 , an E 4 wr origina
lly collctivly
nam th slow- racting su
stancs of anaphylaxis (SRS-A). LTC 4 an LTD 4 ar
1000 tims
mor potnt than histamin in causing incras vascular prma
ility,
ron
choconstriction,
an incras mucous scrtion in small airways. 1,6 In th ints- tins, luko
trins inuc
smooth muscl contraction. Systmically, thy may prouc hypotnsion as
a rsult of
iminish cariac muscl contractility an lssn
loo ow. LTB 4 is a po
tnt
chmotactic factor for nutrophils an osinophils. Th apparanc of osinophil
s is spcially
important as a ngativ f
ack control mchanism. Eosinophils rlas hi
staminas, which
gras histamin, an phospholipas D, which gras PAF. Aitionally, supr
oxis crat in

oth osinophils an nutrophils caus th
rakown of lukotrins. Eosinophil
proucts can
also hav a amaging ffct. Eosinophil cationic pro- tin an osinophil-ri
v nurotoxin
contri
ut to xtnsiv tissu struction. 1,7 PAF is a phospholipi rlas

y monocyts,
macrophags, nutrophils, osinophils, mast clls, an
asophils. Th ff
cts of PAF
inclu platlt aggrgation, chmotaxis of osinophils an nutrophils, incras
 vascular
prma
il- ity, an contraction of smooth muscl in th lungs an ints
tins. 5
Cytokins rlas from mast clls inclu IL-1, IL-3, IL-4, IL-5, IL-6, IL-9, I
L-13, IL-14,
IL-16, GM-CSF, an TNF- . 1,9 Ths cytokins altr th local micronviron- mnt,
laing to an
incras in in ammatory clls such as nutrophils, osinophils, an macrophags. I
L-3 an IL-4
incras IgE prouction to furthr amplify th Th2 rspons. 1,6 In ait
ion, IL-3 is a
growth factor for mast clls an
asophils, an IL-4 rcruits T clls,
a

sophils,
osinophils, an monocyts. 7 IL-5 also rcruits osinophils. A high concntra
tion of TNFmay contri
ut to th symptoms of shock sn in systmic anaphylaxis. Th nw
ly form
miators ar rsponsi
l for a lat phas raction in 4 to 6 hours aftr xposu
r to th
allrgn, uring which numrous clls such as osinophils, nutrophils, Th2 cll
s, mast clls,

asophils, an macrophags xit th circulation an infiltrat th allrgn-fill
 tissu. Thy
rlas furthr miators that prolong th hypractivity an may la to tissu
amag. 7
Clinical Manifstations of Immiat Hyprsnsitivity Typ I hyprsnsitivity is
a wispra
halth pro
lm, as it is stimat that up to 25 prcnt of th population suffrs from som
sort of allrgy. 3,7 Th clinical manifstations caus
y rlas of
oth prfo
rm an nwly
synthsi miators from mast clls an
asophils vary from a locali
 skin raction
to a systmic rspons known as ana- phylaxis. Symptoms pn on such varia
ls
as rout of
xposur, osag, an frquncy of xposur. If an allrgn is inhal, it is mo
st likly to
caus rspiratory symptoms such as asthma or rhinitis. Ingstion of an allrgn
may rsult in
gastrointstinal symptoms, an injction into th
loostram can triggr a syst
mic rspons.
Anaphylaxis is th most svr typ of allrgic rspons,
caus it is an acut
raction that
simultanously involvs multipl organs. It may
 fatal if not trat promptly
. Coin
y

iologists Paul-Juls Portir an Charls Ro
rt Richt in 1902, th trm litra
lly mans
without protc- tion. Anaphylactic ractions ar typically triggr
y glyc
oprotins or
larg polypptis. Smallr molculs, such as pnicillin, ar haptns that may

com
immunognic
y com
ining with host clls or protins. Typical agnts that inuc
anaphylaxis
inclu vnom from
s, wasps, an hornts; rugs such as pnicillin; an foos
such as shllfish, panuts, or airy proucts. 1,7,10 Aitionally, latx snsitivity is
now a signi cant
caus of anaphylaxis among halth-car workrs an in patints who hav ha mult
ipl surgry
procurs, such as chilrn with spina
i a. 11,12 It is stimat that approxim
atly 10
prcnt of all halth- car workrs hav
n snsiti to latx, u t
o th
implmntation of OSHA rgulations rquiring glovs for la
oratory procurs an
 for working
with patints. 11,12 Clinical signs of anaphylaxis
gin within minuts aftr an
tignic challng
an may inclu
ronchospasm an laryngal ma, vascular congstion, skin mani
fstations such
as urticaria (hivs) an angioma, iarrha or vom- iting, an intracta
l sho
ck
caus of th
ffct on
loo vssls an smooth muscl of th circulatory systm. Th svrit

y of th raction
pns on th num
r of prvious xposurs to th antign with consqunt
uil
up of IgE on mast
clls an
asophils. Massiv rlas of ractants, sp- cially histamin, from
th granuls is
rsponsi
l for th nsuing symptoms. Dath may rsult from asphyxiation u to
uppr-airway
ma an congstion, irrvrsi
l shock, or a com
ination of ths symptoms. Rh
initis is th
most common form of atopy, or allrgy. It affcts a
out 15 to 20 prcnt of chil
rn in vlop
1814_Ch13_201-223.qx 7/10/09 2:56 PM Pag 207 208 SECTION 3 Immun Disorrs
countris. 2
Symptoms inclu paroxysmal sning; rhin- orrha, or runny nos; nasal congst
ion; an itching
of th nos an ys. 13 Although th conition itslf is mrly annoying, compl
ications such as
sinusitis, otitis mia (ar infction), ustachian tu
 ysfunction, an slp
istur-
ancs
may rsult. Polln, mol spors, animal anr, an particulat mattr from hous
 ust mits ar
xampls of air
orn forign particls that act irctly on th mast clls in th
 conjunctiva an
rspiratory mucous mm
rans to triggr rhinitis. Particls no largr than 2 to
4 m in iamtr
such as polln, ust, or fums, may rach th lowr-rspiratory tract to caus a
sthma. 1,14
Asthma is riv from th Grk wor for panting or
rathlssnss. It can
 fin

clinically as rcurrnt air ow o
struction that las to intr- mittnt sning,

rathlssnss,
an, occasionally, a cough with sputum prouction. Th air ow o
struction is u t
o
ronchial
smooth muscl contraction, mucosal ma, an havy mucous scrtion. All of th
s changs la
to an incras in airway rsistanc, making it if cult for inspir air to lav
th lungs. This
trapp air crats th sns of
rathlssnss. Foo allrgis ar anothr xam
pl of typ I
immiat hyprsnsitivity ractions. Som of th most common foo allrgis inv
olv cows milk,
panuts, an ggs. 2,13 Symptoms limit to th gastrointstinal tract inclu c
ramping, vomiting, an iarrha, whil spra of antign through th
loostram may caus h
ivs an
angioma on th skin, as wll as asthma or rhinitis. Ecma, an itchy r skin
rash, may

caus
y foo allrgns or xposur to hous ust mits. 13 Tratmnt of Immi
at
Hyprsnsitivity Avoianc of known allrgns is th rst lin of fns. This in
clus
nvironmntal intrvntions such as ncasing mat- trsss an pillows in allrg
n-proof covrs
an not allowing pts insi th hous. 14 Drugs us to trat immiat hyprsnsitivity vary
with th svrity of th raction. Locali allrgic ractions, such as hay fv
r, hivs, or
rhinitis, can
 trat asily with antihistamins an congstants. Asthma is
oftn trat
with a com
ination of thraputic ragnts, incluing antihistamins an
roncho

ilators,
follow
y inhal corticostrois. 13,14 Systmic ractions rquir th us of
pinphrin to
quickly rvrs symptoms. Anothr approach to tratmnt involvs immunothrapy o
r
hyposnsitiation. Vry small quantitis of snsitiing antign ar injct int
o th patint
with th ia of
uil- ing up IgG anti
ois. Iniviuals with morat to sv
r symptoms ar
givn wkly oss of antign that graually incrass in strngth. 15 Th
lock
ing anti
ois
form circulat in th
loo an com
in with antign
for it can rach IgE-c
oat clls. In
aition, it appars that T cllmiat supprssion occurs u to th action of
T rgulatory
clls, thus crasing synthsis of IgE. 1 A nw approach involvs us of an ant
i-IgE monoclonal
anti
oy. This anti
oy com
ins with IgE at th sam sit that IgE woul normal
ly us to
in to
rcptors on mast clls. Blocking of this sit os not allow IgE to
in to mas
t clls, thus
hlping to allviat allrgic symptoms. 1618 Omaliuma
was th rst such anti

oy to

approv for thraputic us in th Unit Stats. 19 In aition to
lock- ing
th rlas of
miators from mast clls, own-rgulation of FcRI rcptors occurs, thus curtail
ing th
rspons. 20 Anti-IgE thrapy has
n succssful in trating morat to svr
asthma an shows
much promis for th futur. 6,16,18,20 Tsting for Immiat Hyprsnsitivity I
n Vivo Skin Tsts
Tsting for allrgis or immiat hyprsnsitivity can
 catgori as in viv
o or in vitro
mthos. In vivo mthos involv irct skin tsting, which is th last xpnsi
v an most
spci c typ of tsting. 21 Cutanous an intrarmal tsts ar th two skin tsts
most oftn
us. In cutanous tsting, or a prick tst, a small rop of matrial is injct
 into th skin
at a singl point. Aftr 15 minuts, th spot is xamin, an th raction is r
cor. A
positiv raction is formation of a whal that is 3 mm gratr in iamtr than
th ngativ
control. 21 A ngativ salin control an a pos- itiv control of histamin shou
l always

inclu. 10,21 Cutanous tsting uss a thousanfol highr concntra- tion tha
n intrarmal
tsts,
ut a lssr amount of antign is us, thus crasing th chancs
of an
anaphylactic rspons. 6,21 Intrarmal tsts us a gratr amount of antign an
 ar mor
snsitiv than cutanous tsting. Howvr, thy ar usually prform only if pr
ick tsts ar
ngativ an allrgy is still suspct. An xtrmity, such as an arm, to which
a tourniqut can

 appli, is us to stop an unxpct sys- tmic raction. A 1 mL tu
r
culin syring
is us to aministr 0.01 to 0.05 mL of tst solution
twn layrs of th s
kin. Th tst
allrgn is ilut 100 to 1000 tims mor than th solution us for cutanous

tsting. Aftr 15
to 20 minuts, th sit is inspct for rythma an whal formation. A whal 3
mm gratr than
th ngativ control is consir a positiv tst. 6 Skin tsting is rlativly
simpl an
inxpnsiv, an it lns itslf to scrning for a num
r of allrgns. Howvr
, antihistamins
must
 stopp 24 to 72 hours
for tst- ing, an thr is th angr that a
systmic raction
can
 triggr. In aition, skin tsting is not rcommn for chilrn un
r 3 yars of ag.
In Vitro Tsts: Total IgE Tsting Principls In vitro tsts involv masurmnt
of ithr total
IgE or antign-spci c IgE. Ths ar lss snsitiv than skin tst- ing
ut usual
ly ar lss
traumatic to th patint. Total IgE tsting has
com mor important as a scr
ning tst
for
a patint is rfrr to an allrgy spcialist,
caus it is a 1814_Ch13_201-22
3.qx 7/10/09
2:56 PM Pag 208 CHAPTER 13 Hyprsnsitivity 209 goo inicator of th likli
hoo of allrgic
isas. 6 Th first tst vlop for th masurmnt of total IgE was th com
ptitiv
raioimmunosor
nt tst (RIST). Th RIST us raiola
l IgE to compt with p
atint IgE for

ining sits on a soli phas coat with anti-IgE. RIST has largly
n rpla
c
y
noncomptitiv soli-phas immunoassays u to th xpns an if culty of workin
g with
raioactivity. 6,19 In noncomptitiv soli-phas immunoassay, antihuman IgE is

oun to a soli
phas such as cllulos, a papr isk, or a microtitr wll. Patint srum is a
 an allow
to ract, an thn a la
l anti-IgE is a to tct th
oun patint IgE.
This la
l is an
nym or a uorscnt tag. Th scon anti-IgE anti
oy rcognis a iffrnt p
itop than that
rcogni
y th first anti
oy. Th rsulting sanwich of soli-phas anti-IgE,
srum IgE,
an la
l anti-IgE is wash, an th la
l is masur. In this cas, th amo
unt of la
l
tct is irctly propor- tional to th IgE contnt of th srum (Fig. 133). A
lthough th
scon incu
ation ou
ls th tim of th assay, th sn- sitivity of this typ
of tsting is
xcllnt, an th rsults ar minimally affct
y th prsnc of nonspcific
srum factors.
19 Total srum IgE tsting is us clinically to ai in iagno- sis of allrgic
rhinitis, asthma,
or othr allrgic conitions that may
 inicat
y patint symptoms. 19 It s
rvs as a scrning tst to trmin if mor spci c allrgy tsting is inicat. It is
also important
in iagnosis of parasitic infc- tions;
ronchopulmonary asprgillosis; an
hypr-IgE
synrom, a conition in which xcssiv amounts of IgE ar prouc. 19 Intrpr
tation of Total
IgE Rsults Total IgE valus ar rport in kilo intrnational units (IU) pr l
itr. On IU is
qual to a concntration of 2.4 ng of protin pr millilitr. IgE concntration

varis with th

ini- viuals ag, sx, an smoking history an his or hr family history of all
rgy. Lvls
also vary as a function of xposur to allrgns. In infants, normal srum lvl
s of IgE ar lss
than 2 k IU/L; incrass up to 10 kIU/L ar inicativ of allrgic isas. 6,14
Aftr th ag of
14 yars, IgE lvls in th rang of 400 kIU/L ar consir to
 a
normally 
lvat. 14 A
cutoff point of 100 IU, howvr, is frquntly us in tsting to intify iniv
iuals with
allrgic tnn- cis. 6 Although two-thirs of aults who hav allrgic N
ONCOMPETITIVE
RIST Spcific
ining Whn su
strat is a, color vlopmnt is proportional
to th amount of
patint anti
oy prsnt. Soli-phas antign NONCOMPETITIVE RAST Spcific
ini
ng
Enym-la
l anti-immunoglo
ulin Bining to patint anti
oy Whn su
strat is
a, color
vlopmnt is proportional to th amount of patint anti
oy prsnt. Enym-la

l
anti-immunoglo
ulin Bining to patint anti
oy
+ + Soli-phas anti-IgE Patint
anti
oy +
Patint anti
oy
+ FIGURE 133. Comparison of RIST an RAST. RIST masurs total I
gE
y
capturing th anti
oy with soli- phas anti-IgE. A scon anti-IgE immunoglo
u
lin with an
nym la
l is us to prouc a visi
l raction. RAST masurs antign-spci c
IgE
y using
soli-phas antign to captur patint anti
oy. Thn a scon anti
oy, nymla
l anti-IgE
immunoglo
ulin, is a. This com
ins with any
oun IgE to prouc a visi
l
raction in th
prsnc of su
strat. 1814_Ch13_201-223.qx 7/10/09 2:56 PM Pag 209 210 SEC
TION 3 Immun
Disorrs symptoms woul fall into this catgory, th othr on-thir might
 i
ncorrctly
iagnos. Aitionally, IgE lvls may
 lvat u to othr conitions, suc
h as hlminth
infc- tions, Wiskott-Alrich synrom, DiGorg synrom, an hypr-IgE synrom
. 19 Thus, total
IgE lvls must
 intr- prt in light of th patints prsonal an family his
tory, an thy
shoul not
 rgar as an a
solut prictor of allrgy. Although masurmnt
of total IgE
lvls can
 a usful scrning tool, allrgn-spci c IgE is mor hlpful in act
ual tratmnt
of th iniviual. Antign-Spci c IgE Tsting Tsting Principls Th original com
mrcial tsting
mtho for trmining spcific IgE was known as th raioallrgosor
nt tst (R
AST), introuc
in 1966. Principls of th tst rmain th sam,
ut nwr tsting mthos invol
v th us of
nym or fluorscnt la
ls rathr than raioactivity. Allrgn-spci c IgE
tsting is
safr to prform than skin tsting an is asir on som patints, spcially ch
ilrn or
apprhnsiv aults, an th snsitivity now approachs that of skin tsting. 15
It is spcially
usful in tcting allrgis to common triggrs such as ragw, trs, grasss

, mols, animal
anr, milk, an gg al
umin. Allrgn-spci c IgE tsting is a noncomptitiv so
li- phas
immunoassay in which th soli phas is coat with spci c allrgn an ract w
ith patint
srum. A car
o- hyrat soli phas, such as papr cllulos isks, agaros
a
s, or
microcrystallin cllulos particls, sms to work
st. Aftr washing to rmov
 un
oun
anti
oy, a la
l anti-IgE is a. Traitional RAST us a raioactiv la
l
, whil nwr
tsts us an nym or fluoromtric la
l. A scon incu
ation occurs, an thn,
aftr a washing
stp, th amount of la
l in th sampl is masur. Th amount of la
l tct
 is irctly
proportional to th amount of spci c IgE in th patints srum. Controls an stan
ars ar run
in paralll with patint srum. Figur 133 shows a comparison of th total an al
lrgn-spci c
IgE tsting. Intrprtation of Allrgn-Spci c IgE Rsults Nwr commrcially a
vaila
l
tsting systms such as Pharmacia an Upjohns ImmunoCAP an Diagnostic Pr
oucts
Corporation IMMULITE hav gratly incras th snsitivity of in vitro allrgy
tsting. 22,23
IgE can now
 masur quantitativly, an concntrations as low as 0.35 KIU/L
can
 tct.
6 It is important to run positiv an ngativ controls to ai in th int
rprtation of
tst rsults. A ngativ tst may
 us to con rm th a
snc of a spci c atopic
isas, an
this has a highr ngativ pr- ictiv valu than total IgE tsting. 19 Howvr
, in a positiv
tst, th quantity of anti
oy masur os not ncssarily corrlat with th
svrity of
isas. 19 This typ of tsting is vry usful to trmin common foo allrgi
s an snsitivity to th vnom of stinging inscts such as hony
s, yllow jackts, wasp
s, an hornts,

ut it is mor if cult to trmin inhalant allrgis. 6 Th cutoff for a positi
v tst also
varis with th particular allrgn. 24 Thus, rsults must
 intrprt in con
junction with
patint symptoms. Microarray Tsting Th nwst gnration of allrgy tsting in
volvs th us of
microarrays. This miniaturiation of iagnostic tsting allows for multiall
rgn iagnosis
with a low sampl volum an a high throughput capacity. 25 Microarrays can
 a
ttach to a
stanar microscop sli, using only a fw picograms of ach allrgn to
 ts
t. 26 In this
mannr, at last 5000 allrgns can
 tst for at onc, using a minimal amoun
t of srum,
approximatly 20L. 3 Th principl is th sam as noncomptitiv immunoas- says,
in that patint
srum with possi
l IgE is allow to ract with th microarray of allrgns, an
 thn an
anti-IgE with a uorscnt tag is a. Aftr carful washing, slis ar ra au
tomatically.
Th prsnc of color inicats a pos- itiv tst for that allrgn. Rcom
inant

DNA tchnology
has gratly contri
ut to th vlopmnt of succssful microarrays. It is now
pos- si
l to
charactri an crat allrgns at th molcular lvl, which nhancs spci cit
y. Up to this
point, allrgy tsting involv us of xtracts of allrgns, which may not hav

n compltly
pur. Inti cation of DNA coing for spci c allrgns has allow cration of puri 
 antigns for
oth tsting an immunothrapy purposs. 26 This sam tsting systm ca
n
 us to look
for incrass in IgG to inicat th f cacy of immunothrapy tratmnt. 3 Us of
microarrays
with rcom
inant allrgns will signi cantly nhanc th valu of spci c IgE tstin
g. TYPE II
HYPERSENSITIVITY Th ractants rsponsi
l for typ II hyprsnsitivity, or cyto
toxic
hyprsnsitivity, ar IgG an IgM. Thy ar trig- gr
y antigns foun on cl
l surfacs. Ths
antigns may
 altr slf-antigns or htroantigns. Anti
oy coats cllular
surfacs an
promots phagocytosis
y
oth opsoniation an activation of th complmnt casc
a. Macrophags,
nutrophils, an osinophils hav FC rcp- tors that
in to th FC rgion of a
nti
oy on targt
clls, thus nhancing phagocytosis. Natural killr (NK) clls also hav FC rcp
tors, an if
ths link to cllular antigns, cyto- toxicity rsults. If th complmnt
casca is
activat, complmnt can triggr cllular struction in two ways: (1)
y coati
ng clls with
C3
, thus facilitating phagocytosis through intraction with spcific rcptors
on phagocytic
clls, or (2)
y complmnt-gnrat lysis if th casca gos to compltion. H
nc, complmnt
plays a cntral rol in th cllular amag that typi s typ II ractions. Transf
usion Ractions
Transfusion ractions ar xampls of cllular struction that rsult from anti

oy com
ining
with htroantigns. Thr ar mor than 29 iffrnt
loo group systms, with
1814_Ch13_201-223.qx 7/10/09 2:56 PM Pag 210 CHAPTER 13 Hyprsnsitivity
211 a total of
mor than 700 iffrnt r cll antigns. 27,28 Som antigns ar strongr than
othrs an ar
mor likly to stimulat anti
oy prouction. Major groups involv in transfusi
on ractions
inclu th ABO, Rh, Kll, Duffy, an Ki systms. 27,29 Crtain anti
ois ar
prouc
naturally with no prior xposur to r
loo clls, whil othr anti-
ois ar
 prouc only
aftr contact with clls carrying that antign. Th ABO
loo group is of
primary
importanc in consiring transfusions. Anti-A an anti-B anti
ois ar n
aturally
occurring anti
ois, or isohmagglutinins, pro
a
ly triggr
y contact with i
ntical
antignic tr- minants on microorganisms. Iniviuals o not form such anti
o
is to thir own
r
loo clls. Thus, a prson who has typ A
loo has anti-B in th srum, an
 a prson with

typ B
loo has anti-A anti
ois. An iniviual with typ O
loo has
oth ant
i-A an anti-B in
th srum,
caus O clls hav nithr of ths two antigns. Th anti
oy form
 typically

longs to th IgM class,
ut IgG may also
 ma. If a patint is givn
loo
for which
anti
ois ar alray prsnt, a transfusion raction occurs. This can rang fr
om acut massiv
intravascular hmolysis to an untct cras in r
loo cll surviva
l. Th xtnt of
th rac- tion pns on th following factors: (1) th tmpratur at which th
 anti
oy is
most activ, (2) th plasma concn- tration of th anti
oy, (3) th particular
immunoglo
ulin
class, (4) th xtnt of complmnt activation, (5) th n- sity of th antign
on th r
loo
cll, an (6) th num
r of r
loo clls transfus. 30 It is most important
to tct
anti
ois that ract at 37C. If a raction occurs only
low 30C, it can
 isr
gar,

caus such antignanti
oy complxs tn to issociat at 37C. Acut hmolytic
transfusion
ractions may occur within minuts or hours aftr rcipt of incompati
l
loo.
In this cas,
th iniviual has
n xpos to th antign
for an has prform anti
oi
s to it.
Ractions that
gin immiatly ar most oftn associat with ABO
loo group
incompati
ilitis, an anti
ois ar of th IgM class. 1,30 As soon as c
lls
aring that
antign ar introuc into th patint, intravascular hmolysis occurs
caus
of complmnt
activation, an it rsults in th rlas of hmo- glo
in an vasoactiv an pro
coagulant
su
stancs into th plasma. This may inuc issminat intravascular coag- ula
tion (DIC),
vascular collaps, an rnal failur. Symptoms in th patint may inclu fvr,
chills, nausa,
lowr
ack pain, tachycaria, shock, an hmoglo
in in th urin. 29,30 Dlay
hmolytic
ractions occur within th first 2 wks following a transfusion an ar cau
s
y a sconary rspons to th antign to which th patint has prviously
n x
pos. Th typ
of anti
oy rsponsi
l is IgG, which was initially prsnt in such low titr th
at it was not
tcta
l with an anti
oy scrn. Antigns most involv in lay ractions
inclu thos in
th Rh, Kll, Duffy, an Ki
loo groups. 1,31 Rh, Kll, an Duffy anti- gns
may also

involv in immiat transfusion ractions. In a lay raction, anti
oycoat r
loo
clls ar rmov xtravascularly in th spln or in th livr, an th patint
may xprinc a
mil fvr, low hmoglo-
in, mil jaunic, an anmia. Intravascular hmolysis
os not tak
plac to any grat xtnt,
caus IgG is not as f - cint as IgM in activating c
omplmnt. (S
Chaptr 4 for furthr tails.) Hmolytic Disas of th Nw
orn Hmolytic isa
s of th nw
orn
(HDN) appars in infants whos mothrs hav
n xpos to
loo-group antigns

on th
a
ys
clls that iffr from thir own. Th mothr maks IgG anti
ois in rspons, a
n ths cross
th placnta to stroy ftal r clls. Svr HDN is call rythro
lastosis f
talis. Th most
common antign involv in svr ractions is th D antign, a mm
r of th Rh

loo group. HDN
u to ABO incompati
ility is actually mor common,
ut th isas is milr

caus th majority of anti
ois form ar IgM, which cannot cross th placnta. 32 Othr anti

ois associat
with HDN inclu anti-c, anti-C, anti-E, anti-, an lss commonly thos asso- c
iat with th
Kll, Duffy, an Ki
loo groups. 33 Exposur usually occurs uring th
irth
procss, whr
ftal clls lak into th mothrs circulation. Typically th first chil is unaff
ct,
ut th
scon an latr chilrn hav an incras risk of th isas u to an anamn
stic rspons.
Th xtnt of th rst ftal-matrnal
l in u- ncs whthr anti
ois will
 p
rouc. If
nough of th
a
ys r clls gt into th mothrs circulation, mmory B clls v
lop. Ths

com activat upon r-xposur to th sam r cll antign, an IgG is thn
prouc. This
crosss th placnta an attachs to th ftal r
loo clls in a su
squnt p
rgnancy.
Dpning on th gr of anti
oy prouction in th mothr, th ftus may
 a

ort,
still
orn, or
orn with vinc of hmolytic isas as inicat
y jaunic.
As r
loo
clls ar lys an fr hmoglo
in rlas, this is convrt to
iliru
in, wh
ich
uils up in
th plasma. Thr is too much of it to
 conjugat in th livr, so it accumul
ats in th
tissus. Biliru
in lvls a
ov 20 mg/L ar associat with position in tissu
 such as th

rain an rsult in a conition known as krnictrus. Tratmnt for svr HDN
involvs an
xchang transfusion to rplac anti
oy-coat r clls. If srum anti
oy ti
trations uring
th prgnancy inicat a high lvl of circulating anti
oy, intrautrin tra
nsfusions can

 prform. 1,32 To prvnt th consquncs of HDN, all womn shoul
 scr
n at th
onst of prgnancy. If thy ar Rh- ngativ, thy shoul
 tst for th
 prsnc of
anti-D anti
ois on a monthly
asis. In currnt practic, anti-D immun glo
uli
n, call Rhogam,
is aministr prophy- lactically at 28 wks of gstation an within 7
2 hours
following livry. 32,34 It is assum that th immun glo
- ulin inhi
its pro
uction of
anti
oy
y com
ining with an stroying th D r
loo clls, thus prvnting
thm from

ing antignic (Fig. 134). This practic has 1814_Ch13_201-223.qx 7/10/0
9 2:56 PM
Pag 211 212 SECTION 3 Immun Disorrs ramatically ruc th num
r of womn
who form anti- D
anti
ois, now affcting a littl ovr 1 prcnt. 35 Autoimmun Hmolytic Anmi

a Autoimmun
hmolytic anmia is an xampl of a typ II hyprsnsitivity raction irct
against
slf-antigns,
caus iniviuals with this isas form anti
ois to thir ow
n r
loo
clls. Symptoms inclu malais, lightha- nss, waknss, an possi
ly m
il jaunic.
Such anti
ois can
 catgori into two groups: warm rac- tiv anti
ois,
which ract
at 37C, an col ractiv anti
ois, which ract only
low 30C. Autoim
mun
hmolytic anmia occurs in approximatly 1 in 80,000 ini- viuals. 32 Warm auto
immun hmolytic
anmia, accounting for mor than 70 prcnt of autoimmun anmias, is charac- t
ri
y
formation of IgG anti
oy, which racts most strongly at 37C. 32 Som of ths an
ti
ois may

pri- mary with no othr isas association, or thy may
 sconary t
o anothr isas
procss. Associat isass may inclu som of th following: viral or rspira
tory infctions, such as infctious mononuclosis, cytomgalovirus, or chronic activ hpa
titis, or
immunoprolifrativ isass such as chronic lymphocytic lukmia an lymphomas.
32,36 In
aition, crtain rugs can
 asor
 onto r
loo clls an ar capa
l of
stimulating
anti
oy prouction. Exampls inclu actaminophn, pnicillins, cphalosporins
, rifampin,
sulfonamis, an mthylopa. 1,37,38 Oftn, how- vr, th unrlying caus
of anti
oy
prouction is unknown, so this is rfrr to as iiopathic autoimmun
hmolytic anmia.
Typically, th patint xhi
its symptoms of anmia
caus of claranc of anti

oy-coat r

loo clls
y th livr an spln. Hmolysis is primarily xtravascular,
cau
s IgG is not as
f cint as IgM in activating complmnt,
ut Anti-Rh srum Rh Rh Rh Rh Rh+ Rh+ Rh+ F
IGURE
134. Passiv immu- niation to prvnt hmolytic isas of th nw
orn. An Rh- n
gativ mothr
is xpos to Rh antigns uring prgnancy with an Rh-positiv ftus. If passiv
ly immuni with
anti-Rh sra at 7 months an on livry, th matrnal anti-Rh immun rspons i
s supprss. A
su
squnt prgnancy (uppr right) is uncomplicat
y HDN. If passiv immuniat
ion is not
practic, th mothr may vlop anti-Rh anti
ois, an a scon Rh- positiv
ftus is likly
to xhi
it struction of r
loo clls charactristic of HDN. (From Barrtt,
JT. Txt
ook of
Immunology, . 5. Mos
y, St. Louis, 1988 p. 339, with prmission.) 1814_Ch13_20
1-223.qx
7/10/09 2:56 PM Pag 212 CHAPTER 13 Hyprsnsitivity 213 intravascular hmol
ysis can also
occur if complmnt os
com activat. Patints with warm autoimmun h
molytic anmia
ar usually trat with corticostrois or splnctomy in mor srious cass. 3
7 A nwr
tratmnt with anti-CD 20 (rituxima
or almtuuma
) is now avail- a
l for cas

s that ar
rfractory to corticostrois. 37,39 This anti
oy attachs to B clls, causing
own-rgulation
of anti
oy prouction an othr ffctor cll activity. Col autoagglutinins, a
lss frqunt
caus of immun hmolytic anmias, typically ar foun in prsons in thir fifti
s an sixtis.
32 Ths ar thought to
 triggr
y antigns on microorganisms,
caus anti

ois hav
n
known to occur following Mycoplasma pnumonia an infc- tious mononuclosis. 37
Col
autoagglutinins hav also
n sn in such isass as chronic lymphocytic luk
mia, nonHogkins lymphoma, myloysplastic synrom, or connctiv tissu isass su
ch as systmic
lupus ryth- matosus. 32 In chilrn, such anti
ois hav
n associat with
virus an
rspiratory infctions. Ths col-racting anti
ois
long to th IgM class,
an most ar
spci c for th Ii
loo groups on r clls. 31,37 Ths anti
ois usually ont c
aus clinical
symptoms. Ractions ar sn only if th iniviual is xpos to th col an t
h tmpratur in
th priphral circulation falls
low 30C. Priphral ncrosis may rsult. Altho
ugh complmnt activation
gins in th col, it can proc at
oy tmpratur. If th
 ntir
complmnt squnc is activat, intravascular hmolysis may occur. 32,37 If r

loo clls

com coat with C3
, this hlps macrophags to
in, an ths clls ar
rapily clar
in th livr, furthr crasing th num
r of circulating r
loo clls. A
sim- pl
tratmnt is to kp th patint warm, spcially th hans an ft, to prvnt
complmnt
activation. Typ II Ractions Involving Tissu Antigns All th ractions that h
av
n
iscuss so far al with iniviual clls that ar stroy whn a sp
cific
antignanti
oy com
ination taks plac. Som typ II ractions involv structi
on of tissus

caus of com
i- nation with anti
oy. Organ-spci c autoimmun isass in which
anti
oy is
irct against a particular tissu ar in this catgory. Goopasturs synrom
is an xampl
of such a isas (s Chaptr 14 for tails). Th anti
oy prouc uring th
cours of this
isas racts with
as- mnt mm
ran protin. Usually th glomruli in
th kiny an
pulmonary alvolar mm
rans ar affct. 1 Anti
oy
ins to glomrular an al
volar
capillaris, triggring th complmnt casca, which provoks in ammation. An v
nly
oun
linar position of IgG in glomrular
as- mnt mm
ran, as tct
y uorsc
nt-la
l
anti-IgG, is inicativ of Goopasturs synrom. Tratmnt usually involvs th
us of
corticostrois or othr rugs to supprss th immun rspons. Othr xampls o
f ractions to
tissu antigns inclu som of th organ-spci c autoimmun isass such as Hash

imotos
isas, myasthnia gravis, an insulin-pnnt ia
ts mllitus. Immunologic
manifstations
an tction of ths isass ar prsnt in Chaptr 14. Tsting for Typ II
Hyprsnsitivity
As iscuss in Chaptr 9, Coom
ss iscovry of th antiglo
ulin tst in
1945 ma
possi
l th tction of anti-
oy or complmnt on r
loo clls. Dirct an
tiglo
ulin
tsting (DAT) is prform to tct transfusion ractions, hmolytic isas
of th nw
orn,
an autoimmun hmolytic anmia. (Rfr to th xrcis at th n of th chap
tr for tails.)
Polyspcific antihuman glo
ulin is a mixtur of anti
ois to IgG an complmnt
componnts such
as C3
an C3, an it is us for initial tsting. If th tst is positiv, th
n it shoul

rpat using monosp- ci c anti-IgG, anti-C3
, an anti-C3 to trmin which o
f ths is
prsnt. 31 If an autoimmun hmolytic anmia is caus
y IgM anti
oy, only th
 tst for
complmnt com- ponnts woul
 positiv. Th inirct Coom
s tst is us in th
 crossmatching
of
loo to prvnt a transfusion raction. It is us ithr to trmin th
prsnc of a
particular anti
oy in a patint or to typ patint r
loo clls for spc
i c
loo group
antigns. In vitro
ining of anti
oy to r clls, rathr than in vivo
ining
, is tct
y
this mtho. This is a two-stp procss, in which r
loo clls an anti
oy a
r allow to
com
in at 37C, an thn th clls ar car- fully wash to rmov any un
oun a
nti
oy.
Antihuman glo
ulin is a to caus a visi
l raction if anti
oy has
n sp
cifically
oun.
Any ngativ tsts ar confirm
y quality-control clls, which ar coat with
anti
oy. Rfr
to Chaptr 9 for aitional tails on inirct antiglo
ulin tsting. TYP
E III
HYPERSENSITIVITY Typ III hyprsnsitivity ractions ar similar to typ II rac
- tions in that
IgG or IgM is involv an struction is complmnt miat. Howvr,
in th cas of
typ III is- ass, th antign is solu
l. Whn solu
l antign com
ins with
anti
oy,
complxs ar form that prcipitat out of th srum. Normally such complxs
ar clar
y
phago- cytic clls,
ut if th immun systm is ovrwhlm, ths complxs p
osit in th
tissus. Thr thy
in compl- mnt, causing amag to th particular tissu.
Dposition of
antignanti
oy complxs is in unc
y th rlativ concntration of
oth compon
nts. If a
larg xcss of anti- gn is prsnt, sits on anti
oy molculs
com fill

for
cross-links can
 form. In anti
oy xcss, a lat- tic cannot
 form
ca
us of th
rlativ scarcity of antignic trminant sits. Th small complxs that rsul
t in ithr of
th prcing cass rmain suspn or may pass irctly into th urin. Prcip

itating
complxs, on th othr han, occur in mil antign xcss, an ths ar th on
s most likly to
posit in th tissus. Sits in which this typically occurs inclu th glomru
lar
asmnt
mm
ran, 1814_Ch13_201-223.qx 7/10/09 2:56 PM Pag 213 214 SECTION 3 Immun
Disorrs
vascular nothlium, joint linings, an pulmonary alvo- lar mm
rans. 1 Compl
mnt
ins to
ths complxs in th tissus, causing th rlas of miators that incras v
asoilation an
vasoprma
ility, attract macrophags an nutrophils, an nhanc
ining of ph
agocytic clls
y
mans of C3
posit in th tissus. If th targt clls ar larg an can- no
t
 ngulf
for phagocytosis to tak plac, granul an lysosom contnts ar rlas

y a procss
known as xocytosis. This rsults in th amag to host tissu that is typifi

y typ III
ractions. Long-trm changs inclu loss of tissu lmnts that cannot rgnr
at an accumulation of scar tissu. Arthus Raction Th classic xampl of a locali typ I
II raction is
th Arthus raction, monstrat
y Mauric Arthus in 1903. Using ra

its that
ha
n
immuni to prouc an a
un- anc of circulating anti
ois, Arthus show tha
t whn ths
ra

its wr challng with an intrarmal injction of th antign, a locali
 in ammatory
raction rsult. This raction, charactri
y rythma an ma, paks wit
hin 3 to 8
hours an is follow
y a hmorrhagic ncrotic lsion that may ulcra
t. 1 Th
inflammatory rspons is caus
y antignanti
oy com
ination an su
squnt for
mation of
immun complxs that posit in small rmal
loo vssls. Complmnt is x, a
ttracting
nutrophils an causing aggrgation of platlts. Nutrophils rlas toxic pro
ucts such as
oxygn-containing fr rai- cals an protolytic nyms. Activation of complm
nt is ssntial
for th Arthus raction,
caus th C3a an C5a gnrat activat mast clls t
o rlas
prma
ility factors; consquntly, immun complxs locali along th nothlial cll

asmnt mm
ran. Th Arthus raction is rar in humans (Fig. 135). Srum Sickn
ss Srum
sicknss is a gnrali typ III raction that is sn in humans, although
not as
frquntly as it us to
. Srum sicknss rsults from passiv immuniati
on with animal
srum, usually hors or
ovin, us to trat such infctions as iphthria, tt
anus, an
gangrn. Vaccins an
 stings may also triggr this typ of ractio
n. 1 Gnrali
symptoms appar in 7 to 21 ays aftr injction of th animal srum an inclu
haach, fvr,
nausa, vomiting, joint pain, rashs, an lymphanopathy. Rcovry taks
tw
n 7 an 30 ays.
In this isas, th snsitiing an th shocking os of antign ar on an th

 sam,
caus
anti
ois vlop whil antign is still prsnt. High lvls of anti
oy form
immun complxs
that posit in th tissus. Usually this is a
nign an slf-limiting isas,

ut prvious
xposur to animal srum can caus cariovascular collaps on rx- posur. Ths
 symptoms hav
occurr with monoclonal anti
oy tratmnt using mous anti
ois to human cll
s. Now, howvr,
monoclonal anti
ois ar gntically nginr human anti
ois, an such
ractions o not
occur. Autoimmun Disass Typ III hyprsnsitivity ractions can
 trig
gr
y ithr
autologous or htrologous antigns. Svral of th autoimmun isass fall int
o this catgory.
Systmic lupus rythmatosus (SLE) an rhumatoi arthritis ar two such x
ampls. In SLE,
anti
ois ar irct against constitunts such as DNA an nuclohistons, whi
ch ar foun in
most clls of th
oy. Immun com- plx position involvs multipl organs,
u
t th main amag
occurs to th glomrular
asmnt mm
ran in th kiny. In rhumatoi arthriti
s, an anti
oy
call rhumatoi factor is irct against IgG. Immun complx position occu
rs in th
mm
rans of in am joints. Complmnt nhancs tissu struction in
oth isas
s. S Chaptr
14 for a mor tail iscussion of ths two conitions. Skin surfac Circulat
ion contains
anti
ois Local injction of antign Immun complxs form on vssl walls, act
ivat complmnt
Anaphylatoxins caus vascular ilatation, accumulation of nu- trophils, scap
of protins, tc.
THE ARTHUS PHENOMENOM A B C FIGURE 135. Th Arthus phnomnon. (A) Antign is inj
ct into th
skin of an iniviual who has circulating anti
oy of that spci- city. (B) Immun
 complxs ar
form an posit on th walls of
loo vssls, activating complmnt. (C) Com
plmnt fragmnts
caus ilation an incras prma
ility of
loo vssls, ma, an accumulat
ion of
nutrophils. (From Wimann, FK. An Introuction to Clinical Immunology. FA Davis
, Philalphia,
1989, p. 228, with prmission.) 1814_Ch13_201-223.qx 7/10/09 2:56 PM Pag 21
4 CHAPTER 13
Hyprsnsitivity 215 Tsting for Typ III Hyprsnsitivity In spcific isas
s such as SLE an
rhumatoi arthritis, th prsnc of anti
oy can
 tct
y agglutination
ractions using
antign-coat carrir particls, incluing r
loo clls or latx particls,
or nym
immunoassays. Fluorscnt staining of tissu sctions has also
n us to t
rmin position
of immun complxs in th tissus. Th staining pattrn sn, an th particula
r tissu
affct, hlps to intify th isas an trmin its svrity. A mor gnr
al mtho of
trmining immun com- plx isass is
y masuring complmnt lvls. Dcras
 lvls of
iniviual componnts or cras functioning of th pathway may
 inicativ

of
antignanti
oy com
i- nation. Th rsults must
 intrprt with rgar to oth
r clinical
nings, howvr. Rfr to Chaptr 6 for a iscus- sion of complmnt tsting. TYP
E IV
HYPERSENSITIVITY Typ IV, or lay, hyprsnsitivity was rst scri
 in 1890

y Ro
rt
Koch, who o
srv that iniviuals infct with Myco
actrium tu
rculosis
(Mt
) vlop
a locali in ammatory rspons whn injct intrarmally with a ltrat from th
organism. 1
Typ IV hyprsnsitivity iffrs from th othr thr typs of hyprsnsi
tivity in that
snsiti T clls, usually a su
population of Th1 clls, play th major rol in
its
manifstations. Anti
oy an complmnt ar not irctly involv. Thr is an i
nitial snsitiation phas of 1 to 2 wks that taks plac aftr th rst contact with anti
gn. Thn, upon
su
squnt xposur to th antign, symptoms typically tak svral hours to v
lop an rach a
pak 48 to 72 hours aftr xposur to antign. 40 Th raction cannot
 tra
nsfrr from
on animal to anothr
y mans of srum,
ut only through transfr of T lympho
cyts. Langrhans
clls in th skin an macrophags in th tissu captur an prsnt antign to T
hlpr clls of
th Th1 su
class. Th1 clls ar activat an rlas cytokins, inclui
ng IL-3,
intrfron gamma (IFN- ., tumor ncrosis factor-
ta (TNF- ), an tumor ncrosis fac
tor- alpha
(TNF- ), that rcruit macrophags an nutrophils, prouc ma, promot fi
rin 
position, an
gnrally nhanc an in ammatory rspons. 1,40 Cytotoxic T clls ar also rcruit
, an thy

in with antign-coat targt clls to caus tissu struction. Allrgic skin
ractions to

act- ria, viruss, fungi, an nvironmntal antigns such as poison ivy typify
this typ of
hyprsnsitivity. Contact Drmatitis Contact rmatitis is a form of lay hyp
rsnsitivity
that accounts for a signi cant num
r of all occupationally acquir illnsss. It
is stimat
that 15 to 20 prcnt of th Wstrn-worl population is allrgic to on or mor
chmicals in th
nvironmnt. 41,42 Ractions ar usually u to low-molcular-wight compouns t
hat touch th
skin. Th most common causs inclu poison ivy, poison oak, an poison sumac, a
ll of which giv
off urushiol in th plant sap an on th lavs. Allrgic rmatitis u to cont
act with ths
thr plants affcts
twn 10 an 50 million Amricans vry yar. 43 O
thr common
compouns that prouc allrgic skin manifstations inclu nickl; ru

r; form
alhy; hair
ys an fa
ric nishs; cosmtics; an mications appli to th skin, such as t
opical
ansthtics, antisptics, an anti
iotics. 41,44 In aition, latx allrgy has

n rport to
affct
twn 3.8 an 17 prcnt of all halth-car workrs in th Unit Stat

s. 12 Most of
ths su
stancs pro
a
ly function as haptns that
in to glyco- protins on
skin clls. Th
Langrhans cll, a skin macrophag, functions as th antign-prsnting cll
at th sit of
antign contact. 42 It appars that Langrhans clls may migrat to rgional lym
ph nos an
gnrat snsi- ti Th1 clls thr. This snsitiation procss taks svral
ays,
ut onc it
occurs, its ffcts last for yars. 1 Aftr rpat xposur to th antign, cyto
kin prouc- tion
causs macrophags to accumulat. A skin ruption charactri
y rythma, sw
lling, an th
formation of papuls appars anywhr from 6 hours to svral ays aftr th xp
osur. Th
papuls may
com vsicular, with
lis- tring, pling, an wping. Thr is
usually itching
at th sit (Fig. 136). Th rmatitis is rst limit to skin sits xpos to th
antign,
ut
thn it spras out to ajoining aras. Th uration of th raction pns upo
n th gr of
snsitiation an th concntration of antign a
sor
. Drmatitis can last for
3 to 4 wks
aftr th antign has
n rmov. 42 Simpl rnss may fa of its own accor
within svral
ays. If th ara is small an locali, a topical stroi may
 us for tra
tmnt. Othrwis,
systmic corticostrois may
 aministr. Th patint also ns to avoi co
n- tact with th
offning allrgn. Hyprsnsitivity Pnumonitis Rcnt vinc shows that hyp
rsnsitivity
pnumonitis is miat prominantly
y snsiti T lymphocyts that FIGURE 136
. Contact
hyprsnsitivity. Formation of papuls aftr xposur to poison ivy. 1814_Ch13_2
01-223.qx
7/10/09 2:56 PM Pag 215 216 SECTION 3 Immun Disorrs rspon to inhal
allrgns. IgG
an IgM anti
ois ar form,
ut ths ar thought to play only a minor part
. This is an
allrgic isas of th lung parnchyma, charactri
y in ammation of th alvo
li an
intrstitial spacs. It is caus
y chronic inhalation of a wi varity of ant
igns, an it is
most oftn sn in mn
twn th ags of 30 an 50 yars. 45 Dpning on th
occupation an
th particular antign, th isas gos
y svral nams, incluing farmrs lung
, pigon

rrs isas, an humi- ifir lung isas. Th raction is most lik
ly u to
microorganisms, spcially
actrial an fungal spors, that iniviuals ar xp
os to from
working with moly hay, pigon roppings, compost, moly to
acco, infst our, a
n moly
chs, to nam just a fw xampls. Symptoms inclu a ry cough, shortnss of

rath, fvr,
chills, wight loss, an gnral malais, which may
gin within 4 to 8
hours aftr
xposur to a high os of th offning allr- gn. 7,45 Alvolar macrophags a
n lymphocyts
triggr a chronic conition charactri
y intrstitial
rosis with alvolar in

flammation.
Systmic corticostroi thrapy is us for tratmnt. Tu
rculin-Typ Hyprsns
itivity Tsting
for xposur to tu
rculosis is a classic xampl of a lay hyprsnsitivity
raction. This is

as on th prin- cipl that solu
l antigns from Myco
actrium tu
rculosis i
nuc a raction
in popl who hav or hav ha tu
rcu- losis. Whn challng with antign
intrarmally,
prviously snsiti iniviuals vlop an ara of rythma an inuration at
th injction
sit. This is th rsult of in l- tration of T lymphocyts an macrophags i
nto th ara.
Th
loo vssls
com lin with mononuclar clls, an th raction rachs
a pak
y 72
hours aftr xposur. Th tu
rculin skin tst uss a Myco
actrium tu
rculo- s
is antign
prpar
y making a puri  ltrat from th cll wall of th organism. This purifi
 protin
rivativ (PPD) is injct unr th skin, an th raction is ra at 48 to 7
2 hours. A
positiv tst inicats that th iniviual has prviously
n xpos to Myco

actrium
tu
rculosis or a rlat organism,
ut it os not ncssarily man thr is a
prsntly activ
cas. Tsting for Dlay Hyprsnsitivity Th patch tst is consir th gol
stanar in
tsting for contact rmatitis. 42,44 This must
 on whn th patint is fr
of symptoms or
whn h or sh at last has a clar tst sit. A nona
sor
nt ahsiv patch con
taining th suspct allrgn is appli on th patints
ack, an th skin is chck for a r
action ovr th
nxt 48 hours. Rnss with papuls or tiny
listrs is consir a positiv t
st. Final
valuation is conuct at 96 to 120 hours. All ra- ings shoul
 on
y a s
kill valuator.
Fals ngativs can rsult from inaquat contact with th skin. Anothr typ o
f skin tst for
lay hyprsnsitivity, th Mantoux mtho, is prform in much th sam mann
r as tsting for
th prsnc of IgE. Typically, 0.1 mL of th anti- gn is injct intrarmall
y, using a
syring an a fin nl. 7,46 Th tst sit is ra at 48 an 72 hours for th
prsnc of
inuration. An inuration of 5 mm or mor is consir a positiv tst. 46 Anti
gns typically
us for tst- ing ar Cania al
icans, ttanus toxoi, tu
rculin, an fungal
antigns such as
trichophyton an histoplasmin. 46 SUMMARY Hyprsnsitivity ractions ar xaggr
at ractions to
anti- gns that ar typically not harmful. Usually thr is struction
of host tissu in
th procss of attmpting to stroy th antign. Gll an Coom
s vis a syst
m of
classification for such ractions
as on immun mia- tors an th natur
of th
triggring antign. Typ I hyprsnsitivity, or immiat hyprsnsitivity, i
s manifst
within minuts of xposur to antign. Th principl mi- ator is IgE, which o

s not rmain
fr in th srum
ut
ins to mast clls an
asophils. Whn cll-
oun anti-

oy
ins
antign, th rsulting granulation of th ffctor clls rlass miato
rs that nhanc
th in amma- tory rspons. Prform miators that ar rlas inclu histamin
, osinophil
chmotactic factor of anaphylaxis, nutrophil chmotactic factor, an protolyti
c nyms such as
tryptas. Togthr, ths factors ar rsponsi
l for con- traction of smooth mu
scl in th

ronchiols,
loo vssls, an intstins; incras capillary prma
ility; in
cras
concntration of osinophils an nutrophils in th ara; an cras coagula

ility of
loo.
Nwly synthsi miators, such as prostaglanins, lukotrins, an PAF, pot
ntiat th
ffcts of histamin an othr prform miators. Clinical manifstations of i
mmiat
hyprsnsitivity inclu locali whal an flar skin ractions; rhinitis; an
systmic
anaphylactic rsponss, which can
 lif- thratning. Tsts to trmin th p
otntial for
incurring such ractions inclu
oth in vitro an in vivo mthos. Th noncomp
titiv RIST
masurs total IgE
y using rai- ola
l anti-IgE that
ins to patints IgE on
soli phas.
RAST is a noncomptitiv or captur mtho to trmin anti
oy to spci c allrg
ns. In vivo
skin tsts such as th prick or intrarmal tsts ar on
y xposing th pati
nt irctly to a
vry small amount of allrgn injct unr th skin. A positiv tst proucs
a whal an ar
raction. Th ractants rsponsi
l for typ II hyprsnsitivity ar IgG an IgM
. Thy ract with
antigns locat on cllular surfacs an triggr activation of th complmnt c
asca. Targt
clls ar amag or stroy
y th opsoniing ffct of anti
oy or compl
mnt componnts
or
y complmnt-gnrat lysis if th casca gos to compl- tion. Exampl
s of typ II
ractions u to xognous antigns inclu autoimmun hmolytic anmia, tran
sfu- sion
ractions, an HDN. Th DAT is us to scrn for transfusion ractions, autoimm
un hmolytic
anmia, an hmolytic isas of 1814_Ch13_201-223.qx 7/10/09 2:56 PM Pag 2
16 CHAPTER 13
Hyprsnsitivity 217 th nw
orn. Wash patint r
loo clls ar com
in
with antihuman
glo
ulin an o
srv for agglutination, inicating th prsnc of IgG or compl
mnt componnts
on th clls. Goopasturs synrom is an xampl of a typ II rac- tion irct
 against a
slf-antign in tissu. This synrom is th rsult of anti
oy that racts with
th
asmnt
mm-
ran of th glomrulus. Complmnt is activat, causing tissu amag an
vntual loss of
rnal function. In typ III hyprsnsitivity ractions, IgG, IgM, an com- plm
nt ar also
involv,
ut th raction is irct against solu
l rathr than cllular anti

gns. If mil
antign xcss occurs, th antignanti
oy complxs prcipitat out an posit
in th tissus.
Th sits most affct ar th
as- mnt mm
ran in th kinys, th linings
of
loo vssls,
an th joint linings. Whn complmnt
ins to ths complxs, phagocytic cll
s ar attract
to th ara. If th targt clls cannot
 ngulf, a rvrs-phagocytic procs
s call
xocytosis taks plac. Th Arthus raction, charac- tri
y position of an
tignanti
oy
complxs in th
loo vssls, is a classic xampl of a typ III raction. Oth
r xampls
inclu srum sicknss an autoimmun isass such as SLE an rhumatoi arthri
tis. Spci c
tsts can
 prform to trmin th natur of th anti
oy involv. Typ IV
, or lay,
hyprsnsitivity iffrs from th othr thr catgoris in that anti
oy an co
mplmnt o not
play a major rol. Activation of T clls with th su
s- qunt rlas of cyt
okins is
rsponsi
l for th tissu amag sn in this typ of raction. Contact rm
atitis rsulting
from xposur to poison ivy, poison oak, or a mtal such as nickl is an xampl
of haptns
triggring a raction to slf-antigns. Skin tsting an lymphocyt activation a
r us to
trmin snsitivity to particular antigns. All four typs of hyprsnsitivity
rprsnt
fns mch- anisms that stimulat an in ammatory rspons to cop with an ract
to antign
that is sn as forign. In many cass, th antign is not harmful,
ut th rsp
ons to it
rsults in tissu amag. This ncssitats tratmnt to limit th amag. 1814_
Ch13_201-223.qx
7/10/09 2:56 PM Pag 217 218 SECTION 3 Immun Disorrs 1. A 13-yar-ol mal
ha numrous
a
sncs from school in th spring u to col symptoms that inclu ha cong
stion an
cough. H ha
n on anti
iotics twic,
ut h sm to go from on col
to th nxt. A
complt
loo cll (CBC) count show no ovrall incras in whit
loo clls,

ut a mil
osinophilia was prsnt. Bcaus h ha no fvr or othr signs of infction, h
is physician
suggst that a total IgE scrn- ing tst
 run. Th total IgE valu was 250
IU/mL, which was
within high-normal limits. Qustions a. What woul account for th osinophilia
not?
. What
conclusions can
 rawn from th total IgE valu? c. What othr tsts might

inicat? 2. A
55-yar-ol mal wnt to his physician complaining of fling tir an run own
. Two months
prviously, h ha pnumonia an was concrn that h might not hav compltly
rcovr. H
inicat that his symptoms only
com notica
l if h gos out in th col. A
CBC count was
prform, showing that his whit cll count was within normal limits,
ut his r
 cll count was
just
low normal. A DAT prform on r
loo clls was wakly positiv aftr

incu
ating at
room tmpratur for 5 minuts. Whn th DAT was rpat with monospci c ragnt
s, th tu

with anti- C3 was th only on positiv. Qustions a. What os a positiv DAT
inicat?
. What
is th most likly class of th anti
oy caus- ing th raction? c. Why was th
DAT positiv only
with anti-C3 whn monospci c ragnts wr us? CASE STUDIES 1814_Ch13_201-223.
qx 7/10/09
2:56 PM Pag 218 CHAPTER 13 Hyprsnsitivity 219 DIRECT ANTIGLOBULIN TEST Pri
ncipl Th irct
antiglo
ulin tst (DAT) is us to tct in vivo coating of r
loo clls wit
h anti
oy,
usually IgG, or com- plmnt graation proucts such as C3 or C3g. Prsnc
of complmnt
proucts inicats that antignanti
oy com-
ination triggr th complmnt pa
thway an
prouc split proucts. DAT tsting hlps to iagnos autoimmun hmolytic anm
ia, HDN,
transfusion ractions, or othr typ II hyprsnsitivity ractions. Sampl Prpa
ration Collct

loo
y vnipunctur using asptic tchniqu an avoiing hmolysis. Us
thylniaminttraactic aci (EDTA) as th anticoagulant. Ragnts, Matrials
, an Equipmnt
Polyspci c antihuman glo
ulin ragnt (AHG) Bovin al
umin, 22 prcnt 12
75 mm 
isposa
l
glass tst tu
s Disposa
l plastic
loo
ank piptts Procur 1. Prpar a 2
to 5 prcnt
suspnsion of patint r clls
y using salin. 2. Prpar a 6 prcnt suspnsi
on of
ovin
al
umin
y iluting th 22 prcnt suspnsion. To mak 10 ml of a 6 prcnt susp
nsion, a 2.7
mL of 22 prcnt
ovin al
umin to 7.3 mL of salin. 3. Plac on rop of th r
 cll suspnsion
in ach of two la
l tst tu
s. 4. Fill ach tu
 with salin, an cntrifug
in a
loo
ank
cntrifug for approximatly 1 minut. 5. Dcant tu
, a salin to compltly
rsuspn th
cll
utton, an rcntrifug. Rpat this procur thr mor tims for a tota
l of four washs.
6. Compltly cant th nal wash, an immiatly a on or two rops of polysp
cific AHG (as
spcifi
y th manufacturr) to on tu
. A on or two rops of 6 prcnt
o
vin al
umin to
th othr tu
. 7. Mix an cntrifug for approximatly 30 scons. 8. Dislog
th
utton from
th
ottom of th tu

y gn- tly tilting
ack an forth until no clls rmain
at th
ottom of
th tu
. 9. Examin th tu
 carfully for agglutination
y tilting
ack an fo
rth an looking
for clumps. 10. Lav any nonractiv tsts for 5 minuts at room tm- pratur,
an thn
cntrifug an ra again. 11. A on rop of IgG-coat r clls to any nonr
ac- tiv tu
.
12. Cntrifug an xamin th clls for agglutination. Any prviously ngativ
tu
 shoul

positiv whn th control clls ar a. Intrprtation This is a scrning t
st for th
prsnc of immunoglo
u- lin, namly ithr IgG or IgM on r clls. It also t
cts
rakown

proucts of complmnt activation, namly C3, inicating that complmnt activa

tion has takn
plac. C3 is a
rakown prouct of C3
, th most a
unant compl- mnt compon
nt. If C3 is
prsnt, this inicats that antignanti
oy com
ination must hav takn plac
 to initiat
th complmnt casca. In ithr cas, a positiv tst inicats that r clls
hav
n coat
in vivo an that a typ II hyprsnsitivity rac- tion has occurr. Typically,
coating with
anti
oy usually causs an immiat raction, whil if only complmnt proucts
ar prsnt, th
raction may n to
 incu
at an rra in 5 minuts. A ngativ rsult is
con rm
y th
us of chck clls or control clls. Ths r clls hav
n coat with IgG

y th
manufacturr. Whn anti
oy-coat clls ar a to a tu
 that is ngativ, t
h antihuman
glo
ulin that is still prsnt an not
oun will
in to th control clls. Wh
n cntrifug, a
tu
 with control clls shoul show at last a 2 agglutination. This nsurs t
hat th
patint clls hav
n proprly wash to gt ri of any rsiual imm
unoglo
ulins in
th srum. It also con rms that ractiv AHG was a. A positiv DAT woul
 fo
un in th cas
of autoim- mun hmolytic anmia, in which th patint proucs an anti
oy to h
is or hr own r
clls. In th cas of a trans- fusion raction, a positiv DAT inicats that th
 patint ha
prform anti
oy to th r clls that wr trans- fus. If cor
loo clls
ar tst an
foun to
 positiv, this is an inicator of HDN, in which th mothrs anti
ois hav coat
th
a
ys r clls. Positiv rsults with polyspci c antiglo
ulin can
 fol- low
 up
y
rpating th ntir procur with monospci c ragnts (i.., on that is sp
ci c for IgG
an on that racts with C3). A raction with only anti-C3 inicats that th
anti
oy
triggring th raction is likly of th IgM class. EXERCISE 1814_Ch13_201-223.q
x 7/10/09 2:56
PM Pag 219 220 SECTION 3 Immun Disorrs 1. Which of th following is a gnr
al charactristic
of hyprsnsitivity ractions? a. Immun rsponsivnss is prss.
. Anti
o
y is involv in
all ractions. c. Eithr slf-antign or htrologous antign may
 involv. 
. Th antign
triggring th raction is a harmful on. 2. Which of th following is associat
 with an
incras in IgE prouction? a. Transfusion raction
. Activation of Th1 clls c
. Raction to
poison ivy . HDN 3. Which of th following woul caus a positiv DAT t
st? a. Prsnc
of IgG on r clls
. Prsnc of C3
or C3 on r clls c. A transfusion rac
tion u to
prform anti
oy . Any of th a
ov 4. All of th following ar associat wi
th typ I
hyprsn- sitivity xcpt a. rlas of prform miators from mast clls.
.
activation of

complmnt. c. cll-
oun anti
oy
rig
y antign. . an inhrit tnncy

to rspon to
allrgns. 5. Which nwly synthsi miator has a mo of action similar to t
hat of histamin?
a. LTB 4
. Hparin c. ECF-A . PGD 2 6. Which of th following is associat wi
th anaphylaxis?
a. Builup of IgE on mast clls
. Activation of complmnt c. Incras in cytot
oxic T clls .
Larg amount of circulating IgG 7. To trmin if a patint is allrgic to ry
grass, th
st
tst to prform is a. total IgE tsting.
. skin prick tst. c. DAT. . complm
nt xation. 8.
Which conition woul rsult in HDN? a. Builup of IgE on mothrs clls
. Snsit
iation of
cytotoxic T clls c. Exposur to antign foun on
oth mothr an
a
y r clls
. Prior
xposur to forign r cll antign 9. What is th immun mchanism involv in
typ III
hyprsnsitivity ractions? a. Cllular antigns ar involv.
. Dposition of
immun complxs
occurs in anti-
oy xcss. c. Only htrologous antigns ar involv. . Tiss
u amag rsults
from xocytosis. 10. What is th immun phnomnon associat with th Arthus r
action? a. Tissu
struction
y cytotoxic T clls
. Rmoval of anti
oy-coat r
loo clls c
. Dposition of
immun complxs in
loo vssls . Rlas of histamin from mast clls 11. Co
ntact rmatitis
can
 charactri
y all of th following xcpt a. formation of antignanti
o
y complxs.

. gnration of snsiti T clls. c. Langrhans clls acting as antign-prs
nting clls. .
complxing of a haptn to slf-antign. 12. Which of th following conclusions c
an
 rawn a
out
a patint whos total IgE lvl is trmin to
 150 IU/mL? a. Th p
atint  nitly
has allrgic tnncis.
. Th patint may
 su
jct to anaphylactic shock. c.
Furthr
antign-spci c tsting shoul
 on. . Th patint will nvr hav an allrgic
raction.
REVIEW QUESTIONS 1814_Ch13_201-223.qx 7/10/09 2:56 PM Pag 220 CHAPTER 13 Hy
prsnsitivity
221 13. Which of th following xplains th iffrnc
twn typ II an typ
III
hyprsnsitivity ractions? a. Typ II involvs cllular antigns.
. Typ III i
nvolvs IgE. c.
IgG is involv only in typ III ractions. . Typ II ractions involv no anti

oy. 14. A
37-yar-ol woman rciv two units of pack r
loo clls following a surgi
cal procur.
Sh ha
n transfus onc
for. Fiv ays aftr surgry, sh xprinc a
slight fvr an
som hmoglo
in in hr urin, inicating a lay transfusion raction. A DAT t
st on a
loo
sampl was positiv. Which of th fol- lowing statmnts
st scri
s this ra
ction? a. Th
patint ha IgM anti
oy to th r clls transfus.
. Th patints raction wa
s u to an
amnstic rspons. c. Only IgE was coating th transfus r
loo clls. . Th
 anti
oy

prsnt ract
st at room tmpratur. 1814_Ch13_201-223.qx 7/10/09 2:56 P

M Pag 221 222
SECTION 3 Immun Disorrs Rfrncs 1. Kint, TJ, Gols
y, RA, an Os
orn, BA
. Ku
y
Immunology, . 6. WH Frman an Company, Nw York, 2007, pp. 371400. 2. Host, A
, Anras, S,
Charkin, S, Dia-Vaqu, C, t al. Allrgy tsting in chilrn: Why, who, whn,
an how. Allrgy
58: 559569, 2003. 3. Moths, N, Valnta, R, an Spitaur, S. Allrgy tsting: Th
 rol of
rcom
inant allrgns. Clin Chm La
M 44: 125132, 2006. 4. Goul, HJ, Su
tton, BJ,
Bavil, AJ, Bavil, RL, t al. Th
iol- ogy of IgE an th
asis of allrgic i
sas. Ann Rv
Immunol 21:579628, 2003. 5. Kay, AB. Allrgy an allrgic isass, Part I. N Eng
l J M
344:3037, 2001. 6. Hom
rgr, HA. Allrgic isass. In McPhrson, RA, an Pincus
, MR (s):
Hnrys Clinical Diagnosis an Managmnt
y La
oratory Mthos, . 21. Saunrs
Elsvir,
Philalphia, 2007, pp. 961971. 7. Mak, TW, an Saunrs, ME. Th Immun R
spons: Basic
an Clinical Principls. Elsvir Acamic Prss, Burlington, MA, 2006, pp.
923962. 8.
Jutl, M, Blasr, K, an Akis, CA. Histamin in allrgic inflammation
an immun
moulation. Int Arch Allrgy Immunol 137:8292, 2005. 9. A
ramson, J, an Pcht
, I. Rgulation
of th mast cll rspons to th typ 1 Fc psilon rcptor. Immunol Rv 217:2312
54, 2007. 10.
Li, JT. Allrgy tsting. Am Fam Physician 66:621624, 2002. 11. Taylor, JS, an Er
kk, E. Latx
allrgy: Diagnosis an man- agmnt. Drmatol Thr 17:289301, 2004. 12. Zak, HN,
Kast, LM,
Schwarn
rgr, K, t al. Halth-car workrs an latx allrgy. Arch Environ H
alth 55:336347,
2000. 13. Brgr, WE. Monoclonal anti-IgE anti
oy: A novl thrapy for allrgic
airways isas.
Ann Allrgy, Asthma, Immunol 88:152160, 2002. 14. Kay, AB. Allrgy an allrgic 
isass, Part
II. N Engl J M 344:109113, 2001. 15. Platts-Mills, T, Lung, DYM, an Schat, M
. Th rol of
allrgns in asthma. Am Fam Physician 76:675680, 2007. 16. Plaut, M, an Valntin
, MD. Allrgic
rhinitis. N Engl J M 353:193444, 2005. 17. Hughs, ATD. Anti-IgE anti
oy may h
lp trat som
asthma patints. JAMA 284:28592890, 2000. 18. Milgrom, H, Fick, RB Jr., Su, JQ, 
t al. Tratmnt
of allrgic asthma with monoclonal anti-IgE anti
oy. N Engl J M 341(26):196619
73, 1999. 19.
Hamilton, RG. Immunological mthos in th iagnostic allrgy clinical an r
sarch
la
oratory. In Dtrick, B, Hamilton, RG, an Fols, JD (s): Manual of Molc
ular an Clinical
La
oratory Immunology, . 7. ASM Prss, Washington, DC, 2006, pp. 955963.
20. Milgrom, H.
Is thr a rol for tratmnt of asthma with oma- liuma
? Arch Dis Chil 88:7174
, 2003. 21.
Damin, DA, an P
ls, Jr., RS. In vivo iagnostic allrgy tsting. In Dtrick,
B, Hamilton, RG,

an Fols, JD (s): Manual of Molcular an Clinical La
oratory Immunology, .
7. ASM Prss,
Washington, DC, 2006, pp. 947954. 22. Co

art, CM, an Jonkr, GJ. Allrgy
tsting on
th Immulit 2000 ranom-accss immunoanalyra clinical valuation stuy. Clin C
hm La
M
43:772781, 2005. 23. Smits, WM, Lt, KL, Evans, TS, an Gis, JK. Evaluating th
 rspons of
patints unrgoing
oth allrgy skin tsting an in vitro allrgy tsting with
th ImmunoCAP
tchnology systm. J Am Aca Nurs Pract 15:415423, 2003. 24. Flischr, DM, an
Woo, RA. Tsts
for immunological rac- tions to foos. In Dtrick, B, Hamilton, RG, an Fols,
JD (s): Manual
of Molcular an Clinical La
oratory Immunology, . 7. ASM Prss, Washington, D
C, 2006, pp.
975983. 25. Rnault, NK, Mirotti, L, an Alcocr, MJC. Biotchnologis in nw hig
h-throughput
foo allrgy tsts: Why w n thm. Biotchnol Ltt 29:333339, 2007. 26. Harwan
gg, C, Laffr,
S, Hillr, R, Mullr, MW, t al. Microarray rcom
inant allrgns for iagnos
is of allrgy.
Clin Exp Allrgy 33:713, 2003. 27. Garratty, G, Dik, W, Issitt, PD, t al. Trmi
nology for

loo group antigns an gnshistorical origins an guilins in th nw milln
nium.
Transfusion 40:477489, 2000. 28. Storry, JR, an Olsson, ML. Gntic
asis of
lo
o group
ivrsity. Brit J Hamatol 126:759771, 2004. 29. Dickowski, JS, an Anrson, K
C. Transfusion

iology an thrapy. In Kaspr, DL, Braunwal, E, Fauci, AE, Hausr, SL, t al.
(s): Harrisons
Principls of Intrnal Micin, . 16. McGraw-Hill, Nw York, 2005, pp. 662667.
30. Davnport,
RD, an Mint, PD. Transfusion micin. In McPhrson, RA, an Pincus, MR (
s): Hnrys
Clinical Diagnosis an Managmnt
y La
oratory Mthos, . 21. Saunrs Elsvi
r, Philalphia,
2007, pp. 669684. 31. Baling, WV, an Cooling, L. Immunohmatology. In Mc
Phrson, RA,
an Pincus, MR (s): Hnrys Clinical Diagnosis an Managmnt
y La
oratory
Mthos, .
21. Saunrs Elsvir, Philalphia, 2007, pp. 617668. 32. Ry, VVB. Extracor
poral fcts
laing to incras rythrocyt structionimmun causs. In Roak, BF, F
ritma, GA,
an Doig, K (s): Hmatology: Clinical Principls an Applications. Saun
rs Elsvir, St.
Louis, 2007, pp. 325332. 33. Garratty, G, Glynn, SA, an McEntir, R. ABO an Rh(
D) phnotyp
frquncis of iffrnt racial/thnic groups in th Unit Stats. Transfusion
44:703706, 2004.
34. Mak, TW, an Saunrs, ME. Th Immun Rspons: Basic an Clinical Principl
s. Elsvir
Acamic Prss, Burlington, MA, 2006, pp. 696749. 35. Murray, NA, an Ro
rts, IA
G. Hamolytic
isas of th nw
orn. Arch Dis Chil 92:F83F88, 2007. 36. Elghtany, MT, an
Banki, K.
Erythrocytic isorrs. In McPhrson, RA, an Pincus, MR (s): Hnrys C
linical

Diagnosis an Managmnt
y La
oratory Mthos, . 21. Saunrs Elsvir, Phila
lphia, 2007,
pp. 504544. 37. Bunn, HF, an Ross, W. Hmolytic anmias an acut
loo loss. I
n Kaspr, DL,
Braunwal, E, Fauci, AE, Hausr, SL, t al. (s): Harrisons Principls of Intrn
al Micin,
. 16. McGraw-Hill, Nw York, 2005, pp. 607616. 38. Garvy, B. Rituxima
in th
tratmnt of
autoimmun hama- tological isorrs. Brit J Hamatol 141:149169, 2008. 39. DArn
a, G, Taylor,
RP, Cascavilla, N, an Linorfr, MA. Monoclonal anti
ois: Nw thraputic ag
nts for autoimmun hmolytic anmia? Enocr Mta
Immun Disor 8:6268, 2008. 40. Jaco
,
SE, an
Zapolanski, T. Systmic contact rmatitis. Drmatitis 19:915, 2008. 41. Thyssn,
JP, Johansn,
JD, an Mnn, T. Contact allrgy pi- mics an thir controls. Contact Drmat
itis 56:185195,
2007. 1814_Ch13_201-223.qx 7/10/09 2:56 PM Pag 222 CHAPTER 13 Hyprsnsitiv
ity 223 42.
Karl
rg, A, Brgstrom, MA, Borj, A, Luthman, K, t al. Allrgic contact rmat
itisformation,
structural rquir- mnts, an ractivity of skin snsitirs. Chm Rs Toxicol
21:5369, 2008.
43. Glaman, AC. Toxiconron rmatitis: Poison ivy, oak, an sumac. Wilrns
s an Environ M
17:120128, 2006. 44. Jaco
, SE, an Stl, T. Allrgic contact rmatitis: Early
rcognition
an iagnosis of important allrgns. Drmatol Nurs 18:433446, 2006. 45. Mrrill,
W.
Hyprsnsitivity pnumonitis: Just think a
out it. Chst 120:10551058, 2001. 46.
McCormick, T,
an Sharr, W. Dlay-typ hyprsnsitiv- ity skin tsting. In Dtrick, B, Ham
ilton, RG, an
Fols, JD (s): Manual of Molcular an Clinical La
oratory Immunology,
. 7. ASM
Prss, Washington, DC, 2006, pp. 234240. 1814_Ch13_201-223.qx 7/10/09 2:56
PM Pag 223 14
Autoimmunity LEARNING OBJECTIVES Aftr nishing this chaptr, th rar will
 a

l to: 1.
Dscri
 th factors that contri
ut to th vlopmnt of autoimmunity. 2. Dist
inguish
organ-spci c an systmic autoimmun isass, giving an xampl of ach. 3. Dsc
ri
 th
ffcts of systmic lupus rythmatosus (SLE) on th
oy. 4. Discuss th immuno
logic mchanisms
known for SLE. 5. List four typs of autoanti
ois foun in lupus, an scri

th pattrn sn
with ach in immuno uorscnc tsting. 6. Diffrntiat scrning tsts from anti

oy-spci c
tsts for lupus. 7. Discuss th symptoms of rhumatoi arthritis (RA). 8. Dscri


charactristics of th ky anti
ois foun in RA. 9. Discuss scrning tsts fo
r rhumatoi
factor (RF) an anti-CCP, xplaining th limitations of currnt tsting procur
s. 10.
Diffrntiat Hashimotos thyroiitis an Gravs isas on th
asis of la
oratory
nings an
immun mchanisms. 11. List th main anti
ois tst for in Gravs an Hashimot
os isass,

an scri
 tsting procurs. 12. Rlat gntic suscpti
ility to th vlo
pmnt of
insulin-pnnt ia
ts mllitus. 13. Explain immunologic mchanisms known to
caus
struction of clls in typ I ia
ts mllitus. 14. Discuss th immunologic ni
ngs in
multipl sclrosis (MS). 15. Dscri
 how th ning of oligoclonal
ans in cr

rospinal ui
can hlp in iagnosis of multipl sclrosis. 16. Explain how th symptoms that o
ccur in
myasthnia gravis (MG) ar rlat to th prsnc of auotanti
ois. 17. Discus
s how
Goopasturs synrom iffrs from glomrulonphritis u to nonspci c position
of immun
complxs. KEY TERMS Antinuclar anti
oy (ANA) Autoimmun isas Autoimmun th
yroi isas
(AITD) Cntral tolranc Fluorscnt antinuclar anti
oy (FANA) tsting Gravs 
isas
Hashimotos thyroiitis Molcular mimicry Multipl sclrosis (MS) Myasthnia gravi
s (MG)
Priphral tolranc Rhumatoi arthritis (RA) Rhumatoi factor (RF) Systmic l
upus
rythmatosus (SLE) Thyroi-stimulating hormon (TSH) Thyroi-stimulating hormon
 rcptor
anti
oy (TSHRa
) Thyrotoxicosis Typ I ia
ts mllitus 224 1814_Ch14_224-247.
qx 7/10/09
3:48 PM Pag 224 CHAPTER 14 Autoimmunity 225 Autoimmun isass ar conitions
in which amag
to organs or tissus rsults from th prsnc of autoanti
oy or autoractiv c
lls. Such
isass affct 5 to 7 prcnt of th population an ar thought to
 caus
y
th loss or

rakown of slf-tolranc. 1 In th arly 1900s, Ehrlich scri
 this phnom
non as horror
autotoxicus, litr- ally maning far of slf-poisoning. Thus, it was rcogni a
rly on
that unr normal circumstancs, th immun rspons was hl in chck so t
hat slf-antigns
wr not stroy. Slf-tolranc is
liv to

rought a
out
y svral m
chanisms,
incluing clonal ltion of rlvant ffctor clls an activ rgulation

y T clls. As
scri
 in Chaptr 2, uring th maturation procss of T clls, th grat ma
jority of
uniffrntiat lymphocyts that ar procss through th thymus o not sur
viv. It is
thought that this is whr potntially slf-ractiv T-cll clons ar stroy
. Th sam
procss happns with B clls as thy matur in th
on marrow. This struction
of potntially
slf-ractiv lymphocyts is rfrr to as cntral tolr- anc. Th procss is
not totally
ffctiv, howvr, as normal iniviuals o possss slf-ractiv lymphocyts.
Thus, in th
sconary lymphoi organs, priphral tol- ranc is maintain
y a licat
a
lanc
twn th
T hlpr cll typ 1 (Th1) an T hlpr cll typ 2 (Th2) pop- ulations. 1 In an
imal mols of
autoimmun isas, th Th1 clls hav
n implicat as primary miators of a
utoim- mun

isas,
caus thy rlas proinflammatory cytokins. 1 Th raction to f

orign antigns
is typically a Th2 rspons, an th accompanying cytokins o not caus th sam
 typ of
struction as Th1 clls. Rcnt nings inicat that rgulatory T clls (Trgs)
play a cntral
rol in maintaining this
alanc an liminating harmful autoimmun rsponss.
(S Chaptr 5
for tails a
out rgulatory T clls.) Major histocompati
ility complx (MHC)
proucts also
sm to in unc antign rcognition or nonrcognition
y trmining th typ of
pptis that
can
 prsnt to th T clls. Thr is a clos t
twn an antignic pp- ti
 an th
groov of an iniviual MHC molcul, as iscuss in Chaptr 3. Thrfor, inh
ritanc of a gn
coing for a spci c MHC molcul may mak an inivi- ual mor suscpti
l to a p
articular
autoimmun isas. In aition, th xprssion of class II molculs on clls w
hr thy ar not
normally foun may rsult in th prsntation of slf-antigns for which no tol
ranc has
n
sta
lish. In svral of th isass prsnt in this chaptr, thr ar xa
mpls of host
clls that xhi
it class II molculs on thir surfacs aftr an in ammatory rspo
ns. Th
strongst link foun to at
twn crtain HLA molculs an spci c isass is

twn
HLA-B27 an ankylosing sponylitis. Iniviuals who possss HLA-B27 hav a 90 ti
ms gratr
chanc of vloping th isas than th normal popula- tion. 1 Othr associati
ons
twn
inhritanc of crtain MHC gns an th tnncy to vlop particular autoimmun isass ar
iscuss in th chaptr. Svral othr mchanisms ar thought to contri
ut to
autoimmunity:
rlas of squstr antigns, molcular mimicry, an polyclonal B-cll activa
tion. Antigns
that ar protct from ncountring th circulation ar not xpos to potntia
lly ractiv
lymphocyts. Exampls of ths ar mylin
asic protin, normally squstr
y
th
loo
rain

arrir, an sprm. Trauma to th tissu
y mans of an acci- nt or infction
may introuc
such antigns to th gnral circulation. In th cas of sprm, vasctomy
can inuc
autoanti
oy formation. CHAPTER OUTLINE SYSTEMIC LUPUS ERYTHEMATOSUS Clinical Si
gns Immunologic
Finings La
oratory Diagnosis of Systmic Lupus Erythmatosus Tratmnt RHEUMATO
ID ARTHRITIS
Clinical Signs Immunologic Finings La
oratory Diagnosis of Rhumatoi Arthritis
Tratmnt
AUTOIMMUNE THYROID DISEASES Gntic Prisposition Clinical Signs an Immunologi
c Finings for
Hashimotos Thyroiitis Clinical Signs an Immunologic Finings for Gravs Disas
La
oratory
Tsting for Autoimmun Thyroi Disass Tratmnt for Autoimmun Thyroi Disas
s TYPE I DIABETES
MELLITUS Immunopathology La
oratory Tsting Tratmnt OTHER DISEASES Multipl Sc
lrosis

Myasthnia Gravis Goopasturs Synrom SUMMARY CASE STUDIES EXERCISE: RAPID SLID
E TEST FOR
ANTINUCLEOPROTEIN ANTIBODY FOUND IN SYSTEMIC LUPUS ERYTHEMATOSUS EXERCISE: SLIDE
AGGLUTINATION
TEST FOR THE DETECTION OF RHEUMATOID FACTOR REVIEW QUESTIONS REFERENCES 1814_Ch1
4_224-247.qx
7/10/09 3:48 PM Pag 225 226 SECTION 3 Immun Disorrs Molcular mimicry rf
rs to th fact
that many inivi- ual viral or
actrial agnts contain antigns that closly r
sm
l
slf-antigns. Exposur to such forign antigns may triggr anti
oy prouction
that in turn
racts with similar slf-antigns. Exampls ar poliovirus VP2 an actylc
holin (ACH)
rcptors, masls virus P3 an mylin
asic protin, an papilloma virus E2 an
insulin
rcptors. 1 Furthr xampls ar iscuss with iniviual isass. Th last
major factor
is polyclonal B-cll activation. B-cll fcts inclu th a
normal xprssi
on or function of
ky signaling molculs, ysrgulation of cytokins, an changs in B-cll vl
opmntal su
sts.
2 On fct in par- ticular, FC rcptor polymorphisms, th rcptor for a
nti
oy that
normally own-rgulats anti
oy prouc- tion, may caus continual B-cll stimul
ation. 2 Ths
fcts may
 nhanc
y organisms such as gram-ngativ
ac- tria an svra
l viruss,
incluing cytomgalovirus an Epstin-Barr virus (EBV), which ar polyclonal act
ivators. Thy
inuc prolifration of numrous clons of B clls that xprss IgM in th a
sn
c of T hlpr
(Th) clls. 1 In this procss, B clls that may
 ractiv to slf-antigns can

 activat.
Bsis th factors mntion prviously, svral oth- rs must
 takn int
o account in
th tiology of autoimmun isass. Som of ths ar fcts in th immun s
ystm, th
influnc of hormons, an nviron- mntal conitions. Dfcts in natural killr
clls, in th
scrtion of cytokins, in apoptosis (or killing of clls), an in complmnt co
mponnts all may
contri
ut to th loss of slf-tolranc. Hormons, spcially strogns, may al
so play a rol,

caus thy ar known to affct cytokin pro- uction an may influnc which T
clls, ithr
Th1 or Th2, ar mor activ in a particular rspons. 3 This xplains in
part why womn
ar mor pron to autoim- mun isass. Thus, thr is not on caus
u
t many
contri
uting factors that ar rsponsi
l for th patholog- ical conitions foun
 in autoimmun
isass. Autoimmun isass can
 classifi as systmic or organ-spci c. Thr
 is oftn a
goo
it of ovrlap
twn th two,
caus som isass that start out
as organspci c latr affct othr organs. Ta
l 141 lists som of th mor important autoi
mmun
isass. Many of th ky isass for which srological tsting is availa

l ar

iscuss in this chaptr. SYSTEMIC LUPUS ERYTHEMATOSUS Systmic lupus rythmat

osus (SLE)
rprsnts th pro- totyp of human autoimmun isass. It is a chronic
systmic
in ammatory isas mark
y altrnating xac- r
ations an rmissions, affc
ting
twn
15 an 50 iniviuals out of 100,000 in th Unit Stats. 4,5 Incinc of th
 isas has
tripl ovr th last four cas. 4 Th pak ag of onst is usually
t
wn 20 an 40
yars. Womn ar much mor likly than mn to
 strickn,
y a margin of appro
ximatly 10 to 1.
1,5 It is also mor common in African Amricans an Hispanics than in whits. 5
With arlir
iagnosis an improv tratmnts, th 5-yar survival rat has incras t
o gratr than 90
prcnt. 6 Th immun rspons is irct against a
roa rang of targt antig
ns, as th
typical patint has an avrag of thr circulating autoanti
ois. 5 It appars
that thr is an
intrplay
twn gntic suscpti
ility an nvironmntal factors. In whits, t
hr is a strong
association with human lukocyt antigns (HLA) DR an DQ. As iscuss in Ta
l
14-1. Spctrum
of Autoimmun Disass SPECIFICITY DISEASE TARGET TISSUE Organ-Spci c Hashimo
tos
thyroiitis Thyroi Gravs isas Thyroi Prnicious anmia clls Gastric parit
al Aisons
isas Arnal glans Typ I ia
ts mllitus Pancras MG Nrv-muscl synaps
s MS Mylin
shath of nrvs Autoimmun hmolytic anmia R
loo clls Iiopathic throm
oc
ytopnic purpura
Platlts Goopasturs synrom Kiny, lungs RA Joints, lungs, skin Sclrorma
Skin, gut,
lungs, kiny Systmic SLE Skin, joints, kiny,
rain, hart, lungs MG myasthni
a gravis; MS
multipl sclrosis; RA rhumatoi arthritis; SLE systmic lupus rythmatosus
1814_Ch14_224-247.qx 7/10/09 3:48 PM Pag 226 CHAPTER 14 Autoimmunity 227 Ch
aptr 3, th HLA
antigns play a rol in prsntation of forign or slf-antigns to T an B cll
s. Aitionally,
lupus has
n associat with inhrit  cincis of compl- mnt componnts C1
q, C2, an C4.
(S Chaptr 6 for a iscussion of complmnt factors.) A
normalitis of Fc rece
ptors on B
cells, macrophaes, dendritic cells, and neu- trophils that
ind IG and prevent
excess immune
reactions have also
een found. 7 Environmental factors include expo- sure to ul
traviolet liht,
certain medications, and infectious aents. 8 Hormones are also important,
ecau
se they may
influence the reulation of transcription of enes that are central to th
e SLE expression.
8 In fact, use of estroen- containin contraceptives or hormone replacement the
rapy has
een
found to dou
le the risk of developin lupus. 5 Clinical Sins The clinical sin
s are extremely
diverse, and nonspecific symptoms such as fatiue, weiht loss, malaise, fever,
and anorexia are
often the rst to appear. Joint involvement seems to
e the most frequently report

ed
manifestation,
ecause over 90 percent of patients with SLE are su
ject to polyarthralias or
arthritis. 9 Typically, the arthritis is symmetric and involves the small joints
of the hands,
wrists, and knees. After joint involvement, the next most common sins are skin
manifestations.
An erythematous rash may appear on any area of the
ody exposed to ultraviolet l
iht. Less common
ut perhaps more dramatic is the appearance of the classic
utter y rash acros
s the nose and
cheeks. This is what is responsi
le for the name lupus, derived from the Latin t
erm meanin
wol ike. This rash appears in only a
out 30 to 40 percent of all patients (Fi. 141).
In
discoid lupus, skin lesions have central atrophy and scarrin. One-half to two-t
hirds of all
patients exhi
it evidence of renal involvement. There are several types of lesio
ns,
ut the most
danerous is diffuse proliferative lomerulonephri- tis (DPGN), in which there i
s cellular
proliferation in at least 50 percent of the lomeruli. 5 Twenty percent of individuals with
DPGN will die or develop end-stae renal disease within 10 years of dianosis. 9
Other conditions
may include deposition of immune complexes in the su
endothe- lial tissue and th
ickenin of the

asement mem
rane. All of these can lead to renal failure, the most frequent cau
se of death in
patients with SLE. Other systemic effects may include cardiac involvement with p
ericarditis,
tachycardia, or ventricular enlarement; pleuritis with chest pain; neuropsychia
tric
manifestations such as seizures, mild conitive dysfunction, psychoses, or depre
ssion; or
hematoloic a
normalities such as anemia, leukopenia, throm
ocytopenia, or ly
mphopenia. 9,10
In order for a clinical dianosis of lupus to
e made, four of eleven specific c
riteria must
e
present: malar rash, discoid rash, photosensitivity, oral ulcers, arthritis, ser
osi- tis, renal
disorders, neuroloical disorders, hematoloic disorders, immunoloic disorders,
and presence of
antinu- clear anti
odies. 5 Within the cateory of immunoloic disorders ar
e anti
odies to
dou
le-stranded DNA, to Smith antien, and to phospholipids. Immunoloic Findin
s The rst clue
in the mystery of lupus was the discovery of the LE cell
y Malcolm Harraves in
1948. The LE
cell is a neutrophil that has enulfed the anti
ody-coated nucleus of another ne
utrophil. This
phenomenon, which mainly appears in vitro, occurs when cells are damaed and rel
ease nuclear
material. Nine years after the LE cell was dis- covered, the rst anti-DNA
anti
ody was
identi ed. Now it is known that SLE is associated with more than 25 autoan- ti
odi
es. Some of
the more common ones are listed in Ta
le 142. The lare num
er of possi
le
autoanti
odies
reflects a eneralized dysreulation of the immune system. 11,12 There is an unc

ontrolled
autoreactivity of T and B cells leadin to the production of autoanti
odies. Thi
s activity may
e
due to a deficiency of reulatory T cells, as discussed in Chapter 2. Reulatory
T cells (Tres)
play a crucial role in the maintenance of self-tolerance in peripheral lym- phoi
d orans, as they
suppress T-cell proliferation. 13 Tres in lupus seem to
e more sensitive to ap
optosis, or prorammed cell death. 13 In addition, altered FC receptors for IG on B cells keep
them from
ein
switched off, so they continue to produce anti
ody. 7 Dysfunctional processin i
n the routine
nonimmunoloic clearin of cellular de
ris may also
e a key to the pathoenesis
of SLE. 12
A
normal apoptosis of certain types of cells may occur, releasin excess amount
s of cellular
constituents such as DNA and ri
onucleic acid (RNA). Individuals who in
herit certain HLA
enes are more likely to respond to such self-antiens. FIGURE 141. Butter y rash i
n SLE.
Characteristic rash over the cheek
ones and forehead is dianostic of SLE. The d
isease often

eins in youn adulthood and may eventually involve many oran systems. (From S
teinman, L.
Autoimmune disease. Sci Am 269:107, 1993, with permission.) 1814_Ch14_224-247.qx
d 7/10/09 3:48
PM Pae 227 228 SECTION 3 Immune Disorders Constant presence of antienic mater
ial triers
poly- clonal activation of B cells, one of the main immunoloic characteristics
in lupus. There
is an accompanyin alter- ation in the function of
oth Th1 and Th2 helper cells
, resultin in
enhanced production of certain cytokines that contri
ute to up-reulation of
anti
ody
production
y B cells. 14 In particular, increased production of interleukin-10
(IL-10), which
normally serves an immunosuppressive role, correlates with increased anti
ody pr
oduction and with
dis- ease activity. IL-10 appears to trier an increase in anti
odies directed
aainst DNA and
stimulate production of platelet- activatin factor. 15,16 Once immune complexes
are formed, they
cannot
e cleared as well from the circulation
ecause of other possi-
le defi
ciencies. These
include defects in complement receptors on phaocytic cells; defects in recep
tors for the FC
portion of immunolo
ulins; or deficiencies of early complement components suc
h as C1q, C2,
or C4. 5,8 Accumulation of IG to dou
le-stranded DNA seems to
e the most path
oenic,
ecause
it forms complexes of an inter- mediate size that
ecome deposited in the
lomerular

asement mem
rane. In addition, anti-DNA anti
ody may also react directly with p
roteins such as
- ctin, found in the b sement membr ne; this enh nces ctiv tion of the complemen
t c sc de,
which contributes to the kidney d m- ge seen with this dise se. 17 Drug-induced
lupus differs
from the more chronic form of the dise se in th t symptoms usu lly dis ppe r onc

e the drug is
discontinued. The most common drugs implic ted re proc in mide, hydr l zine, ch
lorprom zine,
isoni zid, quinidine, nticonvuls nts such s methyldop , nd possi- bly or l co
ntr ceptives.
5,10 Typic lly, this is
milder form of the dise se, usu lly m nifested s feve
r, rthritis, or
r shes; r rely re the kidneys involved. 5 L bor tory Di gnosis of Systemic Lupu
s Erythem tosus
When SLE is suspected, the first test typic lly done is
screening t
est for
ntinucle r
ntibodies (ANA). Fluorescent ntinucle r ntibody (FANA) testing
is the most
widely used nd ccepted test, bec use it detects
wide r nge of ntibodies nd
is positive in
bout 95 percent of p tients with lupus. 5,17,18 This is n extremely sensitive
test nd
rel tively e sy to perform, but it h s low di gnostic speci- ficity, bec use m n
y of the
ntibodies re ssoci ted with other
utoimmune dise ses. 18 Mouse kidney or
hum n
epitheli l HEp-2 cells re xed to
slide nd llowed to re ct with p tient serum
. After c reful
w shing to remove ll unre- cted ntibody,
n ntihum n immunoglobulin wit
h
uorescent
t g or n enzyme l bel such s horser dish per- oxid se is dded. Approxim te
ly 2 percent
of he lthy individu ls nd up to 75 percent of elderly individu ls test positiv
e. 18,19
Conversely, up to 5 percent of SLE p tients test neg tive, so this test c nnot b
e used to
bsolutely rule out SLE. It is now common pr ctice to screen with 1:80 dilu- t
ion (or 1:160 if
the p tient is over 65) to void low positive titers in the norm l popul tion. 1
8 P tients with
SLE usu lly h ve high titers of high- f nity ntibody g inst speci c nti- gens. Pr
o le testing
for individu l ntibodies c n be done s follow-up. A di gr m of the possible u
orescent
p tterns is shown in Figure 142 (see lso Color Pl te 10). Some of the individu l
ntinucle r
ntibodies nd other c tegories of ntibodies re discussed below. T ble 14-2. C
ommon Antinucle r
Antibodies AUTOANTIBODY CHARACTERISTICS OF ANTIGEN IMMUNOFLUORESCENT PATTERN
DISEASE
ASSOCIATION Antids-DNA ds-DNA Homogeneous SLE Antiss-DNA Rel ted to purines nd py
rimidines Not
detected on routine screen SLE, m ny other dise ses Anti-histone Different cl ss
es of histones
Homogeneous Drug-induced SLE, other dise ses Anti-DNP DNA-histone complex Homoge
neous SLE,
drug-induced SLE Anti-Sm Extr ct ble nucle r ntigen Speckled Di gnostic for SL
E (RNA component)
Anti-RNP Proteins complexed with nucle r Speckled SLE, mixed connective tissue
RNA dise ses
Anti-SS-A (Ro) Proteins complexed to RNA Finely speckled SLE, Sjgrens syndrome, o
thers
Anti-SS-B (L ) Phosphoprotein complexed to Finely speckled SLE, Sjgrens syndrome,
others RNA
polymer se Anti-nucleol r RNA polymer se, nucleol r protein Homogeneous st ining

of nucleolus
SLE, systemic sclerosis AntiScl-70 DNA topoisomer se I Atypic l speckled Systemic
sclerosis,
Scleroderm AntiJo-1 Histidyl-tRNA synthet se Fine cytopl smic speckling Polymyos
itis Ad pted
from Br dwell, AR, Hughes, RG, nd K rim, AR. Immuno uorescent ntinucle r ntibod
y tests. In
Detrick, B, H milton, RG, nd Folds, JD (eds): M nu l of Molecul r nd Clinic l
L bor tory
Immunology, ed. 7. ASM Press, W shington, DC, 2006, pp. 99697. DNA deoxyribonucle
ic cid; SLE
systemic lupus erythem tosus; DNP
deoxyribonucleoprotein; RNA
ribonucleic cid;
RNP
ribonucleoprotein 1814_Ch14_224-247.qxd 7/10/09 3:48 PM P ge 228 CHAPTER 14 A
utoimmunity 229
Antinucle r Antibodies FANA Testing Double-str nded DNA (ds-DNA) ntibodies re
the most specific
for SLE, bec use they re m inly seen only in p tients with lupus, nd
levels correl te
with dise se ctiv- ity. 10,18 Although they re found in only 40 to 70 percent
of p tients, the
presence of these ntibodies is considered di gnostic for SLE, especi lly when t
hey re found in
com- bin tion with low levels of complement component C3. 20,21 Antibodies to ds
-DNA typic lly
produce peripher l or homogeneous st ining p ttern on indirect immuno uores- c
ence (IIF). 18
In testing for ntibodies to ds-DNA, puri ed ntigen prep r tion th t is free fr
om
single-str nded DNA (ss-DNA) must be used, s ntibodies to ss-DNA occur in m ny
indi- vidu ls
with other utoimmune or infl mm tory dise ses. One p rticul rly sensitive ss y
for ds-DNA is n
immuno- uorescent test using Crithidi lucili e, hemo gell te, s the substr te.
This
tryp nosome h s circul r ds-DNA in the kinetopl st. A positive test is indic ted
by brightly
st ined kinetopl st with p tient serum nd n ntibody conjug te. This test h s
high degree of
speci city, lthough it is less sensitive th n other FANA tests. 18,20 Other ss y
s th t c n be
used to detect ds-DNA include immunodiffusion, r dioimmuno ss y (RIA), nd
enzyme
immuno ss y (EIA) tests. 20 EIA is now considered more sensitive th n either RIA
or Crithidi l
testing, so it is the most widely used technique. 18,20 A second m jor n
tibody found in
lupus p tients is nti- histone ntibody. Histone is nucleoprotein th t is
m
jor constituent
of chrom tin. It c n be detected in lmost ll p tients with drug-induced lupus.
About 70 percent
of other p tients with SLE h ve elev ted levels of ntihistone nti- bodies, but
the titers re
usu lly f irly low. 5 Presence of ntihistone ntibody lone or combined with n
tibody to ss-DNA
supports the di gnosis of drug-induced lupus. 10,18 Antihistone ntibodies re
lso found in
rheum toid rthri- tis (RA) nd prim ry bili ry cirrhosis, but the levels re us
u lly lower. High
levels of ntihistone ntibodies tend to be ssoci ted with the more
ctive

nd severe
forms of SLE. 10 Antihistone
ntibodies re typic lly detected by immuno uor
escent ss ys,
immunoblotting, nd EIA. On IIF, either
homogeneous p ttern is seen, represent
ing u- orescence
of the entire nucleus, or st ining of the periphery occurs. 18 Antibodies re
lso stimul ted
by DNA complexed to histone, known s deoxyribonucleoprotein (DNP). Immun
o uorescent
p tterns re simil r to those discussed previously. L tex p rticles co ted with
DNP re used in
simple slide gglutin tion test for SLE. Antibody to
prep r tion of extr ct bl
e nucle r ntigen
w s rst described in p tient n med Smith, hence the n me nti-Sm ntibody. Extr
ct ble nucle r
ntigens represent f mily of sm ll nucle r proteins th t re ssoci ted with u
ridine-rich RNA.
The nti-Sm ntibody is specific for lupus, bec use it is not found in other ut
oimmune dise ses.
However, it is found in only 7 to 25 percent of p tients with this dise se. 10,2
1 This ntibody
produces co rsely speckled p ttern of nucle r uorescence on IIF. Titers do not
corre- l te
with dise se ctivity. 21 It c n lso be me sured by immunodiffusion, imm
unoblotting,
immunoprecipit tion, nd EIA. 18 SS-A/Ro nd SS-B/L ntigens lso belong to the
f m- ily of
extr ct ble nucle r ntigens. SS-A/Ro ntigen is RNA complexed to one of two p
roteins with
molecul r weights of 60 kD or 52 kD. Antibody to it ppe rs in pprox
im tely 10 to 50
percent of p tients with SLE. 10 This ntibody is most often found in p tients w
ho h ve cut neous
m nifest tions of SLE, especi lly photosensitivity derm ti- tis. 10 Anti-Ro/SSA
h s been found to
bind to cell surf ce ntigens on neutrophils nd c n c use mild neutropeni , o
ften seen in
p tients with SLE. 22 SS-B/L ntigen is phos- phoprotein th t ppe rs to be b
ound to products
of RNA III polymer se. 10 Anti-SS-B/L is found in only 10 to 20 percent
of p tients
with SLE, nd ll of these h ve nti-SS-A/Ro. 21 Both SS-A/Ro nd SS-B/L
ntibodies re
highly prev lent in p tients with other utoimmune dise ses, not bly scleroderm
nd Sjgrens
syndrome. 5 To detect the presence of these ntibodies on IIF, hum n tissue cult
ure cells such
s HEp-2 (hum n epitheli l) must be used, bec use SS-A/Ro nd SS-B/L nti
gens re not
found in mouse or r t liver nd kidney. A nely speckled p ttern is Homogeneous p
ttern Speckled
p ttern Nucleol r p ttern FIGURE 142. P tterns of immuno uorescent st ining for nt
inu- cle r
ntibodies. Ex mples of predomin nt st ining p tterns obt ined re homogeneousst
ining of the
entire nucleus; speckled p ttern st ining throughout the nucleus; nd nucleol r p
tternst ining
of the nucleolus. (Courtesy of INOVA Di gnostics, S n Diego, CA, with permission
. Color Pl te
10.) 1814_Ch14_224-247.qxd 7/10/09 3:48 PM P ge 229 230 SECTION 3 Immune Diso

rders evident.
These c n lso be detected by immunoprecipit - tion, immunoblotting, nd EIA. 18
An ntibody
th t produces
co rsely speckled IIF p ttern is nti-nRNP ntibody. Ribonu
cleoprotein (RNP)
is protein complexed to p rticul r type of nucle r RNA c lled U1-nRNP (U for ur
idine-rich).
Although it is detected in 20 to 40 percent of p tients with SLE, it i
s lso found
t
high titer in individu ls with mixed connective tissue dise se, syst
emic sclerosis,
Sjgrens syn- drome, nd other utoimmune dise ses. 10,18 Anti-nRNP c n be me sured
by
immunoblotting, immunoprecipit - tion, EIA, nd IIF. 21,22 St ining of the nucle
olus in IIF m y
indic te ntibody to brill rin, dense component of the nucleolus. This is indic ted by
clumpy nucleol r fluorescence. 18 This p ttern is common in systemic sclerosis.
18,21 Homogeneous
st ining of the nucleolus is ssoci ted with myositis nd systemic sclerosis. 18
Other Ass ys for
ANA ANAs c n lso be detected by immunodiffusion. Typic lly, this method is used
to determine the
immunologic speci- ficity of
positive FANA test. Such testing is v lu ble in i
dentifying
specific dise se st tes nd in ruling out those norm l p tients who exhibit
positive FANA
test. Ouchterlony double diffusion detects ntibody to sever l of the sm ll nuc
le r
ribonucleoproteins, or extr ct ble nucle r ntigens (ENA). These include ntibod
ies to the
following ntigens: Sm, nRNP, SS-A/Ro, SS-B/L , Scl-70 (DNA topoisomer se
I), nd Jo-1
( ntigens found in der- m topolymyositis). 21 T ble 142 shows the source of th
ese ntigens
nd ssoci tions with speci c dise ses. A positive re ction is indic ted by immuno
precipit tion
lines of serolog- ic l identity (Fig. 143). (Refer to Ch pter 8 for
description
of the
principles of Ouchterlony immunodiffusion.) Although this type of testing is
not
s
sensitive
s some other techniques, it is highly speci c. 21 Immunoblotting tec
hniques
sep r te out ntigens by me ns of poly cryl mide gel electrophoresis. The p tter
n is then
electrotr nsferred to nitrocellulose sheet, which is incub ted with dilutions
of p tient serum.
Either r diol - beled or enzyme l beled ntibody conjug tes re then used to vis
u lize individu l
b nds. This technique is reli ble, sen- sitive, nd specific, nd it should be c
onsidered the
gold st nd rd for ch r cteriz tion of speci c ntibodies. 10,21 EIA h s lso co
me into wider
use for detection of ntids-DNA, ntihistone ntibodies, nd ntiSS-A nd ntiS
S-B. It
is p rticul rly good for detection of the l tter two ntibodies. 22,23 EIA
testing is
qu ntit tive, is less subjective, nd lends itself well to utom tion. 22,23 It
is more sensitive
th n immunodiffusion, but the specificity is lower. 21 In ddition, it h s

proven to be
more sensitive th n double immunodiffusion for the identi c tion of extr ct ble nu
cle r ntigens.
24 Despite dv nces in EIA technology however, IIF is still considered the bench
m rk in identi c tion of most uto ntibodies ssoci ted with lupus. 18,22 Antiphospholipid Anti
bodies
Antiphospholipid ntibodies re heterogeneous group of ntibodies th t bind to
phospholipid
lone or re complexed with protein. They c n ffect every org n in the body, bu
t they re
especi lly ssoci ted with deep-vein nd rteri l thrombosis nd with morbidity
in pregn ncy.
25,26 They h ve been found in up to 60 percent of p tients with lupus, but they
re ssoci ted
with sever l other dise se st tes. 27 They c n be identi ed by c using f lse-posit
ive results for
syphilis. The lupus ntico gul nt, one of the sever l types of ntiphosp
holipid
ntibodies, w s so n med bec use it pro- duces prolonged ctiv ted p rti l throm
bopl stin time
( PT) nd prothrombin time (PTT). Ironic lly, p tients with this ntibody h ve
n incre sed risk
of clotting nd spont neous bortion. Pl telet function m y lso be ffected. In
ddition to
determining the PT nd PPT, there re sever l EIAs for ntiphospholipid ntibod
ies th t re
sensitive nd rel tively simple to perform. 2527 If these ntibodies re suspecte
d, f ctor
ss ys m y lso need to be performed to rule out ny f ctor de ciencies or f ctorspeci c
inhibitors. Tre tment If fever or rthritis is the prim ry symptom, high dose
of spirin or
other nti-in mm tory drug m y bring relief. For skin m nifest tions, ntim l ri
ls such s
hydroxychloroquine or chloroquine nd topic l steroids re often prescribed. 5 S
ystemic
corticosteroids re used for cute fulmin nt lupus, lupus nephritis, or centr l
nervous system
complic tions, bec use these suppress the immune response nd lower nti- body t
iters. 5 Other
drugs used include cyclophosph mide, Anti-Sm ntibody Sm ENA RNP Anti-RNP ntibo
dy A A B B FIGURE
143. Extr ct ble nucle r ntibody (ENA) immunodiffusion p ttern. A mixture of ext
r ct ble
nucle r ntigens, including RNP, Sm, nd other soluble nucle r ntigens, is pl c
ed in centr l
well in n g rose gel. Sm ntibody nd RNP ntibody re run s positive control
s, nd p tient
s mples re pl ced between the controls. The p ttern of precipitin lines formed
indic tes the
ntibodies present in p tient serum. The rc of serologic l identity formed betw
een Sm nd
p tient A indic tes th t serum A cont ins nti-Sm ntibodies. The rc of p rti l
identity formed
between serum A nd RNP occurs bec use RNP is lw ys found complexed to Sm ntig
en. RNP ntibodies re not present. Serum B cont ins neither Sm nor RNP ntibody. 1814_Ch14_224
-247.qxd 7/10/09
3:48 PM P ge 230 CHAPTER 14 Autoimmunity 231 z thioprine, methotrex te, nd c

hlor mbucil, but

ll of these m y h ve serious side effects such s bone m rrow suppres- sion, n
d they must be
monitored closely. A new ppro ch for p tients whose symptoms c nnot be controll
ed by other me ns
involves utologous stem cell h rvesting nd reinfu- sion fter the p tient unde
rgoes intensive
chemother py. 28 Results indic te th t long-term remission m y be possible using
this method.
Over ll, the present 5-ye r surviv l r te ppro ches 90 percent. 5 RHEUMATOID AR
THRITIS
Rheum toid rthritis (RA) is nother ex mple of
systemic utoimmune diso
rder. It ffects
pproxim tely 1 percent of the popul tion in the Western world. 2931 Wome
n
re three
times s likely to be ffected
s men. Typic lly it strikes individu ls b
etween the ges
of 35 nd 50, lthough it m y occur t ny ge. 29 RA c n be ch r c- terized s
chronic,
symmetric, nd erosive rthritis of the peripher l joints th t c n lso ffect m
ultiple org ns
such s the he rt nd the lungs. 10,30 In ddition to
decline in func- tion l
bility, there is
reduced life expect ncy. 32,33 Progress of the dise se v ries, bec use there m
y be spont neous
remissions or n incre singly ctive dise se in some individ- u ls th t r pidly
progresses to
joint deformity nd dis bility. 34 As in SLE, there ppe rs to be n ssoci tion
of RA with
cert in MHC cl ss II genes. These genes code for the bet ch in, one of the two
ch ins th t m ke
up the HLA cl ss II ntigens (see Ch pter 3). The strongest ssoci tion ppe rs
with cert in
DR4 lleles. These occur in 70 percent of p tients with RA. 29 All of the
lleles involved
ppe r to code for simil r mino cid sequence t positions 67 to 74 in the ch
ain. 29 This
shar pitop on th HLA class II chain may act to facilitat antign prsntatio
n to Th
clls an to B clls. Clinical Signs Diagnosis of RA is
as on th 1987 critr
ia sta
lish
y
th Amrican Collg of Rhumatology. 35 Ky symptoms ar morning stiffnss arou
n th joints
lasting at last 1 hour; swlling of th soft tissu aroun thr or mor
 joints;
swlling of th proximal intrphalangal, mtacarpopha- langal, or wrist joints
; symmtric
arthritis; su
cutanous nouls; a positiv tst for rhumatoi factor (RF); an
rai- ographic
vinc of rosions in th joints of th hans, th wrists, or
oth. At last f
our of ths must

 prsnt for 6 wks or mor for th iagnosis to
 ma. Typically th pati
nt xprincs
nonspci c symptoms such as malais, fatigu, fvr, wight loss, an transint jo
int pain that

gins in th small joints of th hans an ft. Morning stiffnss an joint pa
in usually
improv uring th ay. Joint involvmnt progrsss to th largr joints in a s
ymmtric fashion,
oftn affcting th kns, hips, l
ows, shoulrs, an crvical spin. Th join

t pain
xprinc las to muscl spasm, which,
caus of th consqunt limitation o
f motion, rsults
in prmannt joint formity. A
out 20 prcnt of patints hav nouls ovr th

ons. Nouls
can also
 foun in th myocarium, pricarium, hart valvs, plura, lungs, s
pln, an
larynx. Othr systmic symptoms may inclu anmia, formation of su
cutanous no
uls,
pricaritis, lymphanopathy, splnomgaly, intr- stitial lung isas, or
vasculitis.
Fltys synrom is a com
ination of svral of ths symptoms: chronic
RA coupl with
nutropnia, splnomgaly, an possi
ly throm-
ocytopnia. 29 For many yars, t
hr has
n a
sarch for an infctious agnt or agnts that may
 involv in th tiology of
RA. Numrous
agnts, incluing Mycoplasma, ru
lla, cytomgalovirus, EBV, an parvovirus, h
av
n propos
as possi
l triggring antigns. 29 In fact, an incras titr of anti
oy to E
B an som othr
viruss has
n foun in patints with RA,
ut a causal rlationship has not

n sta
lish.
30 Immunologic Finings Th arlist lsions in rhumatoi joints show an incra
s in clls
lining th synovium an an in ltration of mononu- clar clls, mostly CD4 T lymphoc
yts. 29 CD8
T clls ar scattr throughout, as ar B clls an anti
oy- proucing
plasma clls.
Macrophags an nutrophils ar attract to th ara, an this rsults in th f
ormation of an
organi mass of clls call a pannus, which grows into th joint spac an in
vas th
cartilag. 10,34 Th
alanc
twn proinflammatory an anti- inflammatory
cytokins
appars to
 tipp towar con- tinual inflammation. Proinflammatory cytokins
foun in synovial
flui that contri
ut to inflammation ar intr- lukin-1 (IL-1), intrlukin-6
(IL-6),
intrlukin-8 (IL-8), intrlukin-15 (IL-15), intrlukin-18 (IL-18), an tumor
ncrosis
factor-alpha (TNF-). 34,36 (See Ch pter 5 for com- plete discussion of cytokine
s). TNF- pl ys
key role in the infl mm tory process by inducing continu l secretion of IL-1,
IL-6, nd Il-8.
In ddition, TNF- f cilit tes the tr nsport of white blood cells to the
f
fected re s.
34 Coll gen se nd other tissue-degr ding enzymes re lso rele sed from synovio
cytes nd
chondrocytes th t line the joint c vity. The end result is destruction of connec
tive tissue,
c rtil ge, nd bone. It is not known wh t role uto ntibodies pl y in the ini- t
i tion of the
infl mm tory response. Approxim tely 75 percent of p tients with RA h ve n n
tibody th t h s
been c lled the rheum toid f ctor (RF). 29,30,34 It is most often of the IgM cl
ss nd is
directed g inst the FC por- tion of IgG. However, this ntibody is not speci c fo
r RA, s it
is found in 5 percent of he lthy individu ls nd in 10 to 20 percent of

those over the

ge of 65. In RA, IgM ntibodies combine with IgG, nd these immune comp
lexes become
deposited in the joints, result- ing in
type III (or immune complex) h
ypersensitivity
re ction. The complement protein C1 binds to the immune complexes,
ctiv ting
the cl ssic l
complement c sc de. 1814_Ch14_224-247.qxd 7/10/09 3:48 PM P ge 231 232 SECTI
ON 3 Immune
Disorders During this process, C3 nd C5 re gener ted, which ct s chemot ct
ic f ctors for
neutrophils nd m croph ges. The continu l presence of m croph ges le ds to the
chronic in mm tion usu lly observed which d m ges the synovium itself. Other uto ntib
odies found
include ntiker tin ntibody, ntiperinucle r ntibody, nti l ggrin, nd nti-S
ntibody. All
of these
ntibodies re directed g inst citrullin ted proteins, which re
gener ted by
modi c tion of protein- bound rginine by the enzyme peptidyl rginine deimin se. 3
0,37 This
enzyme is ssoci ted with gr nulocytes, monocytes, nd m croph ges, s well s o
ther types of
cells. De th of gr n- ulocytes nd m croph ges triggers production of citrullin
ted proteins, nd
overexpression of these ntigens m y provoke n immune response. 37 This f mily
of ntibodies is
detected using cyclic citrullin ted peptides (CCP); hence the ntibod- ies re c
lled nti-CCP.
Anti-CCP is now the le d m rker for detection of RA, bec use it is much more spe
ci c th n RF. 38
In ddition, low titers of ntinucle r ntibodies re pres- ent in bout 40
percent of
p tients. The p ttern most identified is the speckled p ttern directed g ins
t ribonucleoprotein. The signi c nce of this group of uto ntibodies rem ins uncle r, bec
use they do not
ppe r to be directly rel ted to p thogenesis. L bor tory Di gnosis of Rheum toi
d Arthritis
Di gnosis of RA is m de on the b sis of
combin tion of clinic l m nifest tions
, r diogr phic
ndings, nd l bor - tory testing. RF is the ntibody th t is most often tested fo
r to id in
m king the initi l di gnosis. Two types of ggluti- n tion tests for RF h ve bee
n developed: one
using sheep red blood cells co ted with IgG nd the other using l tex p rticles
co ted with the
s me ntigen. Bec use the l tex test h s gre ter sensitivity, it is the predom
in nt gglutin tion test used. Agglutin tion tests, however, detect only the IgM isotype, found
in pproxim tely
75 percent of p tients. Thus, neg tive result does not rule out the presence o
f RA. Conversely,
positive test result is not speci c for RA, bec use RF c n be found in other dis
e ses such s
syphilis, SLE, chronic ctive hep titis, tuberculosis, leprosy, infec- tious mon
onucleosis,
m l ri , nd Sjrgrens syndrome. 29,30 Testing for two other ntibody cl sses of RF
, IgG or IgA,
tends to be more specific. These two isotypes re r rely found in other dise se

st tes, which m y
help in m king differenti l di gnosis. The elev tion of IgA e rly in the dise se ppe rs to
be ssoci ted with
poorer prognosis, such s development of bone erosions nd
systemic
m nifest - tions, so this c n be used to predict the dise se outcome. 30 Testing
for IgG nd IgA
isotypes nd IgM c n be performed using EIA techniques. Nephelometric ss ys c n
lso test for
the presence of ll three isotypes. These re b sed on incre ses in light sc tte
ring s immune
complexes ccumu- l te, nd the sensitivity is better th n m nu l gglutin tion
methods. Both
EIA nd nephelometric methods h ve gre ter precision nd sensitivity, nd be
c use they re
uto- m ted, they h ve l rgely repl ced m nu l methods for detecting RF.
30 However, since
RF testing is not specific indic tor of RA, rese rch led to the development of
the nti-CCP
ss y. Second-gener tion nti-CCP EIA ss ys h ve chieved sensitivity of 74 p
ercent nd
specificity of 96 percent, m king it more reli ble indic tor of RA th n the RF
test. 30,38
Approxim tely 35 to 40 percent of RF-neg tive p tients re positive for nti-CCP
, nd studies
h ve found th t presence of nti-CCP precedes the onset of RA by sever l ye rs.
31,37 A new r pid
nti-CCP2 test on the m r- ket ppe rs to be v lid nd reli ble
nd m y
help to
simplify testing. 39 Anti-CCP together with IgA RF testing ppe r to be the best
predictors for
future development of RA. 30,37 Hence, the combin tion of nti-CCP nd RF testing provide
useful inform tion in the e rly nd ccur te di gnosis of RA. Once the di gnosis
is m de,
however, the most helpful tests used to follow progress of the dise se re gener
l indi- c tors
of in mm tion, such s me surement of erythrocyte sediment tion r te, C-re ctive
protein (CRP),
nd comple- ment components. 34 Typic lly, CRP nd sediment tion r tes re elev
ted, nd the
level of complement components is norm l or incre sed, indic ting incre sed synt
hesis. CRP levels
correl te well with dise se ctivity, bec use levels re ect the incre se in proin m
m tory
cytokines. 34 Although not speci c, following CRP levels re ects n incre se in the
in mm tory
response nd is more sensitive me sure of dise se ctivity th n is ch nge in
RF levels.
Tre tment Tr dition l ther py for RA h s included nti-in mm tory drugs such s
s licyl tes
nd ibuprofen to control loc l swelling nd p in. The discovery th t j
oint destruction
occurs e rly on during the dise se h s prompted more ggressive tre tme
nt.
Dise se-modifying ntirheum tic drugs (DMARDS)such s methotrex te, hydroxychlor
o- quine,
sulf s l zine, le unomide, nd penicill mine 32,40,41 re now prescribed if dise se
ctivity
persists fter 4 to 6 weeks of tre tment with nonsteroid l nti-in mm tory drugs.

32,40
Corticosteroids such s prednisone c nnot be used long- term, but they re given
in short or l
courses for dise se fl re-ups. These gents ppe r to h lt the infl mm tory resp
onse nd slow the
progression of joint erosion. A recent development involves tre tment with biolo
gic l gents th t
block the ctivity of TNF-. Agents th t ct g inst TNF- re cl ssi ed into two c te
gories: (1)
mono- clon l ntibody to TNF- (in ixim b nd d limum b) nd (2) TNF- receptors fuse
d to n IgG
molecule (et nercept). All three speci c lly t rget nd neutr lize TNF-, nd they h
ve shown
very promising results in h lting joint d m ge without producing m jor side effe
cts. 40,4244 Any
of the three in combin tion with methotrex te re very effective in preve
nting
r diologic l d m ge. 34 A new gent, rituxim b, specific lly t rgets the CD20 n
tigen on B cells.
Reducing the B-cell popul tion prevents further ntibody form tion, nd this h s
h d modifying
effect on the dise se. 45 However, more rese rch is needed to determine possible
long-term side
effects. 1814_Ch14_224-247.qxd 7/10/09 3:48 PM P ge 232 CHAPTER 14 Autoimmuni
ty 233 AUTOIMMUNE
THYROID DISEASES Autoimmune thyroid dise ses (AITDs) encomp ss sever l differ
ent clinic l
conditions, the most not ble of which re H shimotos thyroiditis nd Gr ves dise s
e. They
represent ex mples of org nic-speci c utoimmune dise ses. Although these conditio
ns h ve
distinctly different symp- toms, they do sh re some ntibodies in common, nd bo
th interfere with
thyroid function. The thyroid gl nd is loc ted in the nterior region of the nec
k nd is norm lly
between 12 nd 20 gr ms in size. It consists of units c lled follicles th t re
spheric l in
sh pe nd lined with cuboid l epitheli l cells. Follicles re filled with m teri
l c lled
colloid. The prim ry constituent of colloid is thyroglobulin, l rge iod- in te
d glycoprotein,
which is the precursor of the thyroid hormones triiodothyronine (T3) nd thyroxi
ne (T4). Under
norm l conditions, thyrotropin-rele sing hor- mone (TRH) is secreted by the hypo
th l mus to
initi te the process th t eventu lly c uses rele se of hormones from the thyroid
. TRH cts on the
pituit ry to induce rele se of thyroid-stimul ting hormone (TSH). TSH, in turn,
binds to
receptors on the cell membr ne of the thyroid gl nd, c using thyroglobulin to be
broken down into
secret ble T3 nd T4. Production of uto ntibodies interferes with this process
nd c uses underor over ctivity of the thyroid. It is believed th t the utoimmune response to t
he thyroid seen
in these dise ses is b sed on combin tion of genetic susceptibility genes coup
led with
environment l triggers. 46 In the c se of H shimotos dise se, one environm
ent l trigger
is high iodine int ke. Genetic Predisposition A genetic predisposition h s

been postul ted

for both H shimotos thyroiditis nd Gr ves dise se, bec use the con- dition is m
ore common in
f milies in which nother member h s utoimmune thyroid dise se. Genes ssoci te
d with
predisposition to thyroid utoimmunity c n be divided into two groups: immune-mo
dul ting genes
nd thyroid-speci c genes. The rst group includes the following: HLA ntigens, cyto
toxic T
lymphocyte ssoci ted f ctor-4 (CTLA-4), nd protein tyrosine phosph t se-22 (PT
PN22). The second
group includes the thyroglobulin gene nd the thyroid- stimul ting hormon
e receptor (TSHR)
genes. The first group of genes enh nces the likelihood of n utoimmune respons
e in sever l
different w ys. The HLA genes code for the HLA ntigens th t re responsible for
presenting
peptides to T cells to initi te n immune response (see Ch pter 3). The ssoci t
ion of HLA DR3
with Gr ves dise se is quite strong. It h s been found th t in the DR1 rgion, an
arginin
su
stitution at position num
r 74 of th
ta chain allows for slf-antigns to

 rcogni.
46,47 Association of Hashimotos thyroiitis with inhritanc of HLA antigns DR3,
DR4, DR5, an
DQw7 (DQ
1*0301) has
n rport,
ut this is not consistnt among iffr- nt
thnic
populations. 46,48 A uniqu fatur of
oth Gravs an Hashimotos isass is that
HLA-DR
antigns ar xprss on th surfac of thyroi pithlial clls, prhaps incr
asing th
autoimmun rspons. Th CTLA-4 gn cos for a rcptor foun on T clls, whic
h is
rsponsi
l for own-rgulation of th T-cll- miat immun rspons. Po
lymorphisms in
this gn ruc supprssion of T clls an incras th liklihoo of autoanti

oy prouction.
47,49,50 PTPN22 is also foun on th surfac of T clls an is involv in inhi

iting T-cll
activa- tion. It is thought that variants in this rcptor ar rsponsi
l for l
owr T-cll
signaling, allowing slf-ractiv T clls to surviv in th thymus an
 rlas
 to priphral
lymph nos. 49 This gn is implicat in
oth Hashimotos an Gravs isas. Las
tly,
mutations in th thyroglo
ulin gn may allow for intraction with HLA-DR antig
ns, rsulting in
antithy- roglo
ulin anti
ois, which can
 foun in Gravs an Hashimotos isas
s. 49
Aitionally, in Gravs isas, moifications in th TSHR gn may contri
ut to
th immun
systm rcogniing th rcptor, with anti
oy prouction rsulting. Clinical Si
gns an
Immunologic Finings for Hashimotos Thyroiitis Hashimotos thyroiitis, also known
as chronic
autoimmun thyroiitis, is most oftn sn in mil-ag womn, although
it may occur
anywhr from chilhoo up to a
out 70 yars of ag, with th mian ag of iag
nosis
ing 60.
10,51,52 Womn ar 5 to 10 tims mor likly to vlop th isas. 53 Patint

s vlop a
com
ination of goitr (or nlarg thyroi), hypothyroiism, an thyroi autoa
n- ti
ois. Th
goitr is irrgular an ru

ry, an immun struction of th thyroi glan
occurs.
Symptoms of hypothyroiism inclu ry skin, cras swating, puffy fac wit
h matous
ylis, pallor with a yllow ting, wight gain, an ry an
rittl hair. 51 T
h thyroi shows
hyprplasia with an incras num-
r of lymphocyts. Cllular typs prsnt in
clu activat T
an B clls (with T clls prominating), macrophags, an plasma clls. Th imm
un rspons
rsults in th vl- opmnt of grminal cntrs that almost compltly rplac
th normal
glanular architctur of th thyroi an progrssivly stroy th thyroi
glan. 51
Anti
ois to thyroglo
ulin prominat, progrssivly stroying thy- roglo
uli
n an proucing
th symptoms associat with hypothyroiism. Clinical Signs an Immunologic Fin
ings for Gravs
Disas Gravs isas, in contrast to Hashimotos thyroiitis, is charactri
y
hyprthyroiism. It is, in fact, th most common caus of hyprthyroiism, affc
ting
approximatly 0.5 prcnt of th population. 54 It is also th most prvalnt au
toimmun isorr
in th Unit Stats toay. 54 Womn 1814_Ch14_224-247.qx 7/10/09 3:48 PM Pa
g 233 234
SECTION 3 Immun Disorrs xhi
it gratr suscpti
ility
y a margin of a
out 1
0 to 1, an thy
most oftn prsnt with th isas
twn th ags of 20 an 50. 51,52 Th is
as is
manifst as thyrotoxicosis, with a if- fusly nlarg goitr that is soft in
sta of ru

ry.
Clinical symptoms inclu nrvousnss, insomnia, prssion, wight loss, hat i
ntolranc,
swating, rapi hart
at, palpitations,
rathlssnss, fatigu, cariac ysrhy
thmias, an
rstlss- nss. 51 Anothr sign prsnt in approximatly 35 prcnt of patints
is xophthalmus,
in which hyprtrophy of th y muscls an incras connctiv tissu in th o
r
it caus th
y
all to
ulg out so that th patint has a larg-y staring xprssion (Fi
g. 144). 55,56
Thr is vinc that or
ital
ro
lasts xprss TSH rcptor-lik protins that
ar affct
y
thyroi-stimulating immunoglo
ulin just as th thyroi is. 55,57 Thus, Gravs is
as is now
sn as a mul- tiorgan auto-immun isorr, causing hyprthyroiism, ophthalmop
athy, an
locali ma in th lowr lgs. Th thyroi shows uniform hyprplasia with a
patchy lymphocytic in ltration. Th follicls hav littl colloi
ut ar ll with hyprplastic
pithlium.
A larg num
r of ths clls xprss HLA-DR antigns on thir surfac in rspon
s to intrfron
(IFN)- prduced by in trating T ces. 54 This aws presentatin f sef-antigen
s such as the
thyrtrpin receptr t activated T ces. B ces, in turn, are stimuated t p
rduce antibdy.

The majr antibdies fund in Graves disease incude thyrid-stimuating hrmne

receptr
antibdy (TSHRab) and antibdies t thyrid perxidase. When antigenantibdy cmb
inatin ccurs,
it mimics the nr- ma actin f TSH and resuts in receptr stimuatin, with
the reease f
thyrid hrmnes t prduce the symptms f hyperthyridism. These have been
shwn t
stimuate prductin f thyrid hrmnes by increasing cycic adensine mnphs
phate (AMP)
eves after bind- ing t receptrs. 51,52,58 Thyrid perxidase antibdies are
actuay fund in
bth cnditins. An additina autantibdy fund in numerus patients is a thyr
trpin
receptrbc ing antibdy that may cexist with thyrid-stimuating antibdy. 52,
57,58 Depending
n the reative activity f bc ing and stimuating autantibdies, patient sym
ptms may vary
frm hyperthyridism t euthyridism t hypthyridism. 57 This may cnfund pat
ient diagnsis.
Labratry Testing fr Autimmune Thyrid Diseases The three majr autantibdie
s present are
antibdies t thy- rgbuin, thyrid perxidase, and TSH receptrs. Antibdies
t thyrgbuin
can be detected in 90 percent f patients with Hashimts thyriditis. 52 Heathy
individuas
may have a w titer f antithyrgbuin antibdy, but in patients with
Hashimts
thyriditis, the titer is cnsideraby higher, s differentiatin can be made in
this manner.
Antibdies t thyrgbuin can be demnstrated by indi- rect immunfurescent
assays, passive
aggutinatin, and EIA. 52,58 Passive hemaggutinatin using tanned red bd ce
s is the mst
cmmny used test. Indirect immun u- rescent testing is ess sensitive than pas
sive
hemaggutinatin, but it can detect nnaggutinating antibdy. 58 Indirect
immun urescent
assays use methan- xed mn ey cry- stat tissue sectins as substrate. 52 Thyrg
buin
antibdies wi create a ccuar r puffy staining pattern. Since antithyrgbu
in antibdies
are nt fund in a patients, a negative test resut des nt necessariy rue
ut Hashimts
disease. Other antibdies that may be present incude cid antibdy (CA2) and
TSH-binding
inhibitry immungbuin. Antibdy activity cntributes t the destructin
f thyrid
ces. Antibdy t CA2 prduces a diffuse r grund-gass appearance. Antibdies
t thyrid
perxidase (TPO) are directed against a 105 D membrane enzyme that catayzes ty
rsine
idinatin. They are fund in apprximatey 90 t 95 percent f patients with th
e disease. 51
They seem t crreate mst csey with disease activity. Antibdies t p
erxidase are
mst cmmny measured by EIA, athugh IIF and par- tice aggutinatin assay
s are as
used. 48,52 Perxidase antibdies prduce staining f the cytpasm and nt the
nuceus f

thyrid ces. These antibdies can be fund in apprximatey 90 percent f pati

ents with
Hashimts dis- ease and 80 percent f patients with Graves disease, s the tw ca
nnt be
distinguished n the basis f this test. 53,58 The third majr autantibdy, ant
i-TSHR antibdy,
is typicay assciated with Graves disease. Hwever, because testing is difficu
t and
expensive, eevatin f the thyrid hrmnes and free triidthyrnine (T3) and
thyrxine (T4) is
chec ed fr rst if this disease is suspected. In additin, TSH eves can be dete
rmined and are
w because f anti- bdy stimuatin f the thyrid. Increased upta e f
radiactive
idine as heps t cn rm the diagnsis. 59 Measurement f thyrid receptr anti
bdies is dne
ny when resuts f these assays are uncear. Tests fr TSHR antibdies are f
tw types:
biassays and binding assays. Biassays require tissue cuture and thus are dif cu
t t perfrm. 48,54,58 Binding assays are based n cmpetitin between radiabeed TSH
and patient
autantibdies fr binding t thyrtrpin receptrs. Apprximatey 80 perce
nt f FIGURE
144. Exphthamus indicative f Graves disease. (Curtesy f CDC/Dr. Seers/Emry
University.)
1814_Ch14_224-247.qxd 7/10/09 3:48 PM Page 234 CHAPTER 14 Autimmunity 235 pa
tients with
Graves hyperthyridism test psitive by this methd. 53,54 This may be due in par
t t the ac
f sensitiv- ity f these assays. 48 A different type f anti-TSHR antibdies ca
n be fund in
Hashimts thyriditis. These antibdies are i ey directed against a different
epitpe than
the abve antibdies, as they serve t bc the receptrs s that the T
SH hrmne
cannt bind. 48 Treatment fr Autimmune Thyrid Diseases Treatment fr Hashim
ts disease
cnsists f thyrid hrmne repacement therapy. TSH eves shud be m
nitred
thrughut treatment. 51 Severa different pr- tcs are used in the treatment
f Graves
disease. In the United States, the first ine f treatment invves radiac- tiv
e idine, which
emits beta partices that are cay destructive within an area f a few
miimeters. This
is given fr 1 t 2 years, and it resuts in a 30 t 50 percent ng-term remis
sin rate. 57
Sme patients, hwever, becme hypthyrida within 5 years, s cntinued mnitring is
essentia. In Eurpe, the patient is first paced n antithyrid medicatins suc
h as methimaze,
carbima- ze, r prpythiuraci with beta bc ers as adjuvant therapy
. 51,57 Surgery
t remve part f the thyrid may as be cnsidered as an aternative. TYPE I D
IABETES MELLITUS
Autimmune diabetes meitus, nw termed type IA dia- betes, is a chrnic au
timmune disease
that ccurs in a geneticay susceptibe individua as a resut f envirnme
n- ta factrs.

60 Apprximatey 10 percent f patients with diabetes meitus have the imm

une-mediated frm
f the disease. 58 It is characterized by insuf cient insuin prduc- tin caused
by seective
destructin f the beta ces f the pancreas. Beta ces are cated in the pan
creas in custers
caed the isets f Langerhans. Uni e ther autimmune endcrine diseases, hr
mne repacement
cannt be given ray, and chrnic vascuar cmpicatins resut in majr mrbi
dity and
mrtaity. Famiy studies indicate that there is an inherited genetic susceptibi
ity t the
disease, prbaby attributabe t mu- tipe genes. Apprximatey 90 percent f
white diabetics
carry the HLA-DR3 r DR4 gene. 61 There appears t be an increased ris when bt
h f these genes
are present. Hwever, recent research indicates that the true suscepti- biity g
enes fr type I
diabetes meitus may ccur in the HLA-DQ regin, especiay in the cding
f the DQ
chain. As iscuss in Chaptr 3, HLA antigns ar ma up of two sparat prot
in chains, nd
. Both of ths chains ar involv in antign rcognition. Within th cha
in,
su
stitutions for th amino aci 57, aspartic aci, ar associat with incrasi
ng risk for
ia
ts. This amino aci position is ajacnt to th antign-
ining groov an
may affct th
structur an function of th
ining sit. 62 This corrlation is strongst wit
h haplotyps
DQA1*0301/ DQB1*0302 an DQA1*0501/DQB1*0201. 61,63 A
out 90 prcnt of th Cauc
asian population
with typ I ia
ts mllitus hav DQB1*0302 an/or DQB1*0201 compar with lss
than 40 prcnt
in th normal population. 63 Environmntal in uncs inclu th possi
ility of vi
ral infctions
an arly xposur to cows milk. 61 Stuis hav attmpt to link virus
s, spcially
ru
lla virus, cytomgalovirus, an Coxsacki B4 virus with ia
ts,
ut most
rsarch is
inconclusiv. Anti
oy prouction coul possi
ly
 initiat as a rsult of mol
cular mimicry,
with a virus as th stimulating antign triggring anti
oy prouction ag
ainst a
slf-antign. Immunopathology Progrssiv in ammation of th islts of Langrhans
in th pancras
las to
rosis an struction of most
ta clls. Th su
clinical prio may la
st for yars,
an only whn 80 prcnt or mor of th
ta clls ar stroy os hypr- gly
cmia
com
vint. Immunohistochmical staining of in am islts shows a prponranc of C
D8 lymphocyts,
along with plasma clls an macrophags. 61 B clls thmslvs may act as antig
n-prsnting
clls, stimulating activation of CD4 lymphocyts. 64 A shift to a Th1 rspons cau
ss prouction of crtain cytokins, incluing TNF-, IFN-, and IL-1. The generaized in amm
atin that
resuts is respnsibe fr the destructin f beta ces. Athugh iset autant
ibdies trigger

the immune respnse, it is nt nwn what re they pay in ce destructin. Th
ere is increasing
evidence that autimmunity t insuin itsef may be centra t disease path- ge
nesis. 64 Ce
death, hwever, is i ey caused by apptsis and attac by cyttxic ymphcyte
s. It is apparent
that autantibdy prductin precedes the devepment f type IA diabetes meit
us by up t
severa years. Autantibdies are present in prediabetic individuas (i.e., ths
e wh are being
mnitred because they have a high ris f deveping diabetes) and in newy dia
gnsed patients.
58 Antibdy prductin diminishes with time, hw- ever. Amng the antibdies fu
nd are antibdies
t tw tyrsine phsphatase-i e transmembrane prteins caed insuinma antig
en 2 (IA-2 r
ICA 512) and IA-2A (phogrin); anti-insulin anti
ois; anti
ois to th nym
 GAD; an
anti
ois to various othr islt cll protins, call islt cll anti
ois (I
CAs). 52,58,65
La
oratory Tsting Although typ I ia
ts mllitus is usually iagnos
y th
prim
charactristic of hyprglycmia, it may
 usful to hav srological tsts to s
crn for
ia
ts
for
ta- cll struction occurs to th xtnt ncssary to
caus symptoms.
Anti
ois to islt clls hav traitionally
n tct
y IIF using fron s
ctions of human
pancras. 52 ICAs hav
n rport in th sra of gratr than 80 prcnt of p
atints nwly
iagnos with typ I ia
ts mllitus. 52,61 Howvr, such assays ar rathr c
um
rsom to
prform, an 1814_Ch14_224-247.qx 7/10/09 3:48 PM Pag 235 236 SECTION 3 Imm
un Disorrs
currnt formats ar availa
l using raioimmuno
ining assays. 58 Anti
oi
s to insulin can

 tct using RIA or EIA mthos. RIA an EIA ar also us to tct anti-G
AD anti
ois an
antiIA-2 anti
ois. 58 Com
in scrning for IA-2A, ICA, an GAD anti
ois app
ars to hav
th most snsitivity an
st positiv prictiv valu for typ IA ia

ts mllitus in
high-risk populations. 52,58 Tratmnt Th us of injct insulin has
n th
mainstay of thrapy for ia
ts. Howvr, nw trials cntr aroun th us of immunosupprssiv
agnts.
Cyclosporin A, aathioprin, an prnison hav all
n us to inhi
it th im
mun rspons.
All hav potntially toxic si ffcts. Limit trials in humans using a small
ppti
slf-antign call a hat shock protin for immuniation inicat that
ta-cl
l struction can

 halt in this mannr. 66 Rsarch using anti-CD20 (rituxima
) to caus B-cl
l pltion has

n conuct an has th potntial to cras anti
oy pro- uction. 67 Growt
h factors or
transplantation of
ta islt clls may also
 of us in th futur. OTHER DISE
ASES Othr
organ-spcific isass that appar to hav an autoimmun tiology inclu
multipl

sclrosis (MS), myas- thnia gravis (MG), an Goopasturs synrom. MS an
MG ar
isorrs of th nrvous systm, whil Goopasturs synrom is rsponsi

l for
glomru- lonphritis. Each of ths is iscuss
ri y. Ta
l 143 lists aitional
isass for
which thr appars to
 an autoimmun caus. Multipl Sclrosis Multipl sclr
osis (MS) is an
in ammatory autoimmun isorr of th cntral nrvous systm, affcting approximatly 350,000
Amricans an 1.1 million iniviuals worlwi. 68,69 It is charactri

y th formation
of lsions call plaqus in th whit mattr of th
rain an spinal cor, rsu
lting in th
progrssiv struction of th mylin shath of axons. Plaqus vary in si from
1 or 2 mm up to
svral cntimtrs. 68 As is th cas for most othr autoim- mun isass, a c
om
ination of
gntic an nvironmntal factors is rsponsi
l for vlopmnt of this coniti
on. Although
thr ar pro
a
ly svral inpnnt loci, MS is most closly associat with
inhritanc of a
particular HLA molcul coing for th
ta chain of DR, namly DRB1*1501. 70 It
has
n
thori that th in ammatory rspons is triggr
y molcular mimicry, in whic
h viral or
othr forign pptis ar prsnt in th groov of sp- ci c HLA class II molc
uls to T
clls. Onc initiat, th immun rspons
coms irct against slf-antign
s that ar
inistinguisha
l from th original forign anti- gn. 69 Som stuis inicat
that patints
with MS hav four tims th
loo concntration of anti
oy to EBV as wa
s foun in a
control population. 71 Othr viruss possi
ly implicat ar masls, hrps sim
plx, variclla,
ru
lla, in una C, human hrps virus-6, an som parain una viruss. 68 Thr a
ppars to

an aitional gntic link to polymorphisms in th gn coing for th intrluk
in-7 rcptor. 72
IL-7 may
 involv in th gnration of autor- activ T clls in this isas
. Damag to th
tissu of th cntral nrvous systm can caus visual istur
ancs, waknss or
iminish
xtrity in on or mor lim
s, locomotor incoorination, iinss, Ta
l 14-3.
Othr Autoimmun
Disass DISEASE ORGAN OR TISSUE IMMUNOLOGIC MANIFESTATIONS Aisons isas
Arnal glans
Anti
oy to arnal clls Autoimmun hmolytic anmia R
loo clls Anti
oy t
o r
loo clls
Autoimmun throm
ocytopnic purpura Platlts Antiplatlt anti
oy Crohns isas
 Intstins
In ammatory in ltrat of T an B clls: mchanism unknown Dissminat ncphalomyl
itis Cntral
nrvous systm Snsiti T clls Prnicious anmia Stomach Parital cll anti
o
y, intrinsic
factor anti
oy Poststrptococcal glomrulonphritis Kiny Strptococcal anti
o
is that
cross-ract with kiny tissu Rhumatic fvr Hart Strptococcal anti
ois th
at cross-ract

with hart tissu Sclrorma Connctiv tissu Antinuclar anti
ois: anti-Scl

-70,
anticntromr anti
oy Sjgrns synrom Eys, mouth Antinuclar anti
ois, RA fa
ctor
antisalivary uct anti
ois, antilacrimal glan anti
ois Sarcoiosis Multisys
tm granulomas,
Activation of T lymphocyts pulmonary manifstations 1814_Ch14_224-247.qx 7/10
/09 3:48 PM
Pag 236 CHAPTER 14 Autoimmunity 237 facial palsy, an numrous snsory a
normal
itis such as
tingling or pins an nls that run own th spin or xtrmitis an as
hs of
light sn on y movmnt. 69 Th isas is most oftn sn in young an mil
-ag aults

twn th ags of 20 an 40, an it is twic as com- mon in womn as in mn. 6
8 Typically, 50
to 70 prcnt of patints altrnat
twn rmissions an rlapss for m
any yars,
whil th rmainr follow a chronic progrssiv cours. 52 Within th plaq
us, T clls an
macrophags promi- nat, an thy ar
liv to orchstrat mylination. 6
8 Anti
oy
ins
to th mylin mm
ran an may initiat th immun rspons, stimulating macroph
ags an spciali phagocyts call microglial clls. 69 Th casca of immunologic vnt
s rsults in
acut inflammation, injury to axons an glia, structural rpair with rcovry of
som function,
an thn postin ammatory nurognration. 69 Activat T clls must pntrat in
to th cntral
nrvous systm to
gin th rspons. Thy may o so
y inucing changs in th
nothlium that
allows thm to pntrat th
loo
rain
arrir, whil vssl walls rmain intact
. 68 Cytokins
IL-1, TNF-, nd IFN- have been thught t be centra t the pathgenesis bserved,
thus
impicating Th1 ces. 68 Hwever, a Th2 respnse, characterized by pr- ductin
f intereu in-4
(IL-4), intereu in-5 (IL-5), and IL-10 may as cntribute t pathgenesis. 68
The tw mst
cmmn tests fr diagnsis f MS are ig- cna banding and the CSF IgG index
. Immungbuin
is increased in the spina uid in 75 t 90 percent f patients with MS, prducing
severa
distinct bands n prtein eec- trphresis that are nt seen in the serum. Thes
e are caed
igcna bands, and there may be as many as eight, but pres- ence f at east
fur bands is
indicative f MS. 73 Iseectric fcusing with immunbtting is as used t id
entify these
bands, and it is thught t be mre sensitive than prtein eectrphresis. 73 A
measure f the
IgG index, which cm- pares the amunt f IgG in spina uid t that f serum, may
as be usefu
in ma ing a diagnsis even thugh it is nt speci c fr MS. Whie there is nt ne
specific
antibdy that is diag- nstic fr MS, a arge percentage f patients pr
duce antibdy
directed against a myein basic prtein peptide. 74 Other antibdies are directe
d against

cmpnents f ig- dendrcytes and against myein membranes, but the maj
rity f
antibdies have yet t be identified. 69 Magnetic resnance imaging that shws d
amage at tw r
mre indi- vidua sites in the centra nervus system is as used t aid in dia
gnsis. Many
heathy individuas have antibdies that bind with w af nity, but antibdy with
high-af nity
binding is fund ny in disease states. There is n ne speci c therapy, but newe
r treatment fr
reapsing-remitting MS centers arund three drugs: IFN-1a (Avonx, R
if), IFN-1

(Btasron),
an glatiramr actat. 68,69 Ths ar
liv to work
y causing own-rgulat
ion of MHC
molculs on antign- prsnting clls an own-rgulating prouction of pr
oin ammatory
cytokins. 68 Acut xacr
ations ar trat with mthylprnisolon,
ut this
cannot
 us on
a long- trm
asis. Othr immunosupprssiv rugs that appar to lssn th s
vrity of
symptoms inclu aathioprin, mthotrxat, an cyclophosphami. 75 Myasthn
ia Gravis
Myasthnia gravis (MG) is an autoimmun isas that affcts th nuromuscular j
unction. It is
charactri
y waknss an fatiga
ility of skltal muscls. It occurs in 20
popl pr
100,000 annually, an th incinc appars to
 incrasing, spcially in ini
viuals ovr th
ag of 60. 7678 Anti
oy-miat amag to th actylcholin rcptors in sklt
al muscl las
to this progrssiv mus- cl waknss. Early signs ar rooping of th ylis a
n th ina
ility
to rtract th cornrs of th mouth, oftn rsult- ing in a snarling apparanc.
76,77,79 Othr
symptoms may inclu if culty in spaking, chwing, an swallowing, an ina
ility
to maintain
support of th trunk, th nck, or th ha. 52 If rspiratory muscl waknss o
ccurs, it can

lif- thratning. 77 Onst of symptoms can
 acut, or thy may vlop an wo
rsn ovr tim.
MG is oftn associat with th prsnc of othr autoimmun isass, s
uch as SLE, RA,
prnicious anmia, an thyroiitis. Early onst isas, which is usually  n a
s occurring

for th ag of 40, appars to
 link to svral HLA antigns, A1, B8, an
DR3. 77,79 In
this ag group, th isas is sn mor frquntly in womn an paks in th th
ir ca of
lif. Lat-onst MG, prsnting in patints olr than 40, is link to HLA anti
gns B7 an DR2
an is mor likly to appar in mn
twn th ags of 30 an 60. 7678 Thymic hy
prplasia is
sn in 75 prcnt of patints, an this can la to thymoma, a tumor of th thy
mus.
Approximatly 80 to 85 prcnt of patints hav anti-
oy to ACH rcptors, an
this appars to

 th main contri
utor to th pathognsis of th isas. 52,76,78,79 Normally
, ACH is rlas
from nrv nings to gn- rat an action potntial that causs th muscl fi

r to contract. It

is
liv that whn th anti
oy com
ins with th rcptor sit,
ining of A
CH is
lock,
an th rcptors ar stroy
caus of th action of anti-
oy an complm
nt 79 (Fig.
145). In th 15 prcnt of patints lacking this anti
oy, othr anti
ois ar p
rs- nt,
incluing antimuscl-spcific kinas (MuSK). 77 Currnt rsarch inicats th
at T rgulatory
clls may
 impair, allowing an immun rspons to occur that is charactri

y Th1 clls
proucing inflammatory cytokins IL-2, IFN-, and TNF-. 79 RIA procedures re use
d to detect
ntibody, b sed on ss ys th t block the binding of receptors by nti-ACH recept
or (ACHR)
ntibody. A r dio-l beled sn ke venom c lled -bung rotoxin is used to irreve
rsibly bind
to ACHRs; precipit tion of receptors c used by combin tion with ntibody is then
me sured. 52
1814_Ch14_224-247.qxd 7/10/09 3:48 PM P ge 237 238 SECTION 3 Immune Disorders
Anticholinester se gents re used s the m in tre t- ment ther py. 76 Thymectom
y is benefici l
to some p tients, especi lly those younger th n 60 with e rly onset dise se, but
there is some
question bout its over ll benefits. 80 Corticosteroids such s z thioprin
e nd
cyclosporine re effective, s is et nercept, which inhibits the ction
of TNF-. 79
Goodp stures Syndrome Goodp stures syndrome is ch r cterized by the presence of u
to ntibody to
glomerul r, ren l tubul r, nd lveol r b sement membr nes, resulting prim rily
in injury to the
glomerulus th t c n r pidly progress to ren l f ilure. This r re disorder occurs
with n nnu l
incidence of pproxi- m tely 0.5 per million. 81 Between 50 nd 70 percent of p
tients lso
exhibit pulmon ry hemorrh ge resulting from inter ction of ntibody with b semen
t membr nes found
in lung tissue. In contr st with other utoimmune dise ses, Goodp stures syndrome
is more likely
to occur in young m les between the ges of 18 nd 35. 8183 Often it follows
v
ir l
infection. The mort lity r te m y be s high
s 50 percent. 83 Severe ne
crosis of the
glomerulus is triggered by n nti- body th t h s speci city for the noncoll genou
s region of the
lph 3 ch in of type IV coll gen. 81,82,84 This uto nti- body re cts with coll
gen in the
glomerul r or lveol r b sement membr nes. Immune deposits ccumul te, nd compl
ement x tion
c uses injury bec use of the rele se of oxygen species nd proteolytic enzymes.
These immune
re ct nts progressively destroy the ren l tubul r, glomeru- l r, nd pulmon ry
lveol r b sement
membr nes. Signs of ren l involvement include gross or microscopic hem turi , pr
oteinuri ,
decre sed 24-hour cre tinine cle r nce, nd n incre se in blood ure
nd s
erum cre tinine
levels. Pulmon ry symptoms include hemorrh ge, dyspne , we k- ness, f tigue, nd
cough. This
syndrome differs from glomerulonephritis s result of the nonspeci c ccumul tio

n of
circul ting immune com- plexes found in other utoimmune dise ses, bec use speci c
ntib sement
ntibodies c n be demonstr ted by form tion of smooth, line r ribbonlike p tte
rn on direct
immuno- uorescent ss y of glomerul r b sement membr ne from p tients with the
dise se. 81
Gre ter th n 90 percent of p tients with Goodp stures nephritis test positive
for pres- ence
of this ntibody. Although little is known bout the circumst nces th t trigge
r the
utoimmune response, 80 percent of those ffected c rry the HLA-DR15 or DR4 ll
eles, indic ting
genetic predisposition. This is the c se in most other utoimmune dise ses. Ci
rcul ting
ntibodies c n be detected by RIA, EIA, nd IIF ss y. The IIF ss y, long held
s the st nd rd,
uses frozen kidney sections th t re incub ted with p tient serum nd then overl
id with
uorescein-l beled nti-IgG. It is often h rd to interpret nd h s
high
percent ge of
f lse-positive nd f lse-neg tive results. 84 A Western blot technique h s been
developed, which
sep r tes out b sement membr ne ntigens by poly cryl mide gel electrophoresis f
ollowed by
tr nsfer to nitrocellulose p per for immunoblot- ting. This is often used s c
onfirm tory test,
bec use it ppe rs to be the most sensitive nd the most specific immun
o ss y. 84 An EIA
technique, which uses the lph 3 subunit to detect ntibody, h s lso been deve
loped, but it is
less sensitive th n the Western blot. 84 About 20 percent of p tients lso h ve
low titers of
ANAs, which usu lly exhibit the perinucle r st ining p ttern. Current tre tment
str tegies focus
on remov l of circu- l ting ntibodies by pl sm pheresis or di lysis, or
use of
immunosuppressive gents such s glucocorticoids, cyclophosph mide, nd z t
hioprine. 81 The
sooner ther py is initi ted, the more likely it is th t ren l f ilure c n be pre
vented. SUMMARY
Autoimmune dise ses result from loss of self-toler nce,
delic te b l nce set
up in the body
to restrict the ctivity of T nd B lymphocytes. Self-toler nce is m int ined in
two m jor w ys:
(1) by deleting potenti lly re ctive B nd T cells s they m ture in the bone m
rrow nd thymus,
respectively, nd (2) by peripher l toler nce, which occurs in the second- ry l
ymphoid org ns.
Peripher l toler nce depends on the ctions of T regul tory cells, which help to
curt il
response to self- ntigens. However, sometimes
combin tion of genetic nd
environment l
f ctors work together to llow re ctions to self- ntigens to occur. Self- ntigen
s become
recognized in conjunction with cert in MHC ntigens, nd it is here th t genetic
f ctors come
into pl y. The sh pe of p rticul r cl ss II MHC molecules llows recognition to
ACH vesicles
Syn pse Postsyn ptic membr ne ACH receptors Axon A Anti-ACH receptor ntibody AC

H vesicles
Postsyn ptic membr ne ACH receptors Axon B FIGURE 145. Mech nism of immunologic i
njury in MG.
(A) Norm l nerve impulse tr nsmission. ACH is rele sed from the xon nd t ken u
p by receptors on
the postsyn ptic membr ne. (B) In MG, ntibodies to the ACH receptors re formed
, blocking
tr nsmission of nerve impulses. 1814_Ch14_224-247.qxd 7/10/09 3:48 PM P ge 23
8 CHAPTER 14
Autoimmunity 239 t ke pl ce. Molecul r mimicry in which foreign vir l or b c- te
ri l gents
resemble self- ntigens m y trigger ntibodies th t in turn re ct with self- ntig
ens. Such gents
m y lso ct s polyclon l ctiv tors, turning on sever l clones of B cells. E c
h clone of B
cells c n m nuf cture different type of ntibody. Thus, environment l f ctors
lso pl y role.
Autoimmune dise ses c n be cl ssi ed s org n-speci c or systemic, depending on whet
her tissue
destruction is loc lized or ffects multiple org ns. SLE nd RA re ex mples of
sys- temic
dise ses, while H shimotos thyroiditis, Gr ves dise se, type I di betes mellitus,
MS, MG, nd
Goodp stures syn- drome re considered org n-speci c dise ses. SLE is chronic in m
m tory
dise se ch r cterized by joint involvement, n erythem tous r sh th t ppe rs on
exposure to
sunlight, nd deposition of immune complexes in the kidney to c use glomerulonep
hritis.
Auto ntibodies include ntids-DNA, nti-DNP, ntihistone, nti-Sm, nti- nRNP, n
ticytopl smic,
ntinucleol r, nd ntiphospholipid ntibodies. E ch of these exhibits speci c st
ining p tterns
in the uorescent ntibody screening test for this dise se. RA ffects the synovi
l lining of
joints; necrotic re s re surrounded by gr nul tion tissue th t eventu lly le d
s to joint
disintegr tion. The m in immunologic nding is the presence of n ntibody c lled
RF. This is
typic lly 19S ntibody directed g inst IgG. Sever l other uto ntibodies cont
ribute to the
infl mm tion observed in the joints, including n uto ntibody c lled nti-C
CP, directed
g inst cyclic citrullin ted proteins. Di gnosis of RA is m de on the b sis of c
linic l
m nifest tions, positive RF test result, nd
positive nti-CCP test. H shimo
tos thyroiditis
is n org n-specific condition th t ffects the thyroid gl nd nd c uses it to b
e enl rged nd
rubbery. Lymphocyte infiltr tion in the thyroid with the presence of ntithyrogl
obulin ntibody
le ds to gr du l thyroid destruction. Clinic l symptoms re consistent with hypo
thyroidism.
L bor tory ss ys focus on detection of two m jor ntibodies, ntithyroglobulin
nd
ntiperoxid se, which re me sured by RIA, IIF, nd gglutin tion. The hyperthyr
oidism th t is
evident in Gr ves dise se produces symptoms such s nervousness, insomni , weight
loss, he t
intoler nce, swe ting, r pid he rtbe t, c rdi c dys- rhythmi s, nd bulging e

yeb lls. Thyroid

stimul ting hormone receptor ntibodies (TSHR b) bind to receptors, c using con
tinuous rele se
of thyroid hormones. Elev ted levels of thyroid hormones re the key l bor tory n
dings, nd
testing for uto ntibodies is done only when results of hormone ss ys re uncle
r. While there
re number of c uses of di betes, type IA di betes mellitus h s n utoimmune
origin. Genetic
sus- ceptibility ppe rs to lie within the DQ region of the MHC group. Vir l i
nfections m y
provide the trigger for the utoimmune response. The progressive destruction
of bet cells in
the p ncre s results in l ck of insulin production nd symptoms of hyperglycem
i . Antibodies to
two tr ns- membr ne proteins, ICA 512 nd IA-2A; to th nym GAD; an to ins
ulin can

tct
y mans of RIA or EIA. Ths can
 important prictors of isas
 in high-risk
populations. MS is charactri
y visual istur
ancs, waknss in th xtrm
itis, locomotor
incoorination, an snsory a
normalitis. T clls initiat a chain of vnts
that inclu

oth anti
oy an cytokin prouction that progrssivly stroys th mylin sh
ath of axons an
amags oligon- rocyts, causing lsions call plaqus. Anti
ois irct
against mylin

asic protin an against mylin oligon- rocyt glycoprotin ar prouc,

ut ths ar
not iagnostic,
caus thy ar not prsnt in all cass of is- as. Protin
lctrophorsis
of cr
rospinal flui that shows two or mor
ans not prsnt in srum is hlp
ful in making a
iagnosis. Th IgG inx, a masur of th ratio of anti
oy in th spinal ui to
that in th
srum, may also
 usful. Anothr isas affcting th nrvous systm is MG. E
xtrm muscl
waknss rsults from th prsnc of anti-
oy to ACH rcptors. By com
ining
with th rcptor
sit, anti
oy
locks th
ining of ACH, an th rcp- tors ar stroy as a
rsult of
complmnt activation. RIA procurs can
 us to tct anti
oy,
as on

ining assays
with ACH rcptors. Goopasturs synrom is charactri
y svr ncro- sis o
f th
glomrulus triggr
y an anti
oy that racts with glycoprotin in th glomru
lar
asmnt
mm
ran. Th rsult is glomrulonphritis, in which immun posits accumulat
an complmnt
fixation causs injury to th tissu, vntually proucing rnal failur. This a
nti
oy can also
ract with
asmnt mm
ran in lung tissu, prouc- ing pulmonary hmorrhag.
Anti
asmnt
anti
oy is usually tct
y IIF, RIA, or EIA. Ths xampls rprsnt a sa
mpling of th
autoimmun isass for which som of th rsponsi
l immunologic mchanisms ha
v
n
iscovr. Thr is a common thra in all of th isass covr in this
chaptr: Thy ar
th rsult of a com
ination of gntic an nvironmn- tal factors. It appars t

hat th MHC

antigns rcogni
y T clls ar a ky factor in whthr a rspons
will
 mount
to a particular slf-antign. Small changs in amino aci squncs chang th s
hap of th
rcptor an allow slf-antign to
 rcogni. Environmntal factors, pro
- a

ly in th natur
of viral or
actrial infctions, triggr an inflammatory rspons that polyclon
ally activats T
an B clls. As mor is larn a
out th mchanisms that trig- gr autoimmun r
sponss, it may

 possi
l to spci cally turn off thos rsponss without affcting th rst of
th immun
systm. 1814_Ch14_224-247.qx 7/10/09 3:48 PM Pag 239 240 SECTION 3 Immun D
isorrs 1. A
25-yar-ol fmal consult hr physician
caus sh ha
n xprincing sym
ptoms of wight
loss, joint pain in th hans, an xtrm fatigu. Hr la
oratory rsults wr
as follows: RA
sli tst positiv at 1:10; ANA sli tst positiv at 1:40; r
loo cll cou
nt 3.5 10 12
pr L (normal is 4.1 to 5.1 10 12 pr L); whit
loo cll count 5.8
10 9 p
r L
(normal is 4.5 to 11
10 9 pr L). Qustions a. What is a possi
l xplanation f
or positiv
rsults on
oth th RA tst an th ANA tst?
. What is th most likly caus o
f th cras
r
loo cll count? c. What furthr tsting woul hlp th physician is- ting
uish
twn RA
an SLE? 2. A 40-yar-ol fmal wnt to hr octor
caus sh was fling tir
 all th tim.
Sh ha gain a
out 10 pouns in th last fw months an xhi
it som facial
puf nss. Hr
thyroi glan was nlarg an ru
-
ry. La
oratory rsults inicat a normal
r an whit

loo cll count,
ut hr T4 lvl was cras, an an assay for antithyroglo

ulin anti
oy was
positiv. Qustions a. What o ths rsults likly inicat?
. What ffct o
antithyroglo
ulin
anti
ois hav on thyroi function? c. How can this conition
 iffrnt
iat from
Gravs isas? CASE STUDIES 1814_Ch14_224-247.qx 7/10/09 3:48 PM Pag 240 CH
APTER 14
Autoimmunity 241 RAPID SLIDE TEST FOR ANTINUCLEOPROTEIN ANTIBODY FOUND IN SYSTEM
IC LUPUS
ERYTHEMATOSUS Principl Anti
ois to DNP, a histon associat with DNA, ar fo
un in ovr 90
prcnt of patints with SLE. Thy ar th most common antinuclar anti
ois n
countr in
lupus. Although thy ar not spcific for lupus, tsting for thir prsnc is a
hlpful
scrning procur. In this tst sys- tm, latx particls ar coat with DNP
xtract from
calf thymus. Whn com
in with patint srum containing anti
oy to DNP, visi
l
 agglutination
occurs. Spcimn Collction Collct
loo
y vnipunctur, using asptic tchniq
u an avoiing
hmolysis. Hmoly or contaminat srum may giv rronous rsults an shoul
not
 us.
Allow th
loo to clot for at last 10 minuts at room tmpratur. Srum shoul


 rmov an
rfrigrat if not tst immiatly. Spcimns can
 kpt at 2C to 8C for up t
o 72 hours.
If longr storag is rquir, th sampl may
 fron an tst at a latr ti
m. Avoi
rpat fring an thawing. Plasma shoul not
 us for this procur. Ra
gnts, Matrials,
an Equipmnt Ragnt tst kit such as Srayn SLE or Immunoscan SLE, which cont
ains th
following: SLE tst ragnt-latx particls coat with DNP SLE positiv control
SLE ngativ
control Glass agglutination sli Capillary piptts Applicator sticks Othr mat
rials rquir

ut not provi: Srological piptts or automatic piptts an tips Timr Phys
iological salin
(0.85 or 0.9 prcnt soium chlori) Disposa
l tst tu
s (12
75 mm) Tst tu

rack CAUTION:
Th ragnt tst kit contains soium ai. Ais ar rport to ract with l
a to form compouns that may tonat on prcussion. Whn isposing of solutions contai
ning soium
ai, flush with larg volums of watr to minimi th
uilup of mtal ai
compouns. Sra
us for controls wr ngativ whn tst for hpatitis B surfac antign an
HIV
y
FDA-rquir tst. Howvr, th controls shoul
 hanl with th sam prcaut
ions as thos
us in hanling human sra. Procur* Qualitativ Tst 1. Bring all ragnts a
n tst sampls
to room tmpratur
for us. 2. Gntly mix th SLE tst ragnt to isprs a
n suspn th
latx particls in th
uffr. Avoi vigorous shaking. 3. Thoroughly clan a gla
ss sli
for
us. Th sli shoul
 wash with trgnt, rins with ioni watr, an
ri with a
lint-fr tissu. 4. Using a capillary piptt provi, plac 1 rop (50 L) of p
atint spcimn
in on sction of th glass sli. Hol th capillary prpnicular to th sli
to livr a
full rop of spcimn. 5. Plac 1 rop of positiv control in th lft sction o
f th sli an 1
rop of ngativ control in th right sction
y invrting th appropriat ropp
r vial an
squing out 1 rop onto th tst sli. 6. Using th roppr, a 1 rop of th
 SLE ragnt to
ach of th ivisions containing spcimn an controls. 7. Mix ach sction w
ith a
isposa
l applicator stick, spraing ach mixtur ovr th ntir sction. U
s a clan
applicator stick for ach sction. 8. Tilt th sli slowly
ack an forth for 3
minuts. Ra
immiatly for agglutination unr irct light. 9. Positiv spcimns shoul

 ilut an
rtst. Quantitativ Tst 1. Bring all ragnts an tst sampls to room tmp
ratur
for
us. 2. Using physiological salin, ilut spcimns 1:2, 1:4, 1:8, 1:16, 1:32,
or as n. 3.
Gntly mix th SLE tst ragnt to isprs an suspn th latx particls in t
h
uffr. Avoi
vigorous shaking. 4. Using a capillary piptt provi, plac 1 rop (50 L) of 

ach ilution on
succssiv ls of th raction sli. Hol th capillary prpnicular to th sl
i to livr
a full rop of spcimn ach tim. 5. Plac 1 rop of positiv control in th l
ft sction of th
sli an 1 rop of ngativ control in th right sction
y invrting th appro
priat roppr
vial an squing out 1 rop onto th tst sli. 6. Mix ach sction with
a isposa
l
applicator stick, spraing ach mixtur ovr th ntir sction. Us a clan a
pplicator stick
for ach sction. * Aapt from th packag insrt from Srayn Sratst for SL
E, Rml, Lnxa,
KS 66215. EXERCISE 1814_Ch14_224-247.qx 7/10/09 3:48 PM Pag 241 242 SECTION
3 Immun
Disorrs 7. Tilt th sli slowly
ack an forth for 3 minuts. Ra immiatl
y for
agglutination unr irct light. Rsults an Intrprtation Th ngativ contro
l shoul giv a
smooth or slightly gran- ular suspnsion with no agglutination, whil th positi
v control shoul
form a visi
l agglutination raction is- tinctly iffrnt from th slight gra
nularity o
srv
with th ngativ control. This tst will
 positiv for ovr 90 prc
nt of patints
with untrat SLE. Howvr, it may also
 positiv with othr isass such as
RA, sclrorma,
chronic livr isas, an progrssiv systmic sclrosis. Antinuclar anti
oi
s can also

foun in a
out 10 pr- cnt of th normal halthy population,
ut typically th
titr is too low
in iniviuals unr th ag of 60 to prouc a positiv agglutination tst. A p
ositiv raction
in this tst inicats that th lvl of antinuclar anti
oy is in th rang co
mmonly foun in
SLE. This is a scrning tst, an clin- ical manifstations an la
oratory rsu
lts must
 takn
into account whn a iagnosis is ma. 1814_Ch14_224-247.qx 7/10/09 3:48 PM
Pag 242 CHAPTER
14 Autoimmunity 243 SLIDE AGGLUTINATION TEST FOR THE DETECTION OF RHEUMATOID FAC
TOR Principl RF
is an IgM anti
oy irct against IgG. It is foun in 70 to 80 prcnt of pati
nts with RA.
Passiv agglutination can
 mploy to tst for th prsnc of th anti
oy,
using a carrir
particl such as shp rythrocyts snsi- ti with IgG. Whn patint sr
um containing
RF is mix with th snsiti ragnt clls, visi
l agglutina- tion occurs.
Spcimn
Collction Collct
loo
y vnipunctur, using asptic tchniqu an avoiing
hmolysis. Allow
th
loo to clot for at last 10 minuts at room tmpratur. Loosn th
clot with a
woon applicator stick, an cntrifug at 1000 g for 10 minuts or unti
l th suprnatant
is fr of clls. Srum shoul
 rmov an tst immiatly. Spcimns may

 kpt
rfrigrat for up to 24 hours. For longr storag, fr spcimns at 20C. Avoi
 rpat
fring an thawing. Plasma shoul not
 us. Ragnts, Matrials, an Equipm

nt Ragnt tst

kit such as Rhumaton Kit, which contains th following: Sta
ili shp rythr
ocyts snsiti
with ra

it gamma glo
ulin Positiv control Ngativ control Cali
rat capillar
y tu
s with
ru

r
ul
s Glass sli Othr matrials n: Disposa
l stirrrs Distill w
atr or isotonic
salin Tst tu
s, 12
75 mm Cntrifug capa
l of 1000 g Srological piptts or
automatic
piptts an tips Timr CAUTION: Th ragnt tst kit contains soium ai. Ai
s ar rport
to ract with la to form com- pouns that may tonat on prcussion. Wh
n isposing of
solutions containing soium ai, flush with larg volums of watr to minimi
th
uilup of
mtal ai compouns. Sra us for controls wr ngativ whn tst for hpa
titis B surfac
antign an HIV
y FDA-rquir tst. Howvr, th controls shoul
 hanl wi
th th sam
prcautions as thos us in hanling human sra. PROCEDURE* 1. Bring all ragn
ts an tst
sampls to room tmpratur
for us. 2. Fill a capillary to th mark with pat
int srum, an
mpty it into th cntr of th mil sction of th sli. 3. Plac 1 rop of
positiv control
in th lft sction of th sli an 1 rop of ngativ control in th right sc
tion. 4. A 1
rop of wll-shakn ragnt to ach sction of th sli. 5. Mix with a isposa

l stirrr,
spraing ach mixtur ovr th ntir sction. Us a clan isposa
l stirrr f
or ach mixtur.
6. Rock th sli gntly with a rotary motion for 2 minuts an immiatly o
s
rv for
agglutination. If th tst is positiv for agglutination, ilut th spcimn 1:
10 with istill
watr or isotonic salin, an rpat th tst. Quantitativ Procur If a titr
is sir, th
following procur can
 us: 1. La
l ight 12 75 mm tst tu
s 1 through 8 a
n plac in a
tst tu
 rack. 2. A 1.8 mL of salin to tu
 1 an 1.0 mL of salin to tu
s
2 through 8. 3.
A 0.2 mL of spcimn to tu
 1, mix, an transfr 1.0 mL of this mixtur to tu

 2. Mix th
contnts of tu
 2 an transfr 1.0 mL of this mixtur to tu
 3. Cont
inu srially
iluting in this mannr through tu
 8. Starting with a 1:10 ilution in th rst
tu
, th ilution in th nal tu
 will
 1:1280. 4. Tst ach ilution as scri
 in th qu
alitativ
proc- ur. Th last ilution to show positiv agglutination is rport as th
titr. Rsults
an Intrprtation Positiv spcimns show raily visi
l agglutination, whil
ngativ
spcimns show no agglutination or a nly gran- ular pattrn. If agglutination is
positiv in
only th unilut spcimn, this rprsnts a vry low titr that might
 prs
- nt in a
varity of othr isass. Som of ths othr isass ar SLE; nocaritis; t
u
rculosis;
syphilis; viral infctions; an isass of th livr, lung, or kiny. In ait

ion, low titr

can
 foun in approximatly 1 prcnt of halthy ini- viuals. Thus, a positi
v tst is not
spci c for RA. Convrsly, a ngativ tst os not rul out RA,
caus approxim
atly 25
prcnt of patints with th isas tst ngativ for RF. Th rsults of this s
impl scrning
tst for RA must
 intrprt in th light of clinical symptoms. * Aapt fro
m th packag
insrt for th Rhumaton Tst with prmission from Invrnss Mical, Waltham, M
A. EXERCISE
1814_Ch14_224-247.qx 7/10/09 3:48 PM Pag 243 244 SECTION 3 Immun Disorrs
1. All of th
following may contri
ut to autoimmunity xcpt a. clonal ltion of slf-ract
iv T clls.
.
molcular mimicry. c. nw xprssion of class II MHC antigns. . polyclonal act
ivation of B
clls. 2. Which of th following woul
 consir an organ- spci c autoimmun
isas? a. SLE

. RA c. Hashimotos thyroiitis . Goopasturs synrom 3. SLE can
 istinguish
 from RA on
th
asis of which of th following? a. Joint pain
. Prsnc of antinuclar an
ti
ois c.
Immun complx formation with activation of com- plmnt . Prsnc of anti-s
DNA anti
ois 4.
Which of th following woul support a iagnosis of rug-inuc lupus? a. Antih
iston anti
ois

. Anti
ois to Smith antign c. Prsnc of RF . Anti
ois to SS-A an SS-B
antigns 5. A
homognous pattrn of staining of th nuclus on IIF may
 caus
y which of
th following
anti
ois? a. Anti-Sm anti
oy
. Anti-SSA/Ro anti
oy c. Antihiston anti
oy
.
Antiou
l-stran DNA 6. Which of th following is charactristic of RA? a. Ass
ociation with
crtain HLA-DR gns
. Joint involvmnt that is symmtric c. Prsnc of anti

oy against IgG
. All of th a
ov 7. Which of th following
st scri
s th sli agglutination tst for
RF? a. It is spci c for RA.
. A ngativ tst ruls out th possi
ility of RA. c
. It is a
snsitiv scrning tool. . It tcts IgG ma against IgM. 8. Hashimotos thyro
iitis can
st

 iffrntiat from Gravs isas on th
asis of which of th following? a.
Dcras in
thyroi hormon lvls
. Prsnc of thyroi proxias anti
ois c. Enlargm
nt of th thyroi
. Prsnc of lymphocyts in th thyroi 9. Which of th following woul
 con
sir a signi cant ning in Gravs isas? a. Incras TSH lvls
. Anti
oy to TSHR c. Dcr
as T3 an
T4 . Antithyroglo
ulin anti
oy 10. Immunologic finings in typ I ia
t
s mllitus
inclu all of th following xcpt a. prsnc of CD8 T clls in th islts of
Langrhans.
.
anti
oy to colloi. c. anti
oy to insulin. . anti
oy to GAD. 11. Dstruction
of th mylin
shath of axons caus
y th prsnc of anti
oy is charactristic of which i
sas? a. MS
.
MG c. Gravs isas . Goopasturs synrom 12. Bloo was rawn from a 25-yar-o

l woman with
sus- pct SLE. A FANA scrn was prform, an a spckl pattrn r
sult. Which
of th following actions shoul
 takn nxt? a. Rport out as iagnostic for
SLE
. Rport
out as rug-inuc lupus c. Prform an anti
oy pro l . Rpat th tst REVIEW
QUESTIONS
1814_Ch14_224-247.qx 7/10/09 3:48 PM Pag 244 CHAPTER 14 Autoimmunity 245 R
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1814_Ch14_224-247.qxd 7/10/09 3:48 PM Pae 247 15 Immunoproliferative Disease
s Donald C.
Lehman, EdD, and Maureane Hoffman, MD, PhD LEARNING OBJECTIVES After nishin this
chapter, the
reader will
e a
le to: 1. Contrast leukemias and lymphomas. 2. Compare the diff
erent hypotheses
explainin the transformation of normal cells into malinant cells. 3. Discuss t
he clonal
hypothesis of malinancy. 4. Descri
e how surface (CD) antiens re ect the differe
ntiation of
hematopoietic cells. 5. Discuss the evidence linkin Epstein-Barr virus to Hodk
ins lymphoma. 6.
Descri
e how uncontrolled proliferation of lymphoid cells and overpro- duction o
f anti
ody can
lead to clinical manifestations. 7. Differentiate multiple myeloma from Waldenst
rms
macrolo
ulinemia. 8. Discuss how la
oratory tests can
e used to dianose and f
ollow the
proression of immunoproliferative disorders. 9. Descri
e how la
oratory tests a
re used to
dianose and classify leukemias and lymphomas. 10. Compare the la
oratory result
s seen for
monoclonal ammopathy and polyclonal increase in immunolo
ulins. KEY TERMS Aneu
ploidy Cold
alutinin Cryolo
ulins Hairy cell leukemia Hodkins lymphoma (HL) Non-Hodkins l
ymphoma (NHL)
Leukemia Lymphoma Monoclonal ammopathy Multiple myeloma Oncoene Paraprotein (M

protein) Plasma
cell dyscrasias Waldenstrms macrolo
ulinemia 248 CHAPTER OUTLINE CONCEPTS OF MALI
GNANT
TRANSFORMATION LYMPHOMAS Hodkins Lymphoma Non-Hodkins Lymphoma LYMPHOBLASTIC LEU
KEMIAS Acute
Lympho
lastic Leukemia Chronic Lymphoid Leukemia/Lymphoma Hairy Cell Leukemia PL
ASMA CELL
DYSCRASIAS Multiple Myeloma Waldenstrms Macrolo
ulinemia ROLE OF THE LABORATORY I
N EVALUATING
IMMUNOPROLIFERATIVE DISEASES Immunophenotypin
y Flow Cytometry Evaluation of I
mmunolo
ulins
Serum Protein Electrophoresis Immuno xation Electrophoresis Evaluation of Genetic
and Chromosomal
A
normalities SUMMARY CASE STUDY EXERCISE: DETECTION OF URINARY BENCE JONES PROT
EINS REVIEW
QUESTIONS REFERENCES 1814_Ch15_248-261.qxd 7/10/09 3:50 PM Pae 248 CHAPTER 1
5
Immunoproliferative Diseases 249 This chapter focuses on malinancies of the imm
une sys- tem,
particularly cells of lymphoid lineae. The lymphoid malinancies are
roadly cl
assified as
leukemias and lym- phomas. In leukemias, the malinant cells are primarily prese
nt in the
one
marrow and peripheral
lood. In lym- phomas, the malinant cells arise in lympho
id tissues, such
as lymph nodes, tonsils, or spleen. There can
e an overlap
etween the sites af
fected
y
leukemias and lymphomas, especially when the malinancy is far advanced. However
, it is enerally
most useful to classify the malinancy accord- in to the site where it rst arose
, rather than
the sites it can ultimately involve. The plasma cell dyscrasias (disorders) prim
arily include
multiple myeloma and Waldenstrms macrolo
ulinemia. These commonly involve the

one
marrow, lymphoid orans, and other nonlymphoid sites. They are considered
iolo
ically distinct
and not classi ed as either leukemias or lymphomas. However, plasma cells may
e f
ound in the

lood late in the course of myeloma. This phenomenon is sometimes referred to as
plasma cell
leukemia. The chapter will also cover the dianosis and monitorin of lymphoid m
alinancies
y
the clinical la
oratory. This chap- ter is not intended as a comprehensive
treatment of
hematopoietic malinancy
ut rather as an introduction to the malinancies most
commonly
evaluated
y the clinical la
o- ratory. Benin hyperactivity or inappropriate ac
tivity of the
immune system (autoimmunity) is discussed in Chapter 14. CONCEPTS OF MALIGNANT T
RANSFORMATION
Malinancy is characterized
y an excess accumulation of cells. In some cases th
is is
ecause of
rapid proliferation of the cells (i.e., excess production). In other cases, the
cells proliferate
at a normal rate
ut fail to undero apoptosis (prorammed cell death). Cells of
the immune
system, espe- cially lymphoid cells, normally respond to a stimulus
y prolifera
tin. Thus,
malinancy may re ect an initially nor- mal process in which reulatory steps have

failed. In
addition to a failure of rowth reulation, the mutations can result in arrest o
f maturation. The
malinant cells do not develop into properly functionin mature cells
ut are stu
ck at some
earlier stae of differentiation. Malinancies are en- erally multifactorial in
onset. Malinant
transformation may require altered or a
normal enes (mutations); however, this
alone is often
insuf cient. The failure of cellular reulation may
e triered
y a viral infect
ion or other
proliferative stimulus. The cells of the immune system are at reat ris
k for malinant
transformation,
ecause the features that char- acterize the development of mali
nancy are also a
normal part of the immune response: an antienic stimulus results in proliferati
on of lymphoid
cells and a hih rate of mutation durin ene rearranement and af nity maturation
. Malinant and
premalinant proliferation of cells can occur at any stae in the differentiatio
n of the lymphoid
lineaes. Despite sufferin from a
normal reulation, the malinant cells enera
lly retain some
or all of the morpho- loical and functional characteristics of their norm
al
counterpartfor example, their characteristic cell surface antiens or secretion o
f
immunolo
ulin. These character- istics are used to classify lymphoid malinanci
es. Based larely
on animal experiments, a dysreulative theory of lymphoma was developed several
years ao. The
concept of this theory was that lymphomas arise when per- sistent immunostimula
tion coincides
with an immune de ciency. The persistent stimulation provokes continuous prolife
ration and
mutations in lymphoid precursors. The immune deficiency can play two roles. Firs
t, the presence
of an ineffective immune response can permit persistent stimulation
y failin t
o clear an
infection. Second, the immune system is responsi
le for surveillance aainst mal
i- nancy. The
immune system attacks and removes those cells that escape proper reulation. It
is reported that
patients with an immunode ciency have a hiher rate of malinancy, especially mali
nancies linked
to a viral etioloy, than indi- viduals with a normally functionin immune syste
m. The immune
system is naturally diverse and heteroe- neous in preparation for its encounter
s with a wide
rane of potential pathoens. However, malinancies are thouht to arise from th
e excessive
proliferation of a sinle eneti- cally identical line, or clone, of cellst
hat is, all
the malinant cells arise from a sinle mutant parent cell. This is referred to
as the clonal
hypothesis of malinancy. The muta- tion ives the affected cell a survival adva
ntae. It either
proliferates uncontrolla
ly or fails to die appropriately, thus producin a lar
e num
er of
similarly malinant offsprin. Normal immune responses are polyclonal (i.e., cel

ls with different
features such as antien speci city all proliferate in response to an immune stimu
lus).
Therefore, malinancy can often
e dianosed when a population of cells is found
to
e more
uniform than normal. For example,
ecause each plasma cell produces only one typ
e of
immunolo
ulin, the persistent presence of a lare amount of a sinle type of im
munolo
ulin with
a sinle idiotype suests malinancy. Idiotype refers to the antien speci city o
f the
immunolo
- ulin. By contrast, an increase in the amount of total immun
olo
ulin,
without an increase in any one specific class, is characteristic of
enin react
ive
immunoprolifera- tion. Similarly, it is nearly always dianostic of malinancy w
hen the
lymphocytic cells in the
loodstream,
one mar- row, or lymphoid tissues consist
primarily of a
uniform population of cells. Many of the enes involved in reulatin cell rowt
h and
proliferation were oriinally identi ed,
ecause they are the normal cellular coun
terparts of
viral products that stimu- late host-cell proliferation and eventual malin
ant
transformation. These viral products are called viral onco- enes, and the cor
respondin host
enes are called proto-oncoenes. One such proto-oncoene, c-Myc, plays an imp
ortant role in
reulatin cell rowth. C-Myc levels rise early in the process of normal lymphoc
yte activation,
and a 1814_Ch15_248-261.qxd 7/10/09 3:50 PM Pae 249 250 SECTION 3 Immune Dis
orders fall in
c-Myc levels is linked to a return to a nonprolifera- tive state. A failure in r
eulation of
c-Myc levels is seen in some malinancies of lymphocytic cells. 1 One example is
Burkitts
lymphoma, a B-cell malinancy associated with Epstein-Barr virus (EBV) infec
tion. In this
case, a ene translocation involvin the c-Myc locus and the immunolo
- ulin he
avy chain
alters reulation of c-Myc levels. The hih c-Myc levels drive the affected
cells to
continually proliferate. Most cases of follicular lymphoma have a t(14;18) ene
translocation. 2
This results in a rearraned and constitu- tively overexpressed ene called

cl-2. The
cl-2
ene induces production of an inner mitochondrial mem
rane protein that
locks
apoptosis.
Therefore, the cells affected
y this translocation do not die normally. Even th
ouh the
malinant clone may not
e proliferatin at an excessive rate, an excessive num

er of the cells
accumulate,
ecause their survival is enhanced compared to normal cells. Many ca
ses of small
noncleaved cell lymphomas, includin Burkitts lymphoma, also have a ene transloc
ationthe c-Myc
ene, t(8;14). Most mantle cell lymphomas rearrane the
cl-1 ene, t(11;14). Th
e speci c
mutation(s) leadin to malinant transfor- mation are not known for most malina

ncies. However,
more are
ein identi ed every day. This knowlede suests that we may someday
e
a
le to treat
malinancies
y adminis- terin drus that specifically taret the a
normal prot
ein produced
y
the mutant ene in a speci c malinancy. LYMPHOMAS The lymphomas are divided into
Hodkins
lymphoma (HL) and non-Hodkins lymphoma (NHL). The classi- cation of NHL has frust
rated
patholoists and clinicians alike for decades. Many classification schemes have

een proposed,
the oldest of which is the Rappaport classifica- tion, which was developed
efor
e a difference in
B cells and T cells was known. The classi cation scheme is
ased solely on morphol
oical features

y liht microscopy. From this scheme comes the followin terms that are still e
ncountered
occasionally today: well-differentiated lymphocytic lymphoma (WDLL) (small lymph
ocytic lymphoma),
poorly differenti- ated lymphocytic lymphoma (PDDL) (follicular center cell lym
phoma with
primarily small-cleaved cells), and histiocytic lymphoma (lare-cell lymphoma).
By the 1980s,
several different schemes were in use to classify lymphomas. Investiators at th
e National Cancer
Institute reviewed existin lymphoma classi cation schemes. None of them clearly e
mered as
superior. Therefore, they developed the Workin Formulation Classi cation Scheme,
which
ultimately
ecame the standard. It, too, was
ased on the lymph node architectur
e and the
cytoloy of the mali- nant cells
y liht microscopy. It also did not consider
the B- or T-cell
lineae. The scheme classi ed the lymphomas into low-, intermediate-, and hih-ra
de processes,

ased on the aressiveness of the untreated lymphoma. In the 1990s, most invest
iators and
clinicians were usin immunoloic, cytoenetic, and molecular techniques to assi
st in lymphoma
classi cation. Advances in understand- in
asic lymphocyte
ioloy led to major r
ethinkin of
the use of a classi cation scheme
ased solely on morpholoy. In 1994, the Interna
tional Lymphoma
Study Group pro- posed the Revised European-American Lymphoma (REAL) classificat
ion. This list
set forth the dianostic features of lymphomas that the roup mem
ers consid
ered to
e widely
reconized and clearly dianosa
le
y contemporary techniques. 3 The REAL approa
ch represented a
new paradim in lym- phoma classification. The reconized lymphomas are dis
tinct
ioloical
entities de ned
y clinicopatholoic and immunoenetic features. The classi cation i
s
ased on
the principle that a classi cation is a list of real disease enti- ties, which are d
efined
y a
com
ination of cell oriins, morpholoy, immunophenotype, enetic features, and
clin- ical
features. The relative importance of each of these features varies amon
diseases, and
there is no one old standard. In some tumors, morpholoy is paramount; in others,

it is
immunophenotype, a speci c enetic a
normal- ity, or clinical features. The REAL s
cheme divides
NHL into neoplasms of precursor cells and neoplasms of mature cells of B, T, or
natural killer
(NK) cell lineae. An individ- ual entity can exhi
it a rane of morpholoical a
ppearances and a
rane of clinical
ehaviors. The REAL classi cation was validated
y a major multi
-institutional
study involvin 1378 cases, showin that it is
oth reproduci
le and clini- call
y relevant. 4
This REAL scheme is the
asis for the classification scheme adopted
y
the World
Health Oranization (WHO). 5 The REAL classi cation scheme is detailed in Ta
les 1
51 and 152.
HODGKINS LYMPHOMA Hodkins lymphoma (HL) is a hihly treata
le and often cura
le l
ymphoma that
occurs
oth in youn adults and in the elderly. 6 It is characterized
y the pre
sence of ReedStern
er (RS) cells in affected lymph nodes and lymphoid orans. RS cells are t
ypically lare
with a
ilo
ed nucleus and two prominent nucleoli. This ives the cell an owls- e
yes appearance.
Variants of RS cells do not have this typical appearance. Althouh the oriin of
the RS cells has
lon
een a matter of de
ate, a consensus has now
een reached that the malinan
t RS cells are
enerally of B-cell lineae. Ta
le 55-2 Comparison of Tests Used I. Nodular lymp
hocyte
predominant Hodkins lymphoma II. Classic Hodkins lymphoma Nodular sclerosis Hod
kins
lymphoma Lymphocyte-rich Hodkins lymphoma Mixed cellularity Hodkins lymphoma Lym
phocyte
depleted Hodkins lymphoma Ta
le 15-1. Hodkins Lymphoma 1814_Ch15_248-261.qxd 7/
10/09 3:50
PM Pae 250 CHAPTER 15 Immunoproliferative Diseases 251 The REAL/WHO classifi
cation
reconizes a
asic distinction
etween nodular lymphocyte predominant HL (
NLP-HL) and
classic HL, reflectin the differences in clinical presentation, morpholoy, phe
notype, and
molec- ular features. Classic HL is su
divided into four types: lymphocy
te-rich, nodular
sclerosis, mixed cellularity, and lymphocyte depleted. RS cells in all su
types
of classic HL
have a similar immunophenotype. They are all CD30 posi- tive, and a
out 80 perce
nt of the cases
are CD15 positive. Expression of the B-cell marker CD20 is weak or a
sent. Most
of the cells in
affected lymph nodes are reactive and appear to represent a host immune response
to the malinant cells. The more intense the immune response, the
etter the pronosis. RS
cells of
NLP-HL have a different morpholoy compared to those found in classic HL; the
y are referred to
as lymphocytic and histiocytic cells. These cells rarely express CD30 or CD15
u
t instead express
the B-cell antiens CD19 and CD20, which are typically a
sent on Reed-Stern
er
cells of classic
HL. The lymphocyte-rich form represents a
out 5 percent of the cases of HL and t

ends to occur in
slihtly older individ- uals. Nodular sclerosis HL is the most common su
type, r
epresentin a
out
70 percent of cases and havin the
est pronosis. It is characterized
y in ltrat
ion of a
mixture of normal macrophaes, lymphocytes, and ranulocytes in affected tissues
alon with small
num
ers of RS cells. There is also marked
rosis, dividin affected lymph nodes i
nto nodules.
Mixed cellularity HL also has a mixed infiltrate of normal cells
ut with less f
i
rosis and
reater num
ers of RS cells. It accounts for a
out 25 percent of cases
.
Lymphocyte-depleted HL has diffuse
rosis, few in ltrat- in normal cells, the rea
test num
er
of RS cells, and the worst pronosis than the other HL su
types. Epidemioloical
studies suest
that HL has an infec- tious etioloy. Patients with HL have elevated levels of a
nti
ody to
Epstein-Barr virus (EBV), the causative aent of infectious mononucleosis. In ad
dition,
histochemical stains demonstrate EBV in approximately 40 percent of all HLs and
a
out 75 percent
of the cases of mixed-cellularity classic HL. This indicates a role in tumorien
esis and the
potential for EBV-tareted therapy. 7 Detection of EBV-encoded RNAs (EBER1 and E
BER2) is a
sensitive method to determine the presence of EBV in RS cells. 8 This RNA assay
is the most
sensitive method for detectin EBV latent infections in clinical specimens
. History of
infectious mononucleosis is associated with an increased risk of HL, particularl
y in EBV-positive
HL in youner adults, while no increase of risk
etween infectious mononucleosis
and EBV-neative
HL was found. 9 This suests more than one cause of HL. While the specific mech
anism of
tumorienesis is unknown, EBV is known to preferentially infect B cells. In addi
tion, viral
proteins can induce activation of key sinalin pathways, producin phenot
ypic chanes seen
in EBV-infected B cells. NON-HODGKINS LYMPHOMA NHL includes a wide rane of neopl
asms. Overall,
B-cell lymphomas represent the majority (a
out 85 percent in the United States)
of NHL cases. The
most common is diffuse lare B-cell lymphoma, which accounts for 30 to 40 percen
t of NHL. The
next most common type is follicular lym- phoma, characterized
y a much more a
ressive course
than diffuse lare B-cell lymphoma. Marinal-zone B cell (includin those of
mucus-associated
lymphoid tissue [MALT]), peripheral T cell, small B lymphocytic, and man- tle c
ell lymphoma each
constitute
etween 5 and 10 percent of all lymphoma cases. Some of these lymphom
as tend to
e
slowly proressive and compati
le with lon-term sur- vival, while others are ty
pically hihly
aressive and rapidly Ta
le 55-2 Comparison of Tests Used B-Cell Neoplasms I. P
recursor B-cell
neoplasm Precursor B lympho
lastic leukemia/lymphoma II. Mature (peripheral) B-c

ell neoplasms
B-cell chronic lymphocytic leukemia/small B-cell prolymphocytic leukemia lymphoc
ytic lymphoma
Lymphoplasmacytic lymphoma Burkitts lymphoma Hairy cell leukemia Plasma cell
myeloma/plasmacytoma Extranodal marinal zone B-cell lymphoma of mucosa- associa
ted lymphoid
tissue Nodal marinal zone lymphoma Follicle center lymphoma Mantle cell lymphom
a Diffuse
lare-cell B-cell lymphoma Splenic marinal zone B-cell lymphoma T-Cell and NK-C
ell Neoplasms
I. Precursor T-cell neoplasm Precursor T lympho
lastic lymphoma/leukemia II. Mat
ure
(peripheral) T-cell and NK-cell neoplasms T-cell prolymphocytic leukemia T-cell
ranular
lymphocytic leukemia Aressive NK-cell leukemia Adult T-cell lymphoma/leukemia
(Human T
lymphotropic virus 1) Extranodal NK/T-cell lymphoma, nasal type Enteropathy-type
T-cell
lymphoma Hepatosplenic amma-delta T-cell lymphoma Su
cutaneous panniculitis-lik
e T-cell
lymphoma Sezarys syndrome Anaplastic lare-cell lymphoma, T/null cell, primary cu
taneous
type Peripheral T-cell lymphoma, not otherwise characterized Anioimmuno
lastic
T-cell
lymphoma Anaplastic lare-cell lymphoma, T/null cell, primary systemic type NK
N
atural killer
Ta
le 15-2. Non-Hodkin Lymphomas 1814_Ch15_248-261.qxd 7/10/09 3:50 PM Pae
251 252 SECTION 3
Immune Disorders fatal if not treated. The various B-cell lymphoma types can
e
divided into
three
road roups for pronostic purposes: (1) the low-risk roup includes
chronic
lymphocytic leukemia/lymphoma, follicular lymphomas, and MALT lym- phomas; (2) t
he
intermediate-risk roup includes diffuse lare B-cell lymphoma and Burkitts lymph
oma; and (3)
the hih-risk roup includes mantle cell lymphoma and lym- pho
lastic lymphoma.
MALT lymphoma is
often associated with autoimmune conditions such as Sjrren syndrome and Hashimo
to thyroiditis
or certain infections (e.., Helico
acter pylori and hepatitis C virus). In
the stepwise
process of oncoenesis, a lymphoma may proressively develop a more aressive p
henotype over the
course of the disease, a process referred to as lymphoma pro- ression. Chanes
in lymphoma
morpholoy frequently indicate alterations in the clinical and
ioloical
ehav
ior of the
disease. 10 Three characteristics usually identify lymphomas as havin a B-ce
ll oriin: (1)
surface immunolo
- ulin, which is found on no other cell type; (2) other cell s
urface proteins
such as CD19 and CD20 that are
oth sensitive and specific for B cells; an
d (3)
rearraned immunolo
ulin enes. In almost all cases,
oth the surface immunolo

ulin and the
rearraned immunolo
ulin enes have features of clonality. Some lymphomas, such
as small
lymphocytic lymphoma, oriinate from small lymphocytes that are quietly awaitin
their rst

encounter with an immunoen. These lymphomas are indolent
ut inexora
le disease
s that are
compati
le with survival for up to a decade. They proress to prolympho- cytic l
eukemia in 10 to
30 percent of cases and to lare-cell lymphoma or other aressive lymphoid mali
nancies in 10 to
15 percent of cases. Other B-cell lymphomas, such as diffuse lare-cell lym- pho
ma or
lympho
lastic lymphoma, derive from rapidly dividin cells. Lymphoid cells under
o proliferation
at two staes in their development: an early cycle as they first emere
from the
one
marrow and a later cycle in response to immunoen exposure. Thus, rapidly prolif
erative lymphomas can correspond to either early or late staes of normal development. Thes
e lymphomas

ehave aressively, and if untreated, they may kill their victims in less than
a year. The
T-cell lymphomas are more difficult to character- ize than B-cell lymphomas,

ecause in
cases that are morpholoically not clearly malinant, no easy way exists to as
say their
clonality in a similar way to testin for mono- typic liht-chain expression in
B-cell cases.
Also, a num
er of T-cell syndromes proress stealthily from atypical
ut nonclon
al proliferations
into clonal malinancies. In cases that are not clearly malinant
ased on their
morpholoy, two
ancillary methods of esta
lishin clonality are availa
le. The first is to use m
olecular
techniques to detect a clonal rearranement of the T-cell receptor ene. In
eni
n pop- ulations,
each cell exhi
its a slihtly different rearranement,
ut in malinant prolifer
ations, the
population of cells uni- formly expresses the same rearranement. A second metho
d is to
demonstrate
y flow cytometry that the suspicious population of T cells uniforml
y fails to
express an antien that is normally expressed on all T cells. LYMPHOBLASTIC LEUK
EMIAS Leukemias
are enerally classi ed as either acute or chronic. Chronic leukemias are usually
slowly
proressive and com- pati
le with extended survival. However, they are enerally
not cura
le with
chemotherapy. By contrast, acute leukemias are enerally much more rapidly pror
essive
ut have a
hiher response rate to therapy. Acute Lympho
lastic Leukemia Acute lympho
lasti
c leukemia (ALL)
is characterized
y the presence of very poorly differentiated precursor cells (

last cells) in
the
one marrow and peripheral
lood. These cells can also in ltrate soft tissues,
leadin to
oran dysfunction. ALL is usually seen in children,
etween 2 and 10 years of a
e, and is the
most common form of leukemia in this ae roup. ALL is a treata
le disease, with
a remission rate
of 90 percent and a cure rate of 60 to 70 percent in children. The remission and
cure rates are
lower in adults with ALL. Immunoloically, ALL is divided into four types: CALLa
(CD10)-expressin immature B cell ALL, pre-B cell ALL without CALLa (CD10), T

-cell ALL, and

B-cell ALL. CALLa (CD10)-expressin immature B cell ALL is the most common ALL,
while pre-B cell
is the second most com- mon. B cell ALL is rare. Chronic Lymphoid Leukemia/Lymph
oma The chronic
lymphocytic leukemias/lymphomas are a roup of diseases almost exclusively of Bcell oriin. They
include chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), pr
olymphocytic
leukemia, and hairy cell leukemia. The World Health Oranization (WHO) cons
iders CLL/SLL a
sinle disease with different clinical presenta- tions. Both reveal B-cell marke
r CD19
ut weakly
express CD20. CLL is a common hematopoietic malinancy that involves the expansi
on of a clone of
B cells that have the appearance of small mature lymphocytes. In a
out 5 percent
of cases, the
malinant clone is T-cell derived. The cyto- loically normal lymphocytes acc
umulate in the

one marrow and
lood as well as in the spleen, lymph nodes, and other orans. C
LL primarily
occurs in patients over 45 years of ae with a two-to-one male predomina
nce. Patients
usually present with an increase in the
lood lym- phocyte count, which may
e a
n incidental
findin on a routine physical examination. As the malinant lymphocytes continue
to increase in
num
er, replacement of normal elements in the
one marrow leads to anemia and th
rom-
ocytopenia.
Lymph node enlarement is prominent early in the disease. CLL is compati
le
with a lon
survival. Palliative therapytreatment that helps control or reduce symptoms
ut i
s not
curativeis used. 1814_Ch15_248-261.qxd 7/10/09 3:50 PM Pae 252 CHAPTER 15
Immunoproliferative Diseases 253 Hairy Cell Leukemia Hairy cell leukemia is a ra
re, slowly
proressive disease characterized
y in ltration of the
one marrow and spleen
y
leukemic cells,
without involvement of lymph nodes. It has a four-to-one male predominance and i
s seen in individuals over 20 years of ae. Patients usually present with cytopenias
ecause o
f
one marrow
infiltration,
ut the
lood lymphocyte count is usually not very hih. However,
the splenomealy
may
e strikin. The malinant lympho- cytes are round with a
land cytoloical ap
pearance.
They often have irreular hairy cytoplasmic projections from their surfaces, most
easily
visi
le on a wet-mount prepara- tion. The malinant cells stronly express B-cel
l markers CD19,
CD20, and CD22. They characteristically contain tartrate-resistant acid phosphat
ase in their
cytoplasm, which can
e identi ed
y histochemical stainin. PLASMA CELL DYSCRASIA
S The plasma
cell dyscrasias include several related syndromes: multiple myeloma, Waldenstrms
macrolo
ulinemia, and the premalinant conditions monoclonal ammopathy of unde
termined
sini cance (MGUS) and smolderin mul- tiple myeloma (SMM). These conditions are c
haracterized
y
the overproduction of a sinle immunolo
ulin compo- nent called a myeloma prote

in (M protein),
or paraprotein,
y a clone of plasma cells. M protein is rarely associated with
other
lymphoproliferative disorders, such as non-Hodkin lymphoma or primary amyloidos
is. La
oratory
evaluation is important in the dianosis and differentiation of these conditions
. Dianosis and
monitorin of the plasma cell dyscrasias depend heavily on detectin and quantit
atin the M
protein. Multiple Myeloma Multiple myeloma is a malinancy of mature plasma cell
s that accounts
for a
out 10 percent of all hematoloic can- cers. It is the most serious and co
mmon of the
plasma cell dyscrasias. It is usually dianosed in persons
etween 40 and 70 yea
rs of ae with a
peak ae of 67 years. Men are slihtly more likely than women to develop multipl
e myeloma. The
American Cancer Society estimates there are a
out 20,000 new cases of multiple m
yeloma dianosed
each year in the United States, alon with a
out 11,000 deaths. 11 Patients pro
ress from
asymptomatic MGUS to SMM to the symp- tomatic disease multiple myeloma. Between
20 to 25 percent
of individuals with MGUS will proress to multiple myeloma. Patients with
multiple myeloma
typically have excess plasma cells in the
one marrow, a monoclonal immu
nolo
ulin
component in the plasma and/or urine, and lytic
one lesions. The plasma cells i
n ltratin the

one marrow may
e morpholoically normal or may show atypical or
izarre
cytoloical
features. Malinant plasma cells phenotypically express CD38, CD56, and CD138. A
pproximately 20
percent of myeloma cells express CD20. The immunolo
ulin produced
y the malin
ant clone can
e
of any type, with IG
ein the most common (50 percent), followed
y I
A, IM, and
liht chains only. Very often the production of heavy and liht chains
y the ma
linant plasma
cells are not well synchronized, and an excess of liht chains may
e produced.
In a
out 10
percent of cases, the myeloma cells exclusively produce liht chains. The liht
chains are
rapidly excreted in the urine. Bence Jones protein is the name iven to fre
e immunolo
ulin
liht chains ( r ) excreted in the urine. Rarey d myemas prduce IgD, IgE, 
r heavy
chains ny. Very rarey, tw r mre distinct M prteins are prduced, r a ci
nicay typ- ica
myema may prduce n secretry prduct. The eve f nrma immungbuin is
ften decreased
in prprtin t the amunt f abnrma immungbuin (M prtein) present in th
e serum. The
cinica manifestatins f mutipe myema are pri- mariy hematgic, s eeta
, and
immungic. Hematgic prbems are ften reated t the faiure f the bne m
arrw t prduce
a nrma number f hematpietic ces, because myema ces prgressivey rep
ace them. This
eads t ane- mia, thrmbcytpenia, and neutrpenia. High eves f M prtein c

an interfere with
caguatin factrs, eading t abnrma pateet aggregatin and abnrma
pateet
functin. This, cuped with thrmbcytpenia, ma es hem- rrhaging, bruising, a
nd purpura a
cmmn cmpicatin f mutipe myema. Mutipe myema tends t preferentia
y invve bne
and frms mutipe ytic esins, ften eading t bne pain and pathgica fr
actures. Bne
pain, usuay invving the spine r chest, is the mst cmmn presenting sympt
m f mutipe
myema. Hypercacemia is very cmmn, because the myema prmtes bne resrpt
in. In advanced
dis- ease, the hypercacemia itsef can reach ife-threatening eves. When immu
ngbuin eves
in the bd are suf cienty high, the presence f rueaux may be seen n examina
tin f the
periphera bd smear. The excess prductin f the abnrma immungbuin is
accmpanied by a
prgressive decrease in the nrma immungbuins. This eads t a de ciency f n
rma antibdy
respnses and a higher inci- dence f infectius diseases. Hyperviscsity can de
vep when the
eve f M prtein in the pasma is high. Because viscsity depends n the size
f the mecue
in sutin, and IgM is the argest f the immungbuins, hypervis- csity is
mst ften
seen with IgM-prducing tumrs. Hyperviscsity syndrme is as smetimes see
n with an
IgG3-prducing myema, because IgG3 is the argest f the IgG subcasses. The t
ype and severity
f cinica manifestatins depend n the type f immungbuin cmpnent prduc
ed. Up t 15
percent f patients with mutipe myema devep ight-chain depsitin disease
r amyidsis.
These are tw reated disrders in which free ight chains r fragments f immun
gbuin are
depsited in the tissues. Amyid bers stain with the dye Cng red and shw app
e-green birefringence when viewed under a parizing micrscpe. Light 1814_Ch15_248-261.qxd
7/10/09 3:50
PM Page 253 254 SECTION 3 Immune Disrders chains can be identi ed in tissue sect
ins by
immun u- rescence r immunhistchemica staining with specific antibdies. The
depsitin f
antibdy-derived materia resuts in rgan dysfunctin. The idneys are mst ft
en affected, but
every tissue in the bdy can devep the dep- sitin f amyid. Cardimypathy
, periphera
neurpathy, hepatspenmegay, and ecchymses are the mst cmmn manifestatin
s. Patients with
myema can devep either acute r chrnic rena faiure. As many as twthirds f
patients with mutipe myema exhibit sme degree f rena insuf ciency. Patients
with myemas
that prduce ight chains r IgD are much mre i ey t devep rena faiure t
han thse with
ther types. Rena insufficiency, due t Bence Jnes prteins, is seen in abut
50 percent f
patients. After infec- tin, this is the secnd eading cause f death. Bence J

nes prteins are

thught t be directy txic t tubuar epithe- ia ces and can damage the i
dneys by
precipitating in the tubues, causing intrarena bstructin. The median surviva
 fr patients
with mutipe myema is apprximatey 3 years. Prgnsis is generay best with
IgM disease and
wrse with IgG disease. Evidence f a deetin in chrmsme 13 has a signi cant
negative impact
n utcme. 12,13 Criteria fr the diagnsis f mutipe myema incude pasma
ces cmprising
greater than 10 percent f bne marrw ces r a pasmacytma (a sid tumr ma
ss f pasma
ces), pus either serum M prtein greater than 3g/dL, urinary M prtein, r s
teytic bne
esins. Serum M prteins can be detected by serum prtein eectrphre- sis an
d by serum
immunfixatin. It is imprtant t differentiate amng mncna free ight
chains, heavy
chains, and gamma gbuins. Patients with mncna gammpathy can have MGUS,
SMM, mutipe
myema, r ne f severa ther cna expansins f pasma ces r B ces. A
n imprtant
feature supprting the diagnsis f muti- pe myema is the presence f Bence
Jnes prtein in
the urine. Abut 60 t 70 percent f the patients with myema excrete Bence J
nes prtein in
the urine, which can be detected by speci c techniques, such as immuneectr
phre- sis, r
nnspeci c techniques, such as heat precipitatin (see Exercise: Detectin f Urin
ary Bence Jnes
Prteins at the end f the chapter). Wadenstrms Macrgbuinemia Wadenstrms
macrgbuinemia is a maignant prif- eratin f IgM-prducing ymphcytes an
d crrespnds t
ymphpasmacytid ymphma as defined by the WHO. The maignant ces are mre
immature than
pasma ces and have a micrscpic appearance smewhere between that f sma 
ymphcytes and
pasma ces. These ces prduce the pan-B-ce mar ers CD19, CD20, CD22, and F
MC7. In mst
cases, CD10 and CD23 are nt expressed. 14 These pasmacytid ymphcytes infi
trate the bne
marrw, speen, and ymph ndes. The median age f affected patients is
65 years;
Wadenstrms macrgbuinemia is abut 10 t 20 percent as cmmn as mutipe mye
ma, and the
etigy f this disease is un nwn. Hwever, because f famiia custerin
g f
Wadenstrms macrgb- uinemia and ther B-ce disrders (e.g., nn-Hdg in ymp
hma),
genetic factrs are thught t be invved. 15,16 The cinica signs and sym
ptms f
Wadenstrms macrgbuinemia are due t in tratin f the maignant ces int b
ne marrw,
the speen, and ymph ndes with the verprductin f mncna IgM. Symptms
ften incude
wea ness, fatigue, anemia, beeding, and hyperviscsity. Beeding can be du
e t a
cmbinatin f thrmbcytpenia and interference f pateet functin by mnc
na IgM. The

mncna IgM can accumuate in any tissue, frming depsits that ead t
 in ammatin and
tissue damage. Lytic bne esins, hypercacemia, and rena tubuar abnrmaitie
s are rare. The
median ength f surviva fr patients with Wadenstrms macrgbuinemia is nge
r than with
mu- tipe myema5 years versus 3 years. A individuas with Wadenstrms macrg
buinemia
have eevated serum mncna prtein that migrates in the gamma regin. Hweve
r, the
cncentratin varies widey, and it is nt pssibe t de ne a cncentratin that
differentiates
this disease frm ther B-ce ymphpriferative disrders. Serum prtein eec
trphresis
shud be used t evauate the amunt f the mncna prtein, and the cncent
ratin f IgM
shud be cn rmed by immun xatin. In 70 t 80 percent f the patients, the ight
chain is .
Whie Bence Jnes prteinuria is ften present, prtein cncentratins rarey ex
ceed 1.0 g/day.
Serum 2 -microglo
ulin lvls ar gnrally ovr th rfrnc rangs uppr limit
of 3.0 mg/L.
In 10 to 20 prcnt of patints, th IgM paraprotins
hav as cryoglo
ulins. C
ryoglo
ulins
prcipitat at col tmpraturs an can occlu small vssls in th xtrmiti
s in col
wathr. Occlusion of small vssls can la to th vlopmnt of skin sors or
vn ncrosis of
portions of th fingrs or tos. Som of th clinical symptoms ar u to autoan
ti
oy activity
of th monoclonal IgM anti
oy. Som IgM paraprotins hav spci city for i or I a
ntigns an
will agglutinat r
loo clls in th col (col agglutinins). Cryoglo
ulins c
an
 tct
whn a
loo or plasma sam- pl is rfrigrat in th clinical la
oratory. Th
prcipitat that
forms at low tmpratur issolvs upon warming. Approximatly 20 prcnt of
th patints
with Walnstrms macroglo
ulinmia will prsnt with priph- ral nuropathy. 17 I
t appars
that th monoclonal IgM is irct against glycoprotins or glycolipis of th
priphral
nrvs. In aition, IgM can
 monstrat against poly- clonal IgG. This r
sults in
immun complx isas charactri
y vasculitis, affcting small vssls of
th skin,
kinys, livr, an priphral nrvs. ROLE OF THE LABORATORY IN EVALUATING IMMU
NOPROLIFERATIVE
DISEASES Th la
oratory is involv in thr major ways in valuating lymphoprol
ifrativ
isorrs. First, it can assss th 1814_Ch15_248-261.qx 7/10/09 3:50 PM
Pag 254
CHAPTER 15 Immunoprolifrativ Disass 255 immunophnotyp of hmatopoitic cl
ls in th
loo,

on marrow, or lymphoi tissus
y ow cytomtry. This is on
y tcting th a
ntigns on th
surfac of th clls that ar charactristic of a spci c linag an stag of if
frntia- tion.
This tchnology srvs as an xcllnt complmnt to microscop-
as traitiona
l iagnostic

mthos an as istinctiv, iscriminatory capa
ilitis that ar unmatch
y

any othr
iagnostic tchniqu. Applications inclu tction of clonal clls in B-cll l
ymphoma, th
rcognition of antignic xprssion anomalis in B- or T-cll malignan- cis, th
 inti cation
of malignant plasma clls, an th rapi masurmnt of cll cycl fractions. Th
 scon an
pro
a
ly most straightforwar rol of th la
oratory is in valuating th amount
an charactristics of immunoglo
ulins. Bcaus th B-cll linag vlops into plasma clls t
hat prouc
anti
oy, malignancis of crtain B clls ar associat with xcssiv or a
nor
mal anti
oy
prouction. Th amount an charactristics of plasma or urin immunoglo
ulin can

 us to
iagnos an valuat th plasma cll yscrasias. Thir, th la
oratory is i
ncrasingly
involv in th assssmnt of gntic an chromosomal a
normalitis in hmatop
oitic
malignancis. Although this is mostly on in larg rfrral cntrs or rfrnc
 la
oratoris,
gntic tchniqus ar moving progrssivly closr to routin prac- tic. Th po
lymras chain
raction can
 us to tct squncs (mutations) within gns that hav
n
link to
particular isass. Cytogntic stuis ar usful to tct chromosomal a
norm
alitis (.g.,
translocations). Immunophnotyping
y Flow Cytomtry Analysis of cll surfac ma
rkr xprssion
is commonly us in th iagnosis an classification of lukmias an lym- phoma
s. 18 Bcaus th
malignant clls xprss markrs that oftn corrspon to thos of thir normal p
rcursors,
insight into th linag of origin an stag of maturation can oftn
 trmin

y this
tchniqu. Rsarchrs in svral la
oratoris hav inti  many lymphoi surfa
c antigns
an vlop anti
ois to thm. Not surprisingly, anti
ois from iffrnt
la
orato- ris
an
aring iffrnt nams wr oftn foun to tct th sam antign/molcul
. At that point,
th antign woul
 assign a clustr of iffrntiation (CD), or clustr signation num
r,
maning that a known clustr of anti
ois rcogni this antign. Th prsnc
 of CD
antigns on th surfac of hmatopoitic clls is oftn tct
y flow cyt
omtry. Ta
ls
153 an 154 list a fw of th important cll markrs oftn tct
y ow c
ytomtry
(s also Chaptr 12). Sampls of potntially noplastic clls ar incu
at wit
h anti
oy
prparations spcific for rlvant antigns. In many cass, th anti
ois conta
in a fluorscnt
la
l. Thus, clls that xprss a particular antign ar
oun
y th anti
oy a
n
com
uorscnt. This allows th anti- gnic profil, or phnotyp, of th cll populat
ion to

trmin. Flow cytomtry is ial for uis in which clls ar natu- rally susp
n,
ut it is

also usful in lymphoi tissus, from which singl-cll suspnsions can
 asil
y ma. Th
avan- tags of ow cytomtry ar largly
as on its a
ility to vry rapily an
simultanously
analy, vn in small sampls, multipl-cll proprtis, incluing si, gr
anularity, an
Ta
l 55-2 Comparison of Tsts Us CELL TYPE CD MARKER All lymphoi clls CD45,
also call
lukocyt common antign B clls Almost all B clls xprss CD19, CD20, an CD22
. B-cll
lymphomas ar somtims ractiv for CD5 an CD43, which ar othrwis foun on
T clls. T clls
Almost all T clls xprss CD2, CD3, CD5, an CD7. Most T clls ar also positiv
 for ithr CD4
(hlpr clls) or CD8 (supprssor or cytotoxic T clls). Natural killr clls Us
ually xprss
CD16, CD56, or CD57 Ta
l 15-3. CD Makrs Important in Flow Cytomtry, List
y
Cll Typ Ta
l
55-2 Comparison of Tsts Us CD MARKERS CELL SPECIFICITY CD2 T cll CD3 T cll
CD4 T hlpr cll
CD5 T cll an CLL CD7 T cll CD8 T supprssor cll CD10 Burkitts an follicular
lymphoma CD11

Myloi cll CD11c Monocyt an HCL CD13 Myloi cll CD14 Monocyt CD15 Myloi
/monocytic clls
CD16 Natural killr cll CD19 B cll CD20 B cll CD22 B cll an HCL CD23 B cll
an CLL CD38
Plasma cll an prognostic inicator for CLL CD45 All whit
loo clls CD56 Nat
ural killr an
a
normal plasma clls CD103 HCL CLL
chronic lymphocytic lukmia; HCL
hairy cll
lukmia
Ta
l 15-4. CD Markrs Important in Flow Cytomtry, List
y CD Markr 1814_Ch1
5_248-261.qx
7/10/09 3:50 PM Pag 255 256 SECTION 3 Immun Disorrs surfac an intracllu
lar antigns. Th
quantitativ natur of th ata prouc,
oth with rgar to cll population i
s- tri
utions an
to xprssion of iniviual cll antigns, offrs o
jctiv critria for intrpr
tation of
rsults. Evaluation of Immunoglo
ulins As iscuss in Chaptr 4, th
asic immu
noglo
ulin unit
con- sists of two intical havy chains an two intical light chains, covaln
tly link
y
isul 
ons. Th structur of th havy chain  ns th class of th anti
oy (
.g., heavy
chain and IG, heavy chain and IM, etc.). The two types of liht chains ( and ) c
an each
ccur in cmbinatin with any f the types f heavy chains. The heavy and ight
chains each
cntain cnstant and variabe regins. The cnstant regins cntain the sites f
immungbuin
binding t ceuar recep- trs and sites invved in cmpement xatin. The vari
abe regins
are respnsibe fr the antigen speci city f the anti- bdy. The immungbuins
in pasma are
hetergeneus in structure, because they recgnize a variety f different antigens. The
variabiity in their amin acid sequences means that they as vary sighty in
their physica
characteristics, such as mecuar weight and charge. B ces differentiate int
antibdy-prducing pasma ces by maturatin thrugh severa stages. Each B ce

 recg- nizes
ny a singe antigenic site r epitpe. An eary B-ce precursr is stimuated
t priferate
and mature when it encunters an immungen that it recgnizes. When a fr- eign
mecue enters
the bdy, the many different epitpes n it each stimuate a B-ce respnse, e
ading t the prductin f an array f different antibdies. Hwever, in a maignant disrder, t
he cna
priferatin f transfrmed pasma ces eads t verprductin f a singe im
mungb- uin.
This is caed a mncna gammpathy. Quantitative measurement f serum r uri
ne immungbuins is used in the diagnsis f sme ymphpriferative disrders. Hwever,
the evauatin
f a patient fr the pssibiity f a mncna gammpathy requires bth q
ua- itative and
quantitative anaysis f immungbuins. 19 The initia tests used t screen f
r the presence f
a mncna gammpathy are serum immungbuin eves and serum prtein eectr
phresis. A high
cinica index f suspicin, abnrma resuts n immungbuin eves, r ndings
sug- gestive
f a mncna cmpnent n serum prtein eectrphresis wi prmpt add
itina testing.
Serum Prtein Eectrphresis Serum prtein eectrphresis (SPE) is a technique
in which serum
prteins are separated n the basis f their size and eectrica charge, as disc
ussed in Chapter
4. SPE resuts in fur regins: abumin, and apha, beta, and gamma gbuins. I
gG, IgM, IgD, and
IgE migrate in the gamma gbuin regin, whie IgA migrates as a brad band in
the beta and
gamma regins. Figure 151, pane A, shws a styized draw- ing f the prtein dis
tributin in
nrma serum. As can be seen, immungbuins nrmay shw a range f mbiitie
s. The SPE
pattern fr a pycna increase in serum immungbuins and a mnc
na gammpathy
are shwn in panes B and C, respectivey. Pycna increases in serum immung
buins are seen
in a variety f disr- ders, incuding infectins, autimmune disrders, iver d
isease, and sme
immundeficiency states (e.g., hyper- IgM syndrme). Additina evauatin f se
rum
immungbuin is per- frmed if the SPE shws a mncna cmpnent, if there
is a signi cant
quantitative abnrmaity f serum immungb- uins, r if the cinica picture
strngy suggests
a pasma ce dyscrasia. Myema in which ny ight chains are prduced may nt
be detected n
SPE, because the ight chains are rap- idy ceared in the urine. Therefre, add
itina studies
n a randm r 24-hur urine sampe may be indicated even in the presence f a n
rma SPE.
Immun xatin Eectrphresis The next step in evauating a mncna gammpathy
is typicay
either immuneectrphresis (IEP) r immun x- atin eectrphresis (IFE). IEP i
s nt very
sensitive, and the turnarund time is engthy (abut 18 hurs). Due t these im

itatins, IEP has

generay been repaced by IFE. In IFE, serum sampes are eectrphresed, just
as fr SPE, and
then speci c antibdy is appied directy t the separat- ing ge. The antibdies
and antigens
diffuse tward each ther, and the antibdyantigen cmpexes frmed are visu- ai
zed by
staining. Areas f diffuse staining indicate pycna immungbuins, wh
ie mncna
bands pr- duce narrw, intensey stained bands (Fig. 152). IFE is much faster (a
but 1 hur)
than IEP, has much greater sen- sitivity, and is the mst accurate assay fr typ
ing paraprteins.
Hwever, IFE is as abr intensive and expensive. A B C FIGURE 151. Serum pr
tein
eectrphresis f nrma and abnr- ma sampes. The wer prtin f each pane
 is a
representatin f a stained agarse eectrphresis ge. The intensity f staini
ng cr- respnds
t the amunt f prtein in each regin f the ge. In the upper prtin f each
pane is a
densitmeter tracing f a ge simiar t the ne beneath it. In the upper pane
shwing a nrma
serum sampe, the argest pea is abumin. The gbuin regins are as indicated
.
1814_Ch15_248-261.qxd 7/10/09 3:50 PM Page 256 CHAPTER 15 Immunpriferative
Diseases 257
Evauatin f Genetic and Chrmsma Abnrmaities The majrity f B-ce 
ymphmas and
sme T-ce ymphmas are characterized by specific chrmsme transcati
ns. Often these
transcatins can be detected by cytgenetic techniques. Li e mst ces, maig
nant ym- phid
ces can be made t priferate in vitr, and their metaphase chrmsmes can
be examined
fr grssy visibe abnrmaities that crrespnd t characteristic trans
catins. Many
f the transcatins invve the immungbuin r T-ce receptr ci wit
h varius partner
chrmsmes and ead t abnrma prt-ncgene expres- sin. Other characteri
stic
transcatins resut in the prductin f a nve fusin prtein. The
detectin f
transcatins is f particuar vaue in diagnsis f disease. Aneupidy (abnr
ma number f
chrmsmes) and deetin f specific chrmsme regins are cmmn secn
dary chrmsme
events that are rarey speci c t a particuar type f ymphma but prvide vauab
e prgns- tic
infrmatin. Traditina cytgenetic evauatin has been expanded by a technique
nwn as
urescence in situ hybridizatin (FISH). FISH is used t directy identify a spec
i c regin f
DNA in a ce. It invves the preparatin f shrt sequences f singe
-stranded DNA,
caed prbes, which are cmpemen- tary t the DNA sequences f interest. These
prbes bind t
the cmpementary chrmsma DNA and, because they cntain a urescent abe, a
w ne t see
the catin f thse sequences f DNA. Prbes can be used n chrm- smes, int

erphase nucei,
r tissue bipsies. FISH is rapid and quite sensitive and des nt require ce
cuture. Hwever,
it ny prvides infrmatin abut the speci c prbe being tested. Lymphid maig
nancies can
as be evauated by mec- uar genetic techniques. These methds are usuay g
eared tward
nding cna rearrangements f the immungb- uin gene in B-ce maignancies 
r f the T-ce
receptr gene in T-ce maignancies. These rearrangements are t subte t be
detected by
cnventina cytgenetics. As a resut f the Human Genme Prject cmpeted in
2003,
apprximatey 20,000 genes have been identi- fied. 20 It is estimated that the h
uman genme
cntains 20,000 t 25,000 genes; hwever, it wi prbaby ta e severa years be
fre an accurate
gene cunt can be estab- ished. Mst f these genes are ny partiay characte
rized, and the
functins f the vast majrity are sti un nwn. It is i ey that many genes t
hat might be
usefu fr diag- nsis r prgnsticatin f human maignancies have yet t be r
ecgnized. The
advent f cmpementary DNA (cDNA) micrar- ray techngy nw aws the effici
ent measurement
f expressin fr amst every gene in the human genme in a singe vernight hy
bridizatin
experiment. This genmic scae apprach has begun t revea nve mecuar-base
d sub- casses
f many maignancies, incuding ymphma and eu emia. 21 Mecuar techniq
ues, incuding
nuceic acid ampi- catin, have fund their way int rutine cinica practice.
SUMMARY
Maignancies f ymphcytes, bth ymphmas and eu emias, are cmmny enc
untered in
cinica practice. The cassi catin f these disrders depends n the iden- ti cati
n f their
ce f rigin. The B- r T-ce derivatin f a ymphid maignancy is ften de
termined in the
ab- ratry by fw cytmetry. Sme B-ce disrders resut in abnrma immung
buin
secretin. Mutipe myema is a maignancy f pasma ces characterized by pr
ductin f a
mncna immungbuin r paraprtein (M pr- tein). The paraprtein may
be f any
immungbuin cass r may be an immungbuin heavy r ight chain. Wadenstrms
macrgbuinemia is a maignancy f pasmacytid ymphcytes that prduces
an IgM paraprtein. Identificatin and quantificatin f the paraprtein are centra t the di
agnsis and
mnitring f these cn- ditins. SPE is used t detect the presence f a parapr
tein, which is
then characterized by immuneectrphresis r immun xatin eectrphresis. Eva
uatin f
genetic and chrmsma abnrmaities is a rapidy evving area f ab- ratry
practice. FIGURE
152. Agarse ge immun xatin eectrphresis f serum. The same patient sampe is
paced n
a six anes and eec- trphresed. Fwing eectrphresis, antisera are add

ed t each ane
as fws: ane 1antitta serum prtein; ane 2anti-IgG; ane 3anti-IgA; ane 4ant
i-IgM;
ane 5anti-; and ane 6anti-. The patient is exhibiting a mncna IgM immungbuin.
(Curtesy f Heena Labratries, Beaumnt, TX.) See Cr Pate 12. 1814_Ch15_2
48-261.qxd
7/10/09 3:50 PM Page 257 258 SECTION 3 Immune Disrders A 63-year-d mae vis
its his primary
care physician cm- paining f fatigue and shrtness f breath, upper bac pain
, and a cugh
that has becme prductive the ast 2 days. The patient was febrie and appeared
acutey i. A
chest X-ray reveaed pneumnia, and the fwing sig- nificant abratry resu
ts were fund:
red bd ce cunt f 4.1
10 12
/
L (reference range 4.6 t 6.0
10 12
/
L), hemgbin 13 g/dL (reference range 14.0 t
18.0 g/dL), white bd ce cunt 4.8
10 9
/
L (reference range 4.5 t 11.0
10 9
/
L), and an erythrcyte
sedimentatin rate f 12 mm/hr (reference range 0 t 9 mm/hr). Based n
these resuts, the
physician rdered serum immungbuin eves. The fwing resuts were r
eprted: IgG 3250
mg/dL (reference range 600 t 1500 mg/dL), IgM 48 mg/dL (reference range
75 t 150 mg/dL),
and IgA 102 mg/dL (reference range 150 t 250 mg/dL). Questins a. What disease(
s) shud yu
suspect, and what addi- tina tests cud hep cn rm the diagnsis? CASE STUDY
1814_Ch15_248-261.qxd 7/10/09 3:50 PM Page 258 CHAPTER 15 Immunpriferative
Diseases 259
DETECTION OF URINARY BENCE JONES PROTEINS Principe Reagent test strips used t
screen fr
prteinuria are gen- eray nt sensitive t immungbuin r free ight chains
. Therefre,
ther techniques are required t detect Bence Jnes prteins in the urine. One m
ethd expits
the unique heat subiity prperties f these prteins. Bence Jnes pr- teins
precipitate at
temperatures between 40C and 60C and redissve again arund 100C. This apprach ca
n generay detect eves f prtein dwn t abut 30 mg/dL. Specimen Cectin A 24
-hur r randm
urine specimen is cected int a cean cntainer. The sampe may be stred in
a refrigeratr t
prevent bacteria grwth. Reagents, Materias, and Equipment Acetate buffer, 2 m
/L, pH 4.9 Test
tubes Biing water bath NOTE: Fr acetate buffer, add 4.1 mL f gacia acetic
acid t 17.5 g f
sdium acetate trihydrate and then add water t give a tta vume f 100 mL. P
rcedure 1. Pace
4 mL f cear urine in a test tube. Add 1 mL f acetate buffer and mix. 2. Heat
fr 15 minutes at
56 C in a water bath r heating bc . The devepment f turbidity is indi
cative f
Bence Jnes prteins. 3. If the sampe has becme turbid, transfer the test tube
t a biing

water bath fr 3 minutes. The turbidity shud decrease if it is due t th

e presence f
Bence Jnes prteins, which redissve at 100C. 4. If the turbidity f the sampe
increases
after biing, it is because f abumin and gbuins. Fiter the test sampe im
mediatey n
remving frm the biing water bath. If Bence Jnes prteins are present,
the sampe
wi becme cudy as it cs, then cear again as it reaches rm temperature.
If the
precipitate is quite heavy at 56C, it may nt dissve easiy n biing. It is b
est t repeat
the test with a diuted urine specimen. Cmments This is simpy a screening test
fr detectin f
Bence Jnes prteinuria. The best methd f detecting Bence Jnes pr- teins is
prtein
eectrphresis fwed by immun xatin eectrphresis. See the exercise in Cha
pter 16.
EXERCISE 1814_Ch15_248-261.qxd 7/10/09 3:50 PM Page 259 260 SECTION 3 Immune
Disrders 1. In
genera, a myema secreting which type f M pr- tein is mst i ey t cause r
ena faiure? a.
IgG b. IgM c. ight chains d. heavy chains 2. Which f the fwing wud be
the best
indicatr f a maignant cne? a. Overa increase in antibdy prductin b. In
crease in IgG and
IgM ny c. Increase in antibdy directed against a speci c epitpe d. Decrease in
vera
antibdy prductin 3. A f the fwing are features f maignancy except a.
excess
apptsis. b. rapid priferatin. c. cna priferatin. d. chrmsma mutat
ins. 4. A f
the fwing features are
cmmny used t cassify ymphid nepasms
except a. Ce f
rigin b. Presence f gene transcatins c. Expsure f the patient t carcing
ens d.
Mrphgy/cytgy f the maignant ces 5. Hdg ins ymphma is characterized
by a.
priferatin f T ces. b. excess immungbuin prductin. c. an incurabe,
rapidy
prgressive curse. d. the presence f Reed-Sternberg ces in ymph ndes. 6. C
hrnic eu emias
are characterized as a. usuay being f B-ce rigin. b. being curabe with ch
emtherapy. c.
usuay ccurring in chidren. d. fwing a rapidy prgressive curse. 7. Whi
ch best describes
acute eu emias? a. Usuay f B-ce rigin b. Usuay ccurring in chidren c.
Pr respnse t
chemtherapy d. Ces are we differentiated 8. Fw cytmetry resuts n a pat
ient revea a
ac f ces with CD2 and CD3. What des this indicate? a. Lac f B ces b. L
ac f T ces c.
Lac f mncytes d. Lac f natura ier ces 9. Which f the fwing
is true f
Wadenstrms macrgbuinemia but nt mutipe myema? a. Hyperviscsity syndrme
is ften
present. b. A singe prtein-prducing cne is eevated. c. The cancerus ce
is a prepasma
ce. d. Bence Jnes prteins are present in the urine. REVIEW QUESTIONS 1814_Ch
15_248-261.qxd

7/10/09 3:50 PM Page 260 References 1. Ptter, M. Pathgenetic mechanisms in B

-ce
nn-Hdg ins ymphmas in humans. Cancer Res 52:5522s, 1992. 2. Faini, B, and Ma
sn, DY.
Prteins encded by genes invved in chrmsma ateratins in ymphma a
nd eu emia:
Cinica vaue f their detectin by immuncytchemistry. Bd 99(2):409426, 200
2. 3. Harris,
NH, Jaffe, ES, Stein, H, et a. A revised Eurpean- American cassificatin f 
ymphid
nepasms: A prpsa frm Internatina Study Grup. Bd 87:1361, 1994. 4. Th
e nn-Hdg ins
Lymphma Cassi catin Prject: A cin- ica evauatin f the Internatina Lymph
ma Study Grup
cassificatin f nn-Hdg ins ymphma. Bd 89(11): 39093918, 1997. 5. Har
ris, ML,
Jaffe, ES, Diebd, J, et a. The Wrd Heath Organizatin cassificatin f he
matgica
maignancies. Reprt f the Cinica Advisry Cmmittee meeting, Airine Huse,
Virginia,
Nvember 1997. Md Path 13:193, 2000. 6. Pieri, SA, Ascani, S, Lencini, L, e
t a. Hdg ins
ymphma: The pathgists viewpint. J Cin Path 55(3):162176, 2002. 7. Jaffett
, RF. Viruses
and Hdg ins ymphma. Ann Onc 13(Supp 1):2329, 2002. 8. Kapatai, G, and Murray
, P.
Cntributin f the Epstein-Barr virus t the mecuar pathgenesis f Hdg in
ymphma. J Cin
Path 60:13421349, 2007. 9. Hjagrim H, Smedby KE, Rstgaard K, et a. I
nfectius
mnnucesis, chidhd scia envirnment, and ris f Hdg in ymphma. Cance
r Res
67(5):23822388, 2007. 10. Muer-Hermein , HK, Zett, A, Pfeifer, W, et a. Path
gy f
ymphma prgressin. Histpathgy 38(4):285306, 2001. 11. American Cancer Sci
ety. Cancer
Facts and Figures 2008. Atanta: American Cancer Sciety, 2008. Avaiabe nine
at
http://www.cancer.rg/dwnads/STT/2008CAFF nasecure d.pdf. Accessed Juy 10, 20
08. 12.
Chiecchi, L, Prthere, RKM, Ibrahim, AH, et a. Deetin f chrmsme 13 dete
cted by
cnventina cytgenetics is a critica prgnstic factr in myema. Leu emia 2
0:16101617,
2006. 13. Chng, WJ, Santanna-Daviia, R, Van Wier, SA, et a. Prgnstic factrs
fr
hyperdipid-myema: Effects f chrmsme 13 deetins and IgH transcatins.
Leu emia
20:807813, 2006. 14. Owen, RG, Barrans, SL, Richards, SJ, et a. Wadenstrms
macrgbuinemia. Devepment f diagnstic criteria and identificatin f prg
nstic factrs.
Am J Cin Path 116: 420428, 2001. 15. McMaster, M. Famiia Wadenstrms macrgb
uinemia.
Semin Onc 30:146152, 2003. 16. Tren, SP, Hunter, ZR, Aggarwa, A, et a. Chara
cterizatin f
famiia Wadenstrms macrgbuinemia. Annas Onc 17:488494, 2005. 17. Vita, A.
Paraprteinemic neurpathies. Bran Path 11:399407, 2001. 18. Steter-Stevensn,
M, and
Brayan, RC. Fw cytmetric anaysis f ymphmas and ymphpriferative di
srders. Semin

Hemat 38(2):111123, 2001. 19. Guinan, JEC, Kenny, DF, and Gatenby, PA. Detecti
n and typing f
paraprteins: Cmparisn f different methds in a rutine diagnstic abratry
. Pathgy
21:35, 1989. 20. Human Genme Prject. Avaiabe at http://www.rn.gv/
sci/techresurces/Human_Genme/hme.shtm. Accessed Juy 12, 2008. 21. Rsenwad
, A, and Staudt,
LM. Cinica transatin f gene expressin prfiing in ymphmas and eu
emias. Semin
Onc 29(3):258263, 2002. CHAPTER 15 Immunpriferative Diseases 261 1814_Ch15_2
48-261.qxd
7/10/09 3:50 PM Page 261 16 Immunde ciency Diseases Thmas S. Aexander, PhD, D
(ABMLI) and
Maureane Hffman, MD, PhD 262 LEARNING OBJECTIVES After nishing this chapter, the
reader wi be
abe t: 1. Reca the rganizatin and devepment f the ceuar and humra
arms f the
immune system. 2. Expain hw a defect in the B-ce, T-ce, myeid, r cmpe
ment sys- tems
can ead t typica cinica manifestatins. 3. Discuss hw abratry tests can
be used t
diagnse and mnitr the different types f cngenita immunde ciency syndrmes.
4. Distinguish
cmmn variabe immunde ciency frm Brutns X-in ed agammagbuinemia. 5. Discus
s the
manifestatins f the DiGerge anmay, purine-nuceside phsphryase (PNP) de c
iency, severe
cmbined immunde ciency (SCID), Wis tt-Adrich syndrme (WAS), and ataxia-teang
iectasia (AT).
6. Recgnize the assciatin between immunde ciency states and the ris f deve
ping
maignancy. 7. Expain hw the ss f neutrphi functin affects hst defenses
. 8. Describe the
basics f perfrming and interpreting immun xatin eectrphresis KEY TERMS
Ataxia-teangiectasia (AT) Brutns agammagbuinemia Chrnic granumatus disea
se (CGD) Cmmn
variabe immunde ciency (CVI) DiGerge anmay Oxidative burst Purine nuceside
phsphryase
(PNP) de ciency Severe cmbined immunde ciency (SCID) Transient hypgamma- gbuin
emia
Wis tt-Adrich syndrme (WAS) CHAPTER OUTLINE IMMUNODEFICIENCY DISEASES Genera
Observatins
De ciencies f the B-Ce System (Agammagbuinemias) De ciencies f Ceuar Immun
ity Cmbined
De ciencies f Ceuar and Humra Immunity Defects f Neutrphi Functin Cmpe
ment
De ciencies LABORATORY EVALUATION OF IMMUNE DYSFUNCTION Screening Tests Cn rmatry
Tests
Evauatin f Immungbuins Bne Marrw Bipsy SUMMARY CASE STUDIES EXERCISE:
IMMUNOFIXATION
REVIEW QUESTIONS REFERENCES 1814_Ch16_262-277.qxd 7/13/09 2:57 PM Page 262 CH
APTER 16
Immunde ciency Diseases 263 The immune system is a diverse and cmpicated netw
r f many
bichemica and ceuar cmpnents. The antigen- specific, r acquired immun
ity, arm
incudes the T-ce system (ceuar immunity, T ces, and their cyt ines);
and the B-ce
system (humra immunity, B ces, and immungbuins). The innate immunit
y arm incudes

the phagcytic system (mnnucear phagcytes, dendritic ces, and granucytes

); and the
cmpement system. A simpi- ed renditin f the rganizatin f ceuar cmpne
nts f the
immune system is shwn in Figure 161. The rgani- zatin, devepment, and functi
n f the
immune system have been discussed in greater detai in Chapters 2, 4, and 5. Thi
s chapter fcuses
n primary, r inherited, dysfunctins f the immune system. The materia
wi emphasize
the antigen-speci c arm f the immune system and the means by which dysfunctina
states can be
diagnsed and mnitred by the cinica abratry. De ciencies in the innate immu
ne sys- tem
wi be cvered in ess detai. This chapter is nt intended as a cmprehensive
treatment f
abnrmaities f the immune system, but rather as an intrductin t the cnditi
ns mst cmmny
evauated in the cinica abratry. IMMUNODEFICIENCY DISEASES Genera Observat
ins The
cmpnents f the immune system pay unique but verapping res in the hst-de
fense prcess.
Therefre, defects in any ne f the ceuar r sube cmpnents resut in di
stinct cinica
manifestatins. Hwever, because the cmpnents f the immune system interact ex
tensivey thrugh
many reguatry and effectr ps, a defect in ne arm f the system may affect
ther aspects f
immune func- tin as we. In many cases, it appears that de ciency f ne cmpne
nt f the
system is accmpanied by hyperactivity f ther cmpnents. This may be because
persistent infectins cntinuusy stimuate the remaining immune ces, r this is a cmpensat
ry mechanism t
crrect fr the de cient immune functin. In additin, the de cient cmpnent may n
rmay exert
reguatry cntr ver ther cmpnents f the immune systemcntr that is ac
ing in the
de ciency state. Fr instance, as iustrated in Figure 161, T-ce sub- sets each
secrete
cyt ines that reguate the devepment f B ces int pasma ces. A defect i
n T-ce functin
remves r unbaances this reguatry p. Whatever the mechanism, many partia
immundeficiency
states are assciated with aergic r autimmune manifestatins. With the excep
tin f IgA
de ciency, primary immun- de ciency syndrmes are rare, with a cmbined incidence 
f abut 2 in
10,000 ive births. 1,2 Severa f the mst impr- tant immunde ciency syndrmes
shw X-in ed
inheritance and, therefre, affect primariy maes. Others shw auts- ma rec
essive
inheritance. Despite the rarity f primary immundeficiency syndrmes, it i
s imprtant t
cnsider this pssibiity in chidren with recurrent infectins, because eary d
etectin and
treatment can hep prevent the devep- ment f serius, ng-term tissue damage
r verwheming
sepsis. Eary diagnsis can as prvide the pprtunity fr apprpriate
genetic

cunseing, carrier detectin, and prenata diagnsis fr ther famiy members.
3 Mre than 120
different cngenita frms f immund- e ciency have been reprted, incuding defe
cts in ymphid
ces, phagcytic ces, and cmpement prteins. 1 The mecuar mechanism
s have been
determined fr many f these deficiencies. The cinica symptms assciated with
immune
de ciencies range frm very mid r subcinica t severe recurrent infectins r
faiure t
thrive. The types f infectin r symptms can give imprtant cues t the specific
immundeficiency present. In genera, defects in humra immunity (antibdy
prductin)
resut in pygenic bacteria infectins, particuary f the upper and wer res
- piratry
tract. Recurrent sinusitis and titis media are cmmn. The cinica curs
e f infectins
with vira agents is nt signi canty different frm that in nrma hsts, with th
e exceptin f
hepatitis B, which may have a fuminant curse in agammagbuinemic patients. D
efects in T-cemediated immunity resut in recurrent infectins with intraceuar pathg
ens such as
viruses, fungi, and intrace- uar bacteria, and patients with these defects a
mst aways
devep muccutaneus candidiasis. They are as prne t disseminated vira inf
ectins,
especiay with atent viruses such as herpes simpex, varicea zster, and cyt
megavirus. T
ces as pay a crucia re in tumr surveiance. Age- adjusted rates f ma
ignancy in
patients with immunde ciency Hematpietic stem ce Erythrid ineage Mega aryc
ytic ineage
Myemncytic ineage Basphi Esinphi Granucyte Macrphage Mncyte Pre-B
ymphcyte B
ymphcyte Pasma ce Lymphid stem ce Antibdy Cyt ines Heper T ce Reg
uatry T ce
Cyttxic T ce FIGURE 161. Devepmenta reatinships between ceuar cm- p
nents f the
immune system. The etters indicate the stages at which a defect ccurs in sme
f the cngenita
immunde ciency syndrmes. A, adensine deaminase de ciency; B, Brutns agamma- gbu
inemia; C,
cmmn variabe immunde ciency; D, DiGerge anmay; and P, PNP de ciency. 1814_Ch1
6_262-277.qxd
7/13/09 2:57 PM Page 263 264 SECTION 3 Immune Disrders disease are 10 t 200
times greater
than the expected rates. 1 Mst f the maignancies are ymphid and may be rea
ted t persistent
stimuatin f the remaining immune ces, cuped with defective immune reguat
in. The
phagcytic system is part f the innate, antigen- nnspeci c immune respnse and i
ncudes
neutrphis and mnnucear phagcytes. Neutrphis are the rst ine f defense a
gainst invading
rganisms and are imprtant effec- tr ces in antibdy-mediated iing. Defec
ts in neutrphi
functin are usuay re ected in recurrent pygenic bacte- ria infectins r impa
ired wund

heaing. Macrphages within the iver and speen are in cntact with the bd a
nd are
respnsibe fr cearing circuating micrrganisms. Spenectmy is assciated w
ith an increased
ris f ver- wheming bacteria infectin accmpanied by septicemia. Macrphage
s are present
in a tissues and as pay an imprtant re in prcessing and presentin
g antigens t T
ces in cmbinatin with cass II majr histcmpatibi- ity cmpex (MHC) me
cues t initiate
a speci c immune respnse. The cmpement system, as discussed in Chapter 6, is ac
tivated t
prduce bigicay active mecues that enhance infammatin and prm
te ysis f
ces and micrrganisms. Deficiency f cmpement cmpnents resuts in recurr
ent bacteria
infectins and autimmune- type manifestatins. The severity f the syndrme var
ies with the
particuar cmpement cmpnent that is de cient. Speci c defects f each cmpnent
f the immune
system are described in the fwing sectins. De ciencies f the B-Ce System
(Agammagbuinemias) Immungbuins migrate in the gamma regin of the serum pro
tein
electrophoretic pro le (discussed in Chapter 4). Therefore, de ciencies of immu
noglobulins
have been termed agammaglobulinemias. The mechanisms of the agammaglob ulinemia
s include genetic
defects in Bcell maturation or mutations leading to defective interactions betw
een B and T
cells. A wide range of immunoglobulin de ciency states have been reported and invo
lve virtually
all combinations of immunoglobulins and all degrees of severity. In some cases,
only a single
isotype of one immunoglobulin class is de cient, while all the other isotypes are
normal. Only
the more com mon and wellcharacterized syndromes are described here and are su
mmarized in Table
161. In evaluating immunoglobulin deficiency states, it is important to rem
ember that
blood levels of immunoglob ulins change with age. The blood level of IgG at bir
th is about the
same as the adult level, reflecting transfer of maternal IgG across the p
lacenta. The IgG
level drops over the rst 2 or 3 months of life as maternal antibody is catab oli
zed. Levels of
IgA and IgM are very low at birth. The concentrations of all immunoglobulins gra
dually rise when
the infant begins to produce antibodies at a few months of age, in response to e
nvironmental
stimuli. IgM reaches nor mal adult levels rst, around 1 year of age, followed by
IgG at about 5
to 6 years of age. In some normal children, IgA levels do not reach normal adult
values until
adolescence. Therefore, it is important to compare a childs immunoglob ulin leve
ls to
agematched reference ranges. Transient Hypogammaglobulinemia of Infancy All inf
ants experience
low levels of immunoglobulins at approximately 5 to 6 months of age, but in some
babies, the low
levels persist for a longer time. Because these children do not begin synthesizi

ng immunoglobulin
promptly, they can experience severe pyogenic sinopulmonary and skin infec tion
s as protective
maternal IgG is cleared. Cellmediated immunity is normal, and there may be norm
al levels of IgA
and IgM. 4 IgG appears to be the most affected, dropping to at least two standar
d deviations (SD)
below the age adjusted mean with or without a depression of IgM and IgA. 4 Immu
noglobulin levels
usually normalize spontaneously, often by 9 to 15 months of age. The mechanism o
f this transient
hypogammaglobulinemia is not known. These Table 161. Characteristics of Selecte
d Defects of the
BCell System CONDITION DEFICIENCY LEVEL OF DEFECT PRESENTATION Transient
All antibodies,
especially IgG Slow development of helper 26 months; resolves by hypogammaglobul
inemia
function in some patients 2 years of infancy IgA de ciency IgA; some with reduced
IgAB cell
differentiation Often asymptomatic IgG2 also Xlinked agammaglobulinemia All ant
ibody isotypes
reduced PreB cell differentiation Infancy Common variable Reduced antibody; man
y B cell; excess
T suppression Usually 2030 years of age immunode ciency different combinations Isol
ated IgG
subclass Reduced IgG1, IgG2, IgG3, Defect of isotype differentiation Variable
with the class
and de ciency or IgG4 degree of de ciency Immunode ciency with Reduced IgG, IgA, IgE,
Bcell
switching Infancy hyperimmunoglobulin M with elevated IgM 1814_Ch16_262277.qxd
7/13/09 2:57 PM
Page 264 CHAPTER 16 Immunode ciency Diseases 265 patients have normal numbers o
f circulating
CD19 positive B cells. This condition does not appear to be Xlinked, although i
t is more common
in males. The cause may be related to a delayed maturation of one or more compon
ents of the
immune system, possibly T helper cells. 4 XLinked Brutons Agammaglobulinemia Bru
tons
agammaglobulinemia, rst described in 1952, is X chromosomelinked, so this synd
rome affects
males almost exclusively. Patients with Xlinked agammaglobu linemia lack circ
ulating mature
CD19 positive B cells and exhibit a deficiency or lack of immunoglobulins
of all classes.
4,5 Furthermore, they have no plasma cells in their lymphoid tissues, but they d
o have preB
cells in their bone marrow. 6 Because of the lack of B cells, the tonsils and ad
e noids are
small or entirely absent, and lymph nodes lack normal germinal centers. T cells
are normal in
number and function. About half of the patients have a family history of the syn
drome. They
develop recurrent bacterial infections beginning in infancy, as maternal antibod
y is cleared.
They most commonly develop sinopulmonary infections caused by encapsulated organ
isms such as
streptococci, meningo cocci, and Haemophilus influenzae. Other infections seen
include bacterial
otitis media, bronchitis, pneumonia, menin gitis, and dermatitis. 4 Some patien

ts also have a
susceptibility to certain types of viral infections, including vaccineassociate
d poliomyelitis.
In general, live virus vaccines should be avoided in immunode cient patients
. Xlinked
hypogammaglobulinemia results from arrested differentiation at the preB cell st
age, leading to a
complete absence of B cells and plasma cells. The underlying genetic mechanism i
s a deficiency of
an enzyme called the Bruton tyrosine kinase (Btk) in Bcell progenitor cells. 6
Lack of the
enzyme apparently causes a failure of Vh gene rearrange ment. As discussed in C
hapter 4, during
Bcell maturation, there is a rearrangement of V, D, and J segments to create fu
nctioning genes
that code for immunoglobulin heavy and light chains. The syndrome can be effecti
vely treated by
administering intramuscular or intravenous immunoglob ulin preparations and vig
orous
antimicrobial treatment of infections. The syndrome can be differentiated from t
ran sient
hypogammaglobulinemia of infancy by the absence of CD19 positive B cells in the
peripheral blood,
by the abnor mal histology of lymphoid tissues, and by its persistence beyond 2
years of age.
Recently, it has been found that patients with a similar clinical presentation t
o Brutons appear
to have a genetic defect that is inherited in an autosomal recessive manner. 4,7
IgA De ciency
Selective IgA deficiency is the most common congenital immunode ciency, occurring
in about 1 in
500 persons of AmericanEuropean descent. 4 Most patients with a defi ciency o
f IgA are
asymptomatic. Those with symptoms usually have infections of the respiratory
and
gastrointesti nal tract and an increased tendency to autoimmune diseases such a
s systemic lupus
erythematosus, rheumatoid arthritis, celiac disease, and thyroiditis. Allergic d
isorders and
malig nancy are also more common. 5 About 20 percent of the IgAde cient patients
who develop
infections also have an IgG2 subclass deficiency. If the serum IgA is le
ss than 5 mg/mL,
the deficiency is considered severe. If the IgA level is two standard deviations
below the
ageadjusted mean but greater than 5 mg/mL, the de ciency is partial. Although the
genetic defect
has not been established, it is hypothe sized that lack of IgA is caused by imp
aired
differentiation of lymphocytes to become IgAproducing plasma cells. 4 AntiIgA
antibodies are
produced by 30 to 40 percent of patients with severe IgA deficiency. These antib
odies can cause
anaphylactic reactions when blood products contain ing IgA are transfused. 5 Be
cause many
patients with severe IgA de ciency have no other symptoms, the IgA de ciency may not
be detected
until the patient experiences a transfu sion reaction resulting from the p
resence of
antiIgA antibodies. Products for transfusion to known IgAde cient patients shoul

d be collected
from IgAde cient donors, or cellular products should be washed to remove a
s much donor
plasma as possible. Most gamma globulin preparations contain significant amounts
of IgA. However,
replacement IgA therapy is not useful, because the halflife of IgA is short (ar
ound 7 days), and
intravenously or intramuscularly administered IgA is not transported to its norm
al site of
secretion at mucosal surfaces. Furthermore, administration of IgAcontaining pro
ducts can induce
the development of antiIgA antibodies or provoke anaphylaxis in patients w
ho already have
antibodies. Common Variable Immunode ciency Common variable immunode ciency (CVI) is
a hetero
geneous group of disorders with a prevalence of about 1 in 25,000. 8 While this
is a low
incidence, it does make CVI the most common primary immune deficiency with a sev
ere clinical
syndrome. 5 Patients usually begin to have symptoms in their 20s and 30s, b
ut age at
onset ranges from 7 to 71 years of age. The disorder can be congenital or ac
quired, familial
or sporadic, and it occurs with equal frequency in men and women. CVI is charact
erized by
hypogammaglob ulinemia that leads to recurrent bacterial infections, part
icularly
sinusitis and pneumonia. In addition, up to 20 percent of CVI patients develop h
erpes zoster
(shingles), a much higher incidence than in immunologically normal young adults.
There is usually
a de ciency of both IgA and IgG, but selective IgG de ciency may occur. CVI is often
associated
with a spruelike syndrome characterized by malabsorption and diarrhea. CVI
is also
associated with an increased risk of lymphoproliferative disorders, gastric
carcinomas, and
autoimmune disorders. 5 The most com mon autoimmune manifestations of CVI
are immune
thrombocytopenia and autoimmune hemolytic anemia. Other symptoms may include
lymphadenopathy,
spleno megaly, and intestinal hyperplasia. 5 CVI is diagnosed when patients wit
h recurrent
bacterial infections demonstrate a low serum IgG level. Additionally, 1814_Ch16_
262277.qxd
7/13/09 2:57 PM Page 265 266 SECTION 3 Immune Disorders blood group isohemaggl
utinins, or
naturally occurring anti bodies, are typically absent or low. In contrast to X
linked
agammaglobulinemia, most patients with CVI have normal numbers of mature B cells
. However, these
B cells do not dif ferentiate normally into immunoglobulinproducing plasma cel
ls. Three major
types of cellular defects have been found in CVI patients. In some cases, T cell
s or their
products appear to suppress differentiation of B cells into plasma cells. Second
ly, T cells may
fail to provide adequate help to sup port terminal differentiation of B cells.
Finally, there
appears to be a primary defect in the Bcell line in some patients. CVI is often

a diagnosis of
exclusion, where an immunode ciency is present with no speci c genetic defect de ned.
7 CVI can
usually be effectively treated with intramuscu lar or intravenous immunoglobuli
n preparations. 9
However, because of their low levels of secretory IgA, patients are still suscep
tible to
respiratory and gastrointestinal infections, and the clinician should be vigilan
t for these
infections and treat them vigorously with antibiotics. Isolated IgG Subclass De ci
ency There are
four subclasses of IgG in the human. IgG subclass de ciencies, initially d
escribed in
1970, are condi tions where the level of one or more of the IgG subclasses is
more than two
standard deviations below the mean ageappropriate level. 4 About 70 percent
of the total IgG
is normally IgG1, 20 percent IgG2, 6 percent IgG3, and 4 percent IgG4. Therefore
, a de ciency of
a single subclass may not result in a total IgG level below the normal range. In
patients with
recurrent infections, levels of the different subclasses should be measured
if the total
IgG level is normal but the clinical picture suggests immunoglobulin de ciency.
4 Most IgG
antibodies directed against protein antigens are of the IgG1 and IgG3 subclasses
, while most IgG
anti bodies against carbohydrate antigens are IgG2 or IgG4. Thus, de ciencies inv
olving IgG1 or
IgG3 lead to a reduced response capability to protein antigens such as toxins, w
hile selective
deficiencies of IgG2 can result in impaired responses to polysaccharide an
tigens, which
cause recurrent infections with polysaccharideencapsulated bacteria such as Str
eptococcus
pneumoniae and Haemophilus influenzae. 4 A variety of genetic defects have been
associated with
IgG sub class de ciency. These include heavy chain gene deletions and transcripti
onal defects.
The most common subclass de  ciency is IgG4, with IgG1 deficiency being th
e least
common, although IgG4 subclass de ciency may have the least clinical signi cance. 4
De ciencies
of Cellular Immunity Defects in cellmediated immunity can result from abnor ma
lities at many
different stages of Tcell development. Many different molecular defects can res
ult in a similar
clin ical picture (as in severe combined immunodeficiency [SCID]). In som
e cases, a
primary defect in cellmediated immunity can also have secondary effects on humo
ral immu nity.
This is because T cells provide helper functions that are necessary for normal B
cell development
and differen tiation. Some of the more common defects of cellular and combined
cellular and
humoral immunity are summarized in Table 162. In general, defects in cellular imm
unity are more
dif  cult to manage than defects in humoral immunity. When immunoglobulin product
ion is
de cient, replacement ther apy is often very effective. However, there is usually

no Table 162.
Characteristics of Selected Defects of the TCell System and Combined Defects CO
NDITION
DEFICIENCY LEVEL OF DEFECT PRESENTATION DiGeorge anomaly T cells; some secon
dary effects
Embryological development of Neonatal, with hypocalcemia on antibody production
the thymus or
cardiac defects if severe; incomplete forms may present later with infection PNP
de ciency T
cells; some secondary effects PNP, purine metabolism Infancy on antibody produc
tion SCID Both T
and B cells ADA, purine metabolism; Infancy HLA expression; RAG1/2; JAK3; commo
n gamma chain
receptor WAS Reduced IgM and Tcell defect CD43 expression Usually infancy; mild
variants occur
AT Reduced IgG2, IgA, IgE, and DNA instability Infancy T lymphocytes Reticular d
ysgenesis All
leukocytes Stem cell defect Neonatal PNP
purine nucleoside phosphorylase; SCID
s
evere
combined immunode ciency; ADA
adenosine deaminase; HLA human leukocyte antigen; WA
S
WiskottAldrich syndrome; AT
ataxiatelangiectasia 1814_Ch16_262277.qxd 7/13/0
9 2:57 PM
Page 266 CHAPTER 16 Immunode ciency Diseases 267 soluble product that can be adm
inistered to
treat a de ciency of cellmediated immunity. Transplantation of immunolog ically
intact cells,
usually in the form of allogenic bone marrow transplantation, is often re
quired to
reconstitute immune function. Patients with severe defects in cellmediated immu
nity may develop
graftversushost (GvH) disease. Transfused lymphocytes are normally destroye
d by the
recipients Tcell system. However, a severe defect in the Tcell sys tem allows
the donor
lymphocytes to survive, proliferate, and attack the recipients tissues as foreign
. GvH disease
can occur in any individual with a severe defect in cellmediated immunity (e.g.
, in bone marrow
transplant recipients) and can be fatal. Irradiation of cellcontaining blood pr
oducts (platelet
concentrates, packed red blood cells, and whole blood) before transfusion destro
ys the ability of
the donor lymphocytes to proliferate and prevents development of GvH disease in
immunodeficient
recipients. It should be noted that a defect in humoral immunity does not predis
 pose one to GvH
disease. GvH disease also occurs in patients who have received a bone marrow
transplant as
therapy for a congenital immune deficiency. The closer the match betwee
n the genetic
constitution of the patient and the graft donor, the less severe the GvH disease
is likely to be.
Thus, although bone marrow transplantation can potentially cure the immune
defect, it can
also have serious, lifelong complica tions of its own. DiGeorge Anomaly DiGeorg
e anomaly is a
developmental abnormality of the third and fourth pharyngeal pouches that affect
s thymic
development. Speci cally, most patients show a deletion in chromosome 22, region q

11, 10 although
this anomaly is not required for diagnosis. 5 All organs derived from thes
e embryonic
structures can be affected. Associated abnormal ities include mental retardatio
n, absence of
ossi cation of the hyoid bone, cardiac anomalies, abnormal facial devel opment, a
nd thymic
hypoplasia. 5 The severity and extent of the developmental defect can be qu
ite variable.
Many patients with a partial DiGeorge anomaly have only a min imal thymic def
ect and are
thus near normal immune function. 5 However, about 20 percent of children wit
h a defect of the
third and fourth pharyngeal pouches have a severe and persistent decrease in Tc
ell numbers. 11
These children tend to have severe, recurrent viral and fungal infec tions. Se
verely affected
children usually present in the neonatal period with tetany (caused by hypoc
alcemia result
ing from hypoparathyroidism) or manifestations of cardiac defects. The possibili
ty of immune
de ciency can be over looked if the association between the presenting abnormalit
y and a
possible thymic defect is not recognized. The immunodeficiency associated with t
he DiGeorge
anomaly is a quantitative defect in thymocytes. Not enough mature T cells are ma
de, but those
that are present are func tionally normal. The immunodeficiency of DiGeorg
e syndrome can be
treated with fetal thymus transplantation. Bone marrow transplantation has also
been successful
in some patients, as has administration of thymic hormones. Purine Nucleoside Ph
osphorylase
De ciency One immunode ciency state for which a speci c enzymatic basis has been defin
ed is
purine nucleoside phosphory lase (PNP) de ciency. PNP de ciency is a rare autosomal
recessive
trait. 11 The condition presents in infancy with recurrent or chronic pulmonary
infections, oral
or cuta neous candidiasis, diarrhea, skin infections, urinary tract infections,
and failure to
thrive. PNP de ciency affects an enzyme involved in the metabolism of purines. It
produces a
moderate to severe defect in cellmediated immunity, with normal or only mildly
impaired humoral
immunity. 11 The number of T cells progressively decreases because of the accumu
lation of
deoxyguanosine triphosphate, a toxic purine metabolite. The levels of immunoglob
ulins are
generally normal or increased. About twothirds of PNPdeficient patients also h
ave neurological
disorders, but no character istic physical abnormalities have been described. B
ecause of the
relatively selective defect in cellmediated immunity, PNP de ciency can be confus
ed with
neonatal HIV infection. The two conditions can usually be distinguished by speci c
tests for
antiHIV antibody (if the infant is old enough to be pro ducing antibody) and b
y assays for PNP
activity. Combined De ciencies of Cellular and Humoral Immunity Defects in both hu

moral (B cell)
and cellmediated (T cell) immunity can be caused by a defect that affects devel
op ment of both
types of lymphocytes or a defective interaction between the two limbs of th
e immune
system. Because helper T cell functions are necessary for normal differenti at
ion and antibody
secretion by B cells, a severe defect of Tcell function will affect immunoglobu
lin levels as
well. Combined de ciencies are referred to using a shorthand of T
B
NK
with the
superscript
denoting whether or not each cell type is present in the de ciency. 5 S
evere
Combined Immunode ciency The most serious of the congenital immune de ciencies is se
vere combined
immunode ciency (SCID). It is actually a group of related diseases that all affect
T and Bcell
func tion but with differing causes. Xlinked SCID is the most common form of t
he disease,
accounting for approximately 46 percent of the cases in the United States today.
2,12 This occurs
with a frequency of about 1 in 50,000 births. 13 The abnormal gene involved code
s for a protein
chain called the common gamma chain, which is common to receptors for inter leu
kins2, 4, 7, 9,
15, and 21. 12 The gene is referred to as the IL2RG gene and is located on the X
chromosome.
Normal signaling cannot occur in cells with defective receptors, thus halting na
tural maturation.
2,13 This may result in either a 1814_Ch16_262277.qxd 7/13/09 2:57 PM Page 2
67 268 SECTION 3
Immune Disorders T
B
NK
or a T B
NK
phenotype, depending on whether or not there
is
an additional defect in the JAK3 gene. 5 JAK3 is required for processing an inte
rleukin binding
signal from the cell membrane to the nucleus. In such cases, no antibody product
ion or lymphocyte
proliferative response follows an antigen or mitogen challenge. A JAK3 deficienc
y may be found
without the common gamma chain deletion. This results in an autosomal reces siv
e form of SCID,
affecting both males and females. 5 The lack of the intracellular kinase JAK3 me
ans that lympho
cytes are unable to transmit signals from interleukins2 and 4. 2,11 These patie
nts have a T
B
NK phenotype, and symptoms are similar to the Xlinked form of the disease. 5 Th
e JAK3 gene
is located on chromosome 19, region p12. About 15 to 20 percent of the patients
with SCID have an
adenosine deaminase (ADA) deficiency, leading to a T
B
NK phenotype. 5 Th
e ADA gene
is located on chro mosome 1, region q21. Like PNP de ciency, ADA de ciency affects
an enzyme
involved in the metabolism of purines. In ADA de ciency, toxic metabolites of puri
nes accumulate
in lymphoid cells and impair proliferation of both B and T cells. As in PNP de cie
ncy, there is a
progressive decrease in lym phocyte numbers. Several different mutations have b
een found to lead
to ADA de ciency, and the degree of immun odeficiency correlates with the degree
of ADA

deficiency. Patients with only mildly reduced ADA activity may have only a sligh
t impairment of
immune function. 5 Other molecular defects have been identi ed as causes of SCID.
Infants with a
lack of both T and B cells but with func tioning NK cells were found to h
ave a mutation
in a recombinase activating gene (RAG1 or RAG2). 12 These muta tions cause a
profound
lymphocytopenia because of the inability of T and B cells to rearrange the DN
A necessary to
produce functional immunoglobulins or Tcell receptors. 11 This condition has be
en referred to as
Ommens syndrome. 6 Defective expression of human leukocyte antigen (HLA) cl
ass II
antigens, normally found on B cells, leads to bare lym phocyte syndrome. HLA cl
ass II molecules
are intimately involved in antigen presentation; thus, this defect profoundly im
pairs the immune
response. 5,12 A newly identi ed molecu lar defect is a mutation in the ge
ne encoding a
common leukocyte protein called CD45. It is a transmembrane phos phatase that
regulates signal
transduction of T and Bcell receptors. 11,12 Patients with SCID generally pres
ent early in
infancy with infection by nearly any type of organism. Oral candi dal infectio
ns, pneumonia,
and diarrhea are the most common manifestations. The receipt of live vaccine
s can cause
severe illness. Unless immune reconstitution can be achieved by bone marrow tran
splantation or by
speci cally replacing a de cient enzyme, patients with SCID die before they reach 2
years of age.
12 ADA de ciency is a special case, because it presents a good opportunity for enz
yme replacement
therapy or somatic cell gene therapy. Although the ADA is normally located withi
n cells, its
de ciency can be treated by maintaining high plasma levels of ADA. Red blood cell
transfusion has
been used in some patients to raise ADA to near normal levels. However, bovine A
DA conjugated
with polyethylene glycol (PEG) has a longer halflife than native ADA and can be
used to raise
the ADA level up to three times higher than normal. This treatment increases Tc
ell production
and speci c antibody responses. Side effects of ADAPEG administration appear to be
minimal.
However, this therapy is very expensive (about $250,000/year) and is primarily u
sed in patients
for whom a suitable bone marrow donor cannot be found or for those who are too
sick to
undergo marrow transplantation. 14 Human cord blood stem cell transplantation
has been used
with moderate success. 15 Studies are currently under way to attempt to treat AD
A deficiency by
transfecting a normal ADA gene into patients T cells or stem cells. These cells c
ould then be
reinfused into the patient. Theoretically, the gene therapy approach could be cu
rative, but there
are many obstacles to overcome. WiskottAldrich Syndrome WiskottAldrich syndro
me (WAS) is a

rare Xlinked recessive syndrome that probably affects six patients a year in t
he United States.
16 It is de ned by the triad of immun ode ciency, eczema, and thrombocytopenia. WAS
is usually
lethal in childhood because of infection, hemorrhage, or malignancy. Milder vari
ants have also
been described, such as an Xlinked form of thrombocytopenia. The laboratory fea
tures of WAS
include a decrease in platelet number and size with a prolonged bleeding time. 5
The bone marrow
contains a normal or somewhat increased number of megakaryocytes. There are abno
rmalities in both
the cellular and humoral arms of the immune system, related to a general defect
in antigen
processing. This is manifest as a severe de ciency of the naturally occurring anti
bodies to
blood group antigens (isohemagglutinins). Patients with WAS can have a
variety of
different patterns of immunoglobulin levels, but they usually have low levels
of IgM, normal
levels of IgA and IgG, and increased levels of IgE. 5 Absence of isohemagglut
inins (IgM
antibodies against ABO blood group antigens) is the most consistent laboratory nd
ing in WAS and
is often used diagnostically. In addition, these patients have persistently
increased
levels of serum alphafetoprotein, which can also be a use ful diagnostic featu
re. The primary
molecular defect in the syndrome appears to be an abnormality of the integral me
mbrane protein
CD43, which is involved in the regulation of protein glycosylation. 6 The gene r
esponsible for
the defect is called the WASp gene, and it is located on the X chromosome, regio
n p11.
Abnormalities cause defective actin polymerization and affect its signal tr
ansduction in
lymphocytes and platelets. 6 Platelets have a shortened halflife, and T lymphoc
ytes are also
affected, although B lymphocytes appear to function normally. Splenectomy can be
very valuable in
controlling the thrombocytopenia. Current treatment for this immun ode ciency is
bone marrow
transplantation or cord blood stem cells from an HLAidentical sibling. 9 1814_C
h16_262277.qxd
7/13/09 2:57 PM Page 268 CHAPTER 16 Immunode ciency Diseases 269 AtaxiaTelang
iectasia
Ataxiatelangiectasia (AT) is a rare autosomal recessive syndrome characterized
by cerebellar
ataxia and telangiec tasias, especially on the earlobes and conjunctiva. Blood
vessels in the
sclera of the eyes may be dilated, and there may also be a reddish butterfly are
a on the face and
ears. Ninetyfive percent of patients exhibit increased levels of serum alphafe
toprotein. 11 The
incidence is between 1 in 10,000 to 1 in 100,000, although as much as 1 percent
of the population
is heterozygous for the gene. 17 Abnormal genes produce a combined defect of bot
h humoral and
cel lular immunity. 5,17 Antibody response to antigens, especially polysacchari
des, is blunted.

The levels of IgG2, IgA, and IgE are often low or absent, although the pattern c
an be quite
variable. In addition, the number of circulating T cells is often decreased. Dea
th usually occurs
in early adult life from either pulmonary disease or malignancy. 2 Patients with
AT have a defect
in a gene that is appar ently essential to the recombination process for genes
in the
immunoglobulin superfamily. The AT gene is located on chromosome 11, region q22.
This abnormality
results in a defective kinase involved in DNA repair and in cell cycle control.
13 Rearrangement
of Tcell receptor and immunoglobulin genes does not occur normally. 11 Pati
ents lymphocytes
often exhibit chromosomal breaks and other abnormalities involving the Tcell re
ceptor genes in T
cells and immunoglobulin genes in B cells. These are sites of high levels of chr
omosomal
recombination, and errors that occur during gene rearrangements may not be repai
red properly. The
syndrome is associated with an even greater risk of lymphoid malignancy than oth
er
immunode ciency syndromes, presumably because the failure to properly repair DNA d
amage leads to
accumulation of mutations. The only effective therapy for AT is allogenic
bone marrow
transplantation. Defects of Neutrophil Function Neutrophils play a crucial role
in the immediate
and non speci c response to invading organisms by reacting before speci c antibody
and
cellmediated immune responses can be mounted. In addition, neutrophils are even
more effec tive
at ingesting and killing organisms coated with speci c antibody and thus continue
to play an
important role in host defense even after a speci c immune response is established
. To destroy
invading organisms, neutrophils must adhere to vascular endothelial lining cells
, migrate through
the capil lary wall to a site of infection, and ingest and kill the
microbes. Defects
affecting each of these steps have been described, each leading to an increased
susceptibility to
pyo genic infections. Chronic Granulomatous Disease Chronic granulomatous disea
se (CGD) is a
group of disor ders inherited as either an Xlinked or autosomal recessive gene
that affects
neutrophil microbicidal function. The Xlinked disease accounts for 70 percen
t of the cases,
and it tends to be more severe. 5 Symptoms of CGD include recur rent suppurativ
e infections,
pneumonia, osteomyelitis, draining adenopathy, liver abscesses, dermatitis, and
hypergammaglob
ulinemia. Typically, catalasepositive organisms such as Staphylococcus aur
eus, Burkholderia
cepacia, and Chromobacterium violaceumare involved, in addition to fungi such as
Aspergillus and
Nocardia. 5,17 Infections usually begin before 1 year of age, and the syndrome i
s often fatal in
childhood. CGD is the most common and best characterized of the neutrophil abnor
malities. Several

speci c molecular defects have been described in this syndrome, all of which resul
t in the
inability of the patients neutrophils to produce the reac tive forms of oxygen n
ecessary for
normal bacterial killing. There are three different autosomal recessive ge
nes involved, and
all of these affect subunits of nicotinamide adenine dinucleotide phosphate (
NADPH) oxidase.
18 Normally, neutrophil stimulation leads to the production of reactive oxygen m
olecules, such as
hydrogen peroxide (H 2 O 2 ), by NADPH oxidase on the plasma membrane. The plasm
a membrane
enfolds an organism as it is phagocytized, and hydrogen peroxide is generated in
close proximity
to the target microbe. Neutrophil granules fuse with, and release their
contents into,
the forming phagosome. Hydrogen peroxide is then used by the granule enzyme my
eloperoxidase to
generate the potent microbicidal agent hypochlorous acid. 18 The process of gene
rating partially
reduced forms of oxygen by stimulated neutrophils was first detected as an incre
ase in oxygen
consumption. Therefore, this response was originally termed the neutrophil respir
atory burst. A
more correct term is oxidative burst. A genetic defect in any of the several com
ponents of the
NADPH oxidase sys tem can result in the CGD phenotype by making the ne
utrophil
incapable of generating an oxidative burst. CGD was historically diagnosed by me
asuring the abil
ity of a patients neutrophils to reduce the dye nitroblue tetrazolium (NBT). NBT
reduction is
caused by the pro duction of hydrogen peroxide and other reactive forms of oxyg
en. Reduction
converts the nearly colorless NBT into a blue precipitate that can be assessed v
isually on a
micro scope slide. 18 More recently, a ow cytometric assay has been used. In thi
s assay,
neutrophils are labeled with dihydrorho damine (DHR). DHR will uoresce when it i
s reduced. The
neutrophils are then activated using phorbol myristate acetate (PMA), whic
h is mitogenic
for neutrophils. The resultant oxidative burst will reduce the DHR, resulting in
uorescence that
may be quantitated on a ow cytometer. Neutrophils from CGD patients will be unabl
e to undergo
the oxidative burst and will show less uorescence than nor mal neutrophils. 18 T
his technique
is more objective and quantitative than the traditional NBT technique. Although
therapy with
granulocyte transfusions may resolve an acute infectious episode, it is imposs
ible to provide
enough granulocytes to treat the condition on a chronic basis. Administration of
cytokines, such
as interferon, may increase the oxidative burst activity in some patients. Conti
nuous use
1814_Ch16_262277.qxd 7/13/09 2:57 PM Page 269 270 SECTION 3 Immune Disorders
of antibiotics
can greatly reduce the occurrence of severe infections. 13 Bone marrow transplan
tation or use of

peripheral blood stem cells may result in a permanent cure. 18 Other Microbicida
l Defects Several
other recognized defects can result in impaired neu trophil microbicidal activi
ty. Neutrophil
glucose6phosphate dehydrogenase de ciency leads to an inability to generate enou
gh NADPH to
supply reducing equivalents to the NADPH oxidase system. This leads to a def
ect in hydrogen
peroxide production and a clinical picture similar to that of CGD. Myeloperoxida
se de ciency is
relatively common, occur ring in about 1 in 3000 persons in the United States.
De cient patients
may have recurrent candidal infections. Defects of neutrophil secondary granul
es have also
been described. However, the molecular nature of the defects is unknown. 9 Leuk
ocyte Adhesion
De ciency Even if microbicidal activity is normal, neutrophils cannot perform thei
r functions
properly if they fail to leave the vas culature and migrate to a site of incipi
ent infection.
Adhesion receptors on leukocytes and their counterreceptors on endothelia
l cells and
extracellular matrix play important roles in these activities. In leukocyte adhe
sion de ciency
(LAD), a protein (CD18) that is a component of adhesion receptors on neutrophils
and monocytes
(with CD11b or CD11c) and on T cells (with CD11a) is defective. 5,18 The CD18 de c
iency is
transmitted with autosomal recessive inheritance and has variable expression. Th
is defect leads
to abnormal adhesion, motility, aggregation, chemotaxis, and endocytosis by the
affected
leukocytes. The defects are clinically manifested as delayed wound healing, chro
nic skin
infections, intestinal and respiratory tract infections, and periodontitis. A de
fect in CD18 can
be diagnosed by detecting a decreased amount of the CD11/18 antigen on patient l
eukocytes by ow
cytometry. 18 The CD11/18 protein is not the only neutrophil mole cule involve
d in adhesion,
motility, and phagocytosis. Recently, another type of adhesion molecule defici
ency (LAD II) has
been characterized. A carbohydrate molecule involved in adhesive interactions, C
D15s, or sialyl
Lewis X, was found to be de cient. 18 Complement De ciencies Complement consists of
a series of
proteins that work in a cascade to assist in antibody destruction of cells, as d
escribed in
Chapter 6. The complement system is also part of the innate immune system and ca
n work as part of
the in am matory system to directly eliminate a potential pathogen. De ciencies in
each of the
major complement components have been described, leading to various clinical seq
uelae. 19
De ciencies in the early complement components, C1q, 4, and 2, are usually associa
ted with a
lupuslike syndrome. De ciency of C2 is believed to be the most common comple ment
component
de ciency. A C3 de ciency may also have a lupuslike clinical presentation but is mor
e likely to

involve recurrent encapsulated organism infection. De ciencies of the later compon

ents of
complement (C5 through C9) are often associated with recurrent Neisserial infect
ions. A de ciency
of C1 esterase inhibitor has been found in patients with hered itary angioedema
. Most complement
de ciencies appear to be inherited in an autosomal recessive manner and are likely
due to
mutations as opposed to genetic deletions. 19 LABORATORY EVALUATION OF IMMUNE DY
SFUNCTION It is
important in performing diagnostic testing for immun ode ciency that the results
for a patient
be compared with appropriate agematched controls. In tests of cellular func ti
on, the patients
cells need to be tested in parallel with cells from a normal control. If an abno
rmal test result
is obtained, it should be con rmed by repeat testing. Screening Tests Screening te
sts used for
the initial evaluation of suspected immunodeficiency states are summarized in Ta
ble 163. Most of
these tests can be performed routinely in any hospi tal laboratory. The evaluat
ion of possible
immunode ciency starts with a complete blood count and white blood cell d
ifferential,
which may reveal a reduced lymphocyte count. Thrombocytopenia with small plat
elets can be
detected in WAS. Measurement of the levels of serum IgG, IgM, and IgA and levels
of the
subclasses of IgG are used to screen for defects in antibody production. Assay f
or
isohemagglutinins is easily performed by the transfusion service. By the age of
2, a child should
have naturally occurring IgM antibodies Table 552 Comparison of Tests Used SUSP
ECTED DISORDER
TESTS All immunode ciencies Complete blood cell count, white Humoral immunity bloo
d cell
differential count Serum IgG, IgA, IgM levels, IgG subclass levels, isohemagglu
tinin titers
(IgM), IgG antibody response to protein and polysaccharide antigens Cellmediate
d immunity
Delayed hypersensitivity skin tests (i.e., candida, diphtheria, tetanus, PPD) Ch
est Xray (thymus
shadow) Phagocyte defect NBT test IgE level (hyperIgE syndrome) Complement CH 5
0 (classical
pathway) Serum C3 level PPD
Puri ed protein derivative; NBT
nitroblue tetrazolium.
Table
163. Screening Tests for Immunode ciencies 1814_Ch16_262277.qxd 7/13/09 2:57 P
M Page 270
CHAPTER 16 Immunode ciency Diseases 271 against ABO blood group antigens. The ab
sence of these
antibodies suggests an abnormal IgM response. An overall assessment of antibody
mediated immu
nity can be made by measuring antibody responses to antigens to which the popula
tion is exposed
normally or following vaccination. This can be easily done by meas uring the
titer of the
specific antibody produced in response to immunization with a commercial
vaccine such as
diphtheria/tetanus. In an unimmunized child, the development of tetanus or dipht
heria antibodies

is deter mined 2 weeks after immunization. In a previously immunized pat

ient, the
response to a booster injection can be evaluated, normally 4 to 6 weeks postvacc
ination. A wide
range of other protein and polysaccharide anti gens can also be used in these t
ests. This
technique is often used to evaluate a possible IgG subclass deficiency. IgG1 and
3 isotypes
normally respond to protein anti gens, such as tetanus and diphtheria. IgG
2 normally
responds to polysaccharide antigens, such as those in the Haemophilus influenza
and Streptococcus
pneumoniae vaccines. 4 Delayedhypersensitivitytype skin reactions can be used
to screen for
defects in cellmediated immunity. These tests are generally performed by the cl
inician and not
by labora tory personnel. Delayed cutaneous hypersensitivity is a localiz
ed cellmediated
reaction to a speci c antigen. The prototype is the tuberculin skin test. An antig
en to which
most of the population has been exposed, such as candida, mumps, or tetanus toxo
id, is injected
intradermally. The presence of induration 48 to 72 hours later indicates a cell
mediated immune
response. A negative test is not always informative, because the patient may not
have been previ
ously exposed to the test antigen. Screening for complement deficiencies usually
begins with a
CH50 assay. 19 This procedure determines the level of functional complement in a
n individual.
Undetectable CH50 levels may indicate a de ciency of a speci c compo nent. Based up
on the
clinical history, individual component assays would be indicated. The laboratori
an should be
aware, however, that low CH50 levels may be due to complement consumption and do
not, by
themselves, indicate a comple ment de ciency. Defects in neutrophil oxidative bur
st activity may
be detected by a flow cytometric assay, as mentioned above. Neutrophils labeled
with
dihydrorhodamine (DHR) are stimulated to undergo an oxidative burst by exposure
to a mitogen. The
oxidative burst will reduce the DHR, caus ing it to fluoresce. The resulta
nt fluorescence
may be measured objectively by flow cytometry. Flow cytometry looking for the e
xpression of the
CD18 antigen may be used to diagnose LAD, type 1. Con rmatory Tests If the screeni
ng tests detect
an abnormality or if the clini cal suspicion is high, more specialized testing
will probably be
necessary to precisely identify an immune abnormality. Some of the tests used fo
r con rming an
immunode ciency state are summarized in Table 164. Enumeration of classes of lympho
cytes in the
peripheral blood is performed by ow cytometry. Even though types of lymphocytes c
annot be
distinguished morphologically, they exhibit different patterns of antigen
or surface
immunoglobulin expression that correlate with functional characteristics. For ow
cytometric

evaluation, antibodies to antigens specific for different types of lymphocytes a

re labeled with a
uorescent probe. These antigens are gener ally referred to by cluster of differe
ntiation (CD)
number. The antibodies are allowed to react with peripheral blood mononuclear ce
lls. The ow
cytometer is used to count the cells that are labeled with each fluorescen
t antibody.
Lymphocytes can then be assigned to specific types based on antigen expression:
B cells (CD19), T
cells (CD3), T helper cells (CD3/CD4), cytotoxic T cells (CD3/CD8), and NK cells
(CD16 and/or
CD56). Flow cytometry is objec tive and quite reliable. It allows detection of
those defects
that result in a decrease in one or more types of lympho cytes. For example, an
absence or
profound decrease in the number of CD3 cells would be consistent with DiGeorge s
yndrome. An
absence of CD19 positive B cells suggests Brutons agammaglobulinemia. Most of t
he genes
associated with primary immune deficiencies have been identified and loc
alized. Thus,
genetic testing is available for many conditions, including the DiGeorge delet
ion, the
WiskottAldrich gene, and the IL2RG mutations. While genetic testing is useful
to understand
the pathology of the disease, it is often not Table 552 Comparison of T
ests Used
SUSPECTED DISORDER TESTS Humoral immunity B cell counts (total and IgM, IgD, I
gG, IgAbearing)
B cell proliferation in vitro Histology of lymphoid tissues Cellmediated immuni
ty T cell counts
(total and helper supressor subsets) T cell functions in vitro Enzyme assays (A
DA, PNP)
Phagocyte defect Leukocyte adhesion molecule analysis (CD11a, CD11b, CD11c, CD18
) Phagocytosis
and bacterial killing assays Chemotaxis assay Enzyme assays (myeloperoxidase, gl
ucose6phosphate
dehydro genase, components of NADPH oxidase) Complement Other speci c component a
ssays NADPH
Nicotinamide adenine dinucleotide phosphate. Table 164. Specialized Con rmatory T
ests for
Immunode ciencies 1814_Ch16_262277.qxd 7/13/09 2:57 PM Page 271 272 SECTION 3
Immune
Disorders required for making a diagnosis. Genetic testing of family members of
affected patients
may be helpful in determin ing who may be at risk of developing the disease or
passing it on to
offspring. Tcell function can be measured by assessing the ability of isolated
T cells to
proliferate in response to an antigenic stim ulus or to nonspeci c mitogens in cu
lture.
Classically, the Tcell response may be measured by quantitating the uptake of r
adioactive
thymidine, a precursor of DNA. Increased thymidine uptake suggests cell division
and activation.
This assay requires experienced technologists, a radioactive mate rials license
, and
laboratorydetermined reference ranges. More recently, antigen or mitogenstimu
lated Tcell

activation has been measured without the use of radioactive materials. As of Aug
ust 2008, three
such assays have been FDA cleared for diagnostic use. The QuantiFERON TB assay a
nd the TSpot
assay measure an individuals response to Mycobacterium tuberculosis antigens. Fol
lowing
overnight whole blood activation with TB antigens, gamma interferon (secreted by
activated Th1
cells) is quantitated by either an ELISA or ELISpottype procedure. Either of th
ese assays may
be used as an in vitro assessment of exposure to Mycobacterium tubercul
osis. A second
assay, Cylex ImmuKnow, uses the mitogen phyto hemagglutinin (PHA) to activate T
cells. Following
incubation, ATP production is measured by a uorescent immunoassay technique. Prod
uction of ATP
is a general measurement of Tcell function and is often used to monitor individ
uals receiv ing
immunosuppressant therapy. The assay may be used to determine overall Tcell fun
ctional
capabilities in an individ ual suspected of a primary immune de ciency. Evaluatio
n of
Immunoglobulins Quantitative measurement of serum or urine immunoglob ulins is
used in the
workup of both immunode ciency states and some lymphoproliferative disorders. Seru
m protein elec
trophoresis can be quantitative if the total serum protein is determined and the
results are read
using a densitometer. Serum Protein Electrophoresis Serum protein electrophoresi
s (SPE) is a
technique in which molecules are separated on the basis of their size and electr
i cal charge.
SPE allows reproducible separation of the major plasma proteins (see Chapter 4 f
or details). The
serum pro tein electrophoretic pro le is traditionally divided into ve regions: al
bumin and
alpha1, alpha2, beta, and gamma glob ulins. Some laboratories now use six reg
ions, dividing
the beta region into beta1 (transferrin) and beta2 (complement com ponent C3)
regions. IgG,
IgM, IgD, and IgE migrate in the gamma globulin region, while IgA migrates as a
broad band
overlapping the beta and gamma regions. Immunoglobulins normally show a range of
mobilities.
Additional evaluation of serum immunoglobulin is performed if the SPE shows a mo
noclonal
component or if there is a signi cant quantita tive abnormality of serum immunogl
obulins.
Immuno xation Electrophoresis Another method of characterizing immune deficie
ncies is
immunofixation electrophoresis (IFE). In IFE, serum samples are electrophoresed,
just as for SPE,
and then spe ci c antibody is applied directly to the separating gel. The antibod
yantigen
complexes form and are visualized by staining. Polyclonal immunoglobulins are in
dicated by areas
of diffuse staining, while monoclonal bands produce narrow, intensely st
ained bands
(refer to Fig. 152 in Chapter 15.). Lack of bands indicates immunode ciencies of
one or more

immunoglobulin classes. IFE is laborinten sive and expensive, although it is f

airly fast. For
accurate interpretations, specific immunoglobulin isotype levels, determined by
nephelometry, are
necessary. Bone Marrow Biopsy A bone marrow aspirate and biopsy is indicated in
any eval uation
of monoclonal gammopathy or immunodeficiency state. It is important in establish
ing the diagnosis
of such disorders and for excluding other diseases. The bone mar row specimen w
ill be analyzed
microscopically and by ow cytometry. Cytogenetic analysis may also be performed t
o detect the
specific genetic anomalies associated with the primary immune de ciency diseases.
6,12,20 SUMMARY
The immune system consists of a network of intercon nected components. Defect
s in the
development and regulation of individual portions of the immune system are reco
gnized as
causes of clinically distinct syndromes. Defects in antibodymediated immuni
ty lead to
recurrent infections with pyogenic bacteria, particularly of the respi ratory a
nd intestinal
tracts. Defects in T cellmediated immunity generally lead to recurrent infections
with intra
cellular pathogens. Primary defects in Tcell function can also lead to secondar
y abnormalities
in antibody produc tion and an increased risk of malignancy. Defects in
phagocyte
function generally lead to pyogenic bacterial infections, often of the skin. The
clinical
manifestations of these deficiency states have led to a better understanding of
the normal roles
of the different components of the immune system. Immunode ciency states run the g
amut from quite
mild to lethal. It is important to diagnose immunodeficiency states early in lif
e, because
effective therapies are available for many of them. Failure to begin therapy as
soon as possible
can result in permanent organ damage or death caused by infection. The diagnosis
must be
suspected clinically and is evaluated by serological analysis, first by screenin
g tests measuring
leukocyte counts and immunoglobulin levels, then by specialized laboratory testi
ng. Prenatal
diagnosis and carrier detection is possible for some familial immun ode ciency sy
ndromes.
1814_Ch16_262277.qxd 7/13/09 2:57 PM Page 272 CHAPTER 16 Immunode ciency Disea
ses 273 1. A
7monthold male was diagnosed with bacterial meningitis. Previously he had
been
hospitalized with bacterial pneumonia. Laboratory testing results were as follow
s: red cell
count: normal; white cell count: 22
10 9
/
L (normal is 5 to 24 10
9
/
L); differential:
70 percent neutrophils, 15 percent monocytes, 5 percent eosinophils, and
10 percent
lymphocytes; and SPE: no gamma band present. Questions a. What possible conditio
ns do these

results indicate? b. How are these conditions inherited? c. What type of further
testing do you
recommend? 2. A 3yearold female appeared to be developmentally slow. She had s
everal facial
anomalies, including a small jaw and ears that were set farther back than usual.
She seemed prone
to infections, especially yeast infections. Laboratory testing results were as f
ollows: red cell
count was normal; white cell count was low normal; and a dif ferential white
cell count
indicated a decrease in lymphocytes. Flow cytometry results demonstrated that
the decrease in
lymphocyte population was caused by low numbers of T cells. SPE showed a weak ga
mma band present.
Questions a. What immunode ciency do you suspect? b. Are the facial anomalies link
ed to the
immunologic ndings? c. What kinds of treatment are possible? 3. A 37yearold fem
ale presents
with a history of recur rent upper respiratory infections. She states that she
was always a sick
child, usually with respiratory infec tions, but occasional diarrhea would also
occur. She has
received countless antibiotic regimens over the years. The physician orders a se
rum protein
electrophoresis (SPE) and immunoglobulin levels. The SPE is read as a low gamm
a level with
no monoclonal proteins detected. Levels of IgG, IgM, and IgA are below the ref
erence ranges.
The physician then orders an immuno xation assay (Fig. 162). Questions a. Which
pattern
would be most representative of the expected pattern for this patient? b. Explai
n why you chose
this answer. CASE STUDIES FIGURE 162. Four different patient immuno xation patterns
. (See Color
Plate 12.) 1814_Ch16_262277.qxd 7/13/09 2:57 PM Page 273 274 SECTION 3 Immun
e Disorders
IMMUNOFIXATION Principle Immunofixation (IFE) is used to identify a monoc
lonal
immunoglobulin. This assay is a combination of serum protein electrophore
sis and
immunoprecipitation. Serum proteins are separated by agarose gel electrophoresis
. The gel is then
overlayed with antibodies to speci c immunoglob ulin heavy and light chain isotyp
es, each on an
individual electrophoresis pattern or lane. Following an incubation period, the
gel is stained
for antigenantibody complexes. A monoclonal immunoglobulin will appear as a band
in a lane
reacting with a heavy chain antibody (e.g., gamma) and a light chain antibody
(e.g., lambda).
Normal polyclonal immunoglobulins appear as a diffuse region. Urine may be exa
mined by this
method for the presence of Bence Jones protein. Sample Preparation Immunofixatio
n is performed on
both serum and urine. Plasma should not be used due to the presence of a brino g
en. Fibrinogen
appears as a gamma band, which can mimic a monoclonal immunoglobulin. A 24hour
urine specimen is
recommended for Bence Jones protein analysis. Reagents, Materials, and Equipment
Instrument: 1.

At least three companies make immunofixation elec trophoresis equipment: Beckma

nCoulter,
Helena, and Sebia. Although there are some variations in the speci c procedures am
ong these
companies, the basic format is the same, regardless of the equipment the company
uses in a given
laboratory. The guidelines given here will be based primarily on the Sebia proce
dure. a.
Materials and reagents: electrophoresis chamber and power supply b. IFE agarose
gels (options
exist for gels to handle 19 IFE assays) c. Electrophoresis buffer d. Application
tool e.
Isotypespeci c antisera f. Staining reagents Protocol: 1. Ensure that the electro
phoresis buffer
chambers are lled with the appropriate buffer. 2. Allow the gels to reach room te
mperature. 3.
Apply the specimen as directed. a The gels contain six lanes for each specimen.
One lane is used
for a standard protein electrophoresis (SPE) reading. The other five lanes
are used for
immunoglobulin isotype analysis. Serum is normally diluted prior to application.
Each
manufacturer may have a speci c dilution; however, Sebia recommends a 1:3 dilution
for the SPE
lane and the IgG lane and a 1:6 dilution for each additional isotype lane. This
dilution may need
to be adjusted based on the total protein concentration. Concentrated urine spec
imens are run
undiluted in all lanes. 4. Place the gel on the electrophoresis instrument. 5. A
pply power and
electrophorese for the appropriate time. The Sebia automated system uses a
9minute elec
trophoresis time. 6. Overlay each lane with an appropriate antisera. This step m
ay be automated
or performed manually. a. Three of the lanes are covered with antiheavy chain ant
isera
(antigamma, antialpha, and antimu). b. Two of the lanes are covered with antiligh
t chain
antisera (antikappa and antilambda). 7. Following incubation (5 minutes in the S
ebia system), the
gel is washed, stained, and dried. 8. The patterns are read. A pathologist
or clinical
boardcertified PhD (ABMLI or AACC) may sign out the interpretations. Interpretati
on In normal
serum, there are many immunoglobulins, each with its own specific amino acid seq
uence. Thus, each
immunoglobulin molecule will migrate to a slightly dif ferent location during
electrophoresis
than the other immunoglobulins. Therefore, the stained regions in each isotyp
e lane will have
a diffuse appearance in a normal specimen. A monoclonal immunoglobulin will appe
ar as a dense
band in the same electrophoretic location in one of the heavy chains and in one
of the light
chains. For exam ple, a matching band in the gamma lane and lambda lane indicat
es the presence
of a monoclonal IgG lambda pro tein. Multiple monoclonal proteins may be
seen in a
specimen. Biclonal gammopathies with two monoclonal proteins have been described
. IgA and IgM

monoclonal proteins may appear as two or more bands due to the pres ence of m
onomers, dimers,
trimers, and pentamers. Specimens with multiple IgA or IgM monoclonal bands m
ay be reduced
with 2mercaptoethanol and repeated on an IFE gel. This reduction process will b
reak apart the
EXERCISE 1814_Ch16_262277.qxd 7/13/09 2:57 PM Page 274 CHAPTER 16 Immunode cie
ncy Diseases
275 multimers into individual immunoglobulin molecules, and a single band should
result. One may
see a matching monoclonal heavy and light chain band with an additional nonmatch
ing light chain.
This indicates the presence of free serum light chains. The pres ence of a ligh
t chain band with
no associated heavy chain band may indicate either light chain disease or possib
ly an IgD or IgE
monoclonal protein. A repeat IFE with antidelta and antiepsilon sera would be re
commended in that
case. Immunoglobulin levels, determined by nephelometry, should be performed wit
h an
immunofixation. The IFE technique is not quantitative, and the immunoglobu
lin levels along
with the IFE interpretation are useful in assist ing the physician to arrive at
the proper
diagnosis. Urine IFEs are performed to look for the presence of Bence Jones prot
ein or free
monoclonal light chains. It is essential to use antifree light chain antisera in
the urine assay.
A 24hour specimen is preferred due to the low sensitivity of detecting Bence Jo
nes proteins in
random urine speci mens. Urine specimens are routinely concentrated prior to be
ing run in an IFE
procedure. 1814_Ch16_262277.qxd 7/13/09 2:57 PM Page 275 276 SECTION 3 Immun
e Disorders 1.
For which immunode ciency syndrome should patients receive irradiated blood produc
ts to protect
against the development of GvH disease? a. Brutons agammaglobulinemia b. Severe I
gA de ciency c.
SCID d. CGD 2. Tcell subset enumeration by ow cytometry would be most useful in
making the
diagnosis of which disorder? a. Brutons agammaglobulinemia b. Severe IgA de ciency
c. SCID d.
Multiple myeloma 3. What clinical manifestations would be seen in a patient with
myeloperoxidase
de ciency? a. Defective Tcell function b. Inability to produce IgG c. Defective N
K cell function
d. Defective neutrophil function 4. Defects in which arm of the immune system ar
e most commonly
associated with severe illness after adminis tration of live virus vaccines? a.
Cellmediated
immunity b. Humoral immunity c. Complement d. Phagocytic cells 5. Which of the f
ollowing
statements applies to Brutons Xlinked agammaglobulinemia? a. It typically appear
s in females.
b. There is a lack of circulating CD19 positive B cells. c. T cells are abnormal
. d. There is a
lack of preB cells in the bone marrow. 6. DiGeorge anomaly may be characterized
by all of the
following except a. autosomal recessive inheritance. b. cardiac abnormalities. c
. parathyroid

hypoplasia. d. decreased number of mature T cells. 7. A 3yearold boy is hospit

alized because of
recurrent bouts of pneumonia. Laboratory tests are run, and the following ndings
are noted:
prolonged bleeding time, decreased platelet count, increased level of serum alph
a fetoprotein,
and a deficiency of naturally occurring isohemagglutinins. Based on these r
esults, which is
the most likely diagnosis? a. PNP de ciency b. Selective IgA de ciency c. SCID d. WA
S 8. Which of
the following is associated with AT? a. Inherited as an autosomal recessive b. D
efect in both
cellular and humoral immunity c. Chromosomal breaks in lymphocytes d. All of the
above REVIEW
QUESTIONS 1814_Ch16_262277.qxd 7/13/09 2:57 PM Page 276 CHAPTER 16 Immunode ci
ency Diseases
277 References 1. Primary immunodeficiency diseases. Report of a WHO sci enti c g
roup.
Immunode c Rev 3:195236, 1992. 2. Buckley, R. Primary immunode ciency diseases due to
defects in
lymphocytes. N Engl J Med 343:13131324, 2000. 3. Puck, J. Prenatal diagnosis and
genetic
analysis of Xlinked immunode ciency disorders. Pediatr Res, 33(suppl):S29, 1993 4
. Conley, ME.
Primary antibody deficiency diseases. In Detrick, B, Hamilton, RG, and Folds
, JD (eds):
Manual of Molecular and Clinical Laboratory Immunology, ed. 7. ASM Press, Washin
gton, D.C., pp.
906913, 2006. 5. Fleisher, TA. Back to basics: Primary immune deficiencies: Windo
w into the
immune system. Pediatric Reviews. 27(10): 363372, 2006. 6. Fischer, A. Human p
rimary
immunodeficiency diseases. Immunity 27:83545, 2007. 7. Stiehm, RE. The four most
common
pediatric immunode  ciencies. Adv Exp Med and Biol 601:1526, 2007. 8. Fischer, A.
Primary
immunodeficiency diseases: An experi mental model for molecular medicine. Lance
t 357:18631869,
2001. 9. Garcia, JM, et al. Update on the treatment of primary immun ode ciencies
. Allergol
Immunopath 35(5):184192, 2007. 10. Taddei, I, Morishima, M, Huynh, T, and Lindsay
, EA. Genetic
factors are major determinants of phenotypic variability in a mouse model of the
DiGeorge/del22q11 syndromes. PNAS 98(20):1142811431, 2001. 11. Primary immunodef
iciency
diseases. Report of an IUIS Scienti c Committee. International Union of Immuno
logical
Societies. Clin Exp Immunol 118(Suppl 1):128, 1999. 12. Roifman, CM. Approach to
the diagnosis
of severe combined immunodeficiency. In Detrick, B, Hamilton, RG, and Fold
s, JD (eds):
Manual of Molecular and Clinical Laboratory Immunology, ed. 7. ASM Press, Washin
gton, D.C., pp.
895900, 2006. 13. Puck, JM. Primary immunodeficiency diseases. JAMA 278:18351
841, 1997.
14. Hilman, B, and Sorensen, RU. Management options: SCIDS with adenosine deamin
ase de ciency.
Ann Allergy 72, 1994. 15. Gennery, AR, and Cant, AJ. Cord blood stem cell transp
lan tation in
primary immune de ciencies. Curr Opin Allergy Clin Immunol 7:528534, 2007. 16. Peac

ocke, M, and
Siminovitch, KA. WiskottAldrich syn drome: New molecular and biochemical insig
hts. J Am Acad
Dermatol 27, 1992. 17. Cooper, M, and Schroeder, HW, Jr. Primary immune defi ci
ency diseases.
In Braunwald, E, Fauci, AS, Kasper, DL, et al. (eds): Harrisons Principles
of Internal
Medicine, ed. 15, McGrawHill, New York, pp. 18431851, 2001. 18. Holland, SM. N
eutropenia and
neutrophil defects. In Detrick, B, Hamilton, RG, and Folds, JD (eds): Manual
of Molecular and
Clinical Laboratory Immunology, ed. 7. ASM Press, Washington, D.C., pp. 924932, 2
006. 19.
Giclas, PC. Hereditary and acquired complement deficien cies. In Detrick, B, Ha
milton, RG, and
Folds, JD (eds): Manual of Molecular and Clinical Laboratory Immunology, ed. 7.
ASM Press,
Washington, D.C., pp. 914923, 2006. 20. Verbsky, JW, and Grossman, WJ. Cellular a
nd genetic
basis of primary immune deficiencies. Pediatr Clin North Am 53:649684, 200
6.
1814_Ch16_262277.qxd 7/13/09 2:57 PM Page 277 17 Transplantation Immunology
John L. Schmitz,
Ph.D., D(ABMLI, ABHI) 278 LEARNING OBJECTIVES After nishing this chapter, the rea
der will be
able to: 1. List the histocompatibility systems relevant to clinical transplanta
tion. 2. Discuss
the mechanism of alloantigen recognition. 3. Distinguish allograft, autograft, x
enograft, and
syngeneic graft. 4. Discuss the mechanisms of graft rejection. 5. Identify risk
factors for
graftversushost (GvH) disease. 6. Discuss the major classes of immunosuppressi
ve agents. 7.
Discuss methods for HLA typing. 8. Discuss methods for detecting and identifying
HLA antibodies.
KEY TERMS Accelerated rejection Acute rejection Acute GVHD Allograft Autograft C
hronic rejection
Complementdependent cytotoxicity (CDC) Crossmatch Direct allorecognition Graft
versushost
disease (GVHD) Haplotypes HLA antibody screen HLA genotype HLA typing HLA match
HLA phenotype
Hyperacute rejection Immunosuppressive agent Indirect allorecognition Major hist
ocompatibility
antigens Mixed lymphocyte response Syngeneic graft Zenograft 1814_Ch17_278291.q
xd 7/10/09 3:51
PM Page 278 CHAPTER 17 Transplantation Immunology 279 INTRODUCTION Transplant
ation is a
potentially lifesaving treatment for end stage organ failure, cancers, autoimmu
ne diseases,
immune de ciencies, and a variety of other diseases. Over 28,000 solid organ (kid
ney, pancreas,
liver, heart, lung, small intestine) transplants were performed in the United St
ates, 1 and
approx imately 50,000 hematopoietic stem cell transplants were performed
worldwide in
2006. 2 The number of transplants performed is a testament to the numerous devel
opments over the
past few decades in patient management pre and post transplant and in the
technologies for
organ/stem cell acquisition and sharing. Of critical importance has been the c

ontinued
elucidation of the immunologic mechanisms of graft rejection and graftversusho
st disease, in
particular the role of human leukocyte antigens (HLA) and the develop ment of p
harmacological
agents that interfere with various components of the immune system to promote su
stained graft
survival. The HLA system (covered in detail in Chapter 3) is the largest immunol
ogic barrier to
successful allogeneic organ transplantation. It consists of cell surface protein
s that play a
central role in immune recognition and initiation of immune responses. Be
cause of the
ubiquitous presence of these proteins on the surface of nucleated cells and thei
r extensive
degree of polymorphism, an allogeneic response may result in graft rejection in
solidorgan and
stem cell transplantation or graftversushost disease in stem cell transplantat
ion.
HISTOCOMPATIBILITY SYSTEMS The classical (transplant) HLA antigens, also k
nown as major
histocompatibility antigens, include the class I (HLAA, B, and C) and class II
(HLADR, DQ, and
DP) proteins. HLA proteins are encoded by a set of closely linked genes on the
short arm of
chromosome 6 in the major histocompatibility complex (MHC). The HLA gen
es are inherited
as haplotypes from parental chromosomes (Fig. 171). Offspring receive one materna
l and one
pater nal HLA haplotype. Based on this Mendelian inheritance, there is a 25 per
cent chance that
any two siblings will inherit the same two haplotypes (i.e., are HLA identical),
a 50 per cent
chance of being HLA haploidentical (i.e., share one of two HLA haplotypes), and
a 25 percent
chance of being HLA nonidentical (i.e., share neither HLA haplotype). CHAP
TER OUTLINE
INTRODUCTION HISTOCOMPATIBILITY SYSTEMS Minor Histocompatibility Antigens MIC An
tigens ABO Blood
Group Antigens Killer Immunoglobulinlike Receptors ALLORECOGNITION TRANSPLANT R
EJECTION
Hyperacute Rejection Acute Cellular Rejection Chronic Rejection GRAFTVERSUSHOS
T DISEASE
IMMUNOSUPPRESSIVE AGENTS Corticosteroids Antimetabolic Agents Calcineurin Inhibi
tors Monoclonal
Antibodies Polyclonal Antibodies CLINICAL HISTOCOMPATIBILITY TESTING HLA Typing
HLA Phenotyping
HLA Genotyping HLA Antibody Screening and Identi cation SUMMARY CASE STUDY EXERCIS
E: HLA
HAPLOTYPE SEGREGATION ANALYSIS REVIEW QUESTIONS REFERENCES FIGURE 171. HLA genes
are linked and
inherited in Mendelian fashion as haplotypes. One paternal (a or b) and one mate
rnal (c or d)
haplotype is passed to each offspring. Four different combi nations of haplotyp
es are possible
in offspring. Elucidation of haplotype sharing between siblings is an important
assessment in the
search for a transplant donor. A1 B8 A2 B44 a b A1 B8 A3 B14 a c A1 B8 A24 B17
a d A2 B44 A3
B14 b c A2 B44 A24 B17 b d A3 B14 A24 B17 c d 1814_Ch17_278291.qxd 7/10/09

3:51 PM Page
279 280 SECTION 3 Immune Disorders Recombination can take place, resulting in th
e inheritance of
unexpected haplotypic combinations; however, this occurs in less than 1 percent
of families that
are HLA typed. HLA proteins are heterodimeric molecules. Class I proteins
are the product
of the HLAA, B, and C genes and are expressed on the cell surface covalently bo
und to
beta2microglobulin. Class I heterodimers are codomi nantly expressed on virtu
ally all
nucleated cells. Class II heterodimers are the products of the HLADRB1 DRA1, HLA
DQB1 DQA1,
and DPB1 DPA1 genes. Class II heterodimers are codominantly expressed primari
ly on
antigenpresenting cells (e.g., dendritic cells, monocytes, macrophages, B lymph
ocytes). As
discussed in Chapter 3, HLA proteins have critical roles for the development and
functioning of
the innate and adap tive immune systems. They serve as recognition elements for
antigen
receptors on T lymphocytes, thus initiating adaptive immune responses. In additi
on, they serve as
ligands for regulatory receptors on natural killer cells in the innate i
mmune response.
The CD8 molecule on cytotoxic T lym phocytes interacts with class I HLA protein
s in addition to
the HLA
peptide complex, while the CD4 molecule on the T helper cell subset inte
racts with
class II HLA proteins in addition to the HLA peptide complex. A cardinal feature
of the genes
encoding the HLA pro teins is an extensive degree of allelic polymorphism. The
HLA system is the
most polymorphic genetic system in humans (Table 171). This degree of polymorphis
m is believed
to have resulted from the need to bind peptides from an innumerable array of pat
hogenic organisms
and thus generate protective immune responses. While this has successfully enabl
ed populations to
survive infectious chal lenges, it severely restricts the ability to transplant
foreign tissues
or cells between any two individuals. Minor Histocompatibility Antigens A second
set of
transplantation antigens was identi ed based on studies in mice and humans demonst
rating tissue
rejec tion in MCHidentical transplants and based on outcomes of human stem
cell transplants
between HLAidentical siblings in whom graftversushost disease has developed
. 3 Early
experimental studies documented a slower rejection pace mediated by these transpla
ntation
antigens, thus their nameminor histocompatibility antigens (mHAs). mHAs are nonH
LA proteins
that demonstrate polymor phism in amino acid sequence within a species. Both X
linked and
autosomally encoded mHAs have been identified. Introducing a polymorphic va
riant of one of
these proteins from one individual into another individual who possesses a diffe
rent polymorphic
variant (via transplantation of tissue or cells) can induce a recipient immune r

esponse to the
donor variant. The immune response is mediated by CD4 and/or CD8 T cells recogni
zing a variant
protein in the context of the recipient MHC molecule. This response is analogous
to the reaction
to a foreign microbial antigen. Several different types of mHAs have been i
dentified,
including proteins encoded by the male Y chromosome, proteins for which the rec
ipient has a
homozygous gene deletion, proteins that are autosomally encoded, and proteins th
at are
mitochondrial DNA encoded. 3 MIC Antigens The MHC class Irelated chain A (MICA) e
ncodes a cell
surface protein that is involved in gamma/delta Tcell responses. MICA is
polymorphic with
over 50 allelic vari ants. MIC proteins are expressed on endothelial cel
ls,
keratinocytes, broblasts, epithelial cells, dendritic cells, and monocytes, but t
hey are not
expressed on T or B lympho cytes. 4 As such, MICA proteins could serve as targe
ts for allograft
immune responses. Antibodies to MICA antigens have been detected in as many as 1
1 percent of
kidney trans plant patients. 5 MICA antibodies have been associated with reject
ion episodes and
decreased graft survival. ABO Blood Group Antigens The ABO system is the on
ly blood group
system that impacts clinical transplantation. AntiA or antiB antibod ies dev
elop in
individuals lacking the corresponding blood group antigens. ABO blood group inco
mpatibility is a
bar rier to solidorgan transplantation, because these antibodies can bind the
corresponding
antigens that are expressed on the vascular endothelium. Binding activates the c
omplement
cascade, which can lead to hyperacute rejection of the transplanted organ. As su
ch,
recipientdonor pairs must be ABO identical or compatible to avoid this adverse ou
tcome. For
example, an individual of blood group A will possess antiB antibodies and can t
hus receive an
organ only from an ABOA or O donor. Likewise, a Bexpressing individual has ant
iA antibodies
and can receive an organ only from an ABOB or O donor. Recently, approaches usi
ng plasma
exchange and intravenous immunoglobulin administration have allowed successful t
ransplantation of
kidneys from ABOincompatible donors by lowering ABO antibody to levels that all
ow
transplantation to proceed without risk of hyperacute rejection. 6 Table 552 Co
mparison of Tests
Used HLA LOCUS # ANTIGENS # ALLELES A 28 506 B 61 851 C 10 276 DRB1 24 559 DQB1
9 81 DPB1 6 126
Table 171. Approximate Number of HLA Antigens and Alleles De ned at the Six Class
ical Transplant
Loci 1814_Ch17_278291.qxd 7/10/09 3:51 PM Page 280 CHAPTER 17 Transplantatio
n Immunology
281 Killer Immunoglobulinlike Receptors Another polymorphic genetic system that
impacts
allogeneic transplantation is the killer immunoglobulinlike receptor (KIR) syst

em. 7 KIRs are

one of several types of cell surface molecules that regulate the activity of nat
ural killer (NK)
lymphocytes. Within the KIRs are activating and inhibitory receptors that vary i
n number and type
on any individual NK cell. The balance of signals received by the activating and
inhibitory
receptors governs the activity of the NK cell (see Fig. 212). Included among
the functions
of NK cells are secretion of cytokines and nonMHC restricted cytotoxicity. Th
e ligands for the
inhibitory KIRs have been defined as several of the MHC class I molecules, inclu
ding spe cific
HLAA, B, and C proteins. Normally, an NK cell encounters self class I MHC pro
teins as it
circulates in the body. This interaction between MHC protein and the inhibitory
KIR maintains the
NK cells in a quiescent state. If an NK cell encounters a cell lacking or having
decreased class
I expression, inhibitory receptors are not occupied and an inhibitory signal los
s occurs,
resulting in NK cell activation. The regulatory role of KIRs has been exploited
in hap
loidentical stem cell transplantation. 8 Stem cell donors have been selected for
recipients who
lack a corresponding class I MHC protein for the donors inhibitory KIR type. This
results in
alloreactivity by NK cells that repopulate the recip ient after transplant. The
se alloreactive
NK cells have been shown to mediate a graftversusleukemia reaction and p
revent relapse
after transplantation. ALLORECOGNITION Transplantation of cells or tissues betwe
en two
individuals is classi ed by the genetic relatedness of the donor and the recipient
. An autograft
is the transfer of tissue from one area of the body to another of the same indiv
idual. A syn
geneic graft is the transfer of cells or tissues between identical twins
. An allograft is
the transfer of cells or tissue between two individuals of the same specie
s. Lastly, a
xenograft is the transfer of tissue between two individuals of a different speci
es. Most
transplantation falls into the category of allografting. As stated earlier, HLA
disparity between
donor and recipient will result in a vigorous immune response to the for
eign MHC antigens
and is the primary stimulus of graft rejection. The response to foreign MHC ant
igens is
characterized by strong cellular and humoral immune responses. The recipient
immune system
recognizes foreign HLA proteins via two distinct mechanismsdirect and indirect al
lorecognition 9
(see Fig. 172). In direct allorecogni tion, recipient T cells bind and respond d
irectly to
foreign (allo) HLA proteins on graft cells. Although an individual T lymphocyte
can recognize
selfHLA
peptide, foreign HLA proteins may mimic a selfHLA
peptide complex due
to
similarities in structure of the alloHLA protein itself or to structural simila

rities of
alloHLA protein peptide. Either way, direct allorecognition is characterized by
a high
frequency (up to 2 percent) of responding T cells 9 compared to the responder fr
equency in a
typical Tcell response to a foreign antigen. The high frequency of responding T
cells may be due
to several factors, including recognition of multiple amino acid disparities by
multiple Tcell
clones; the presence of multi ple different peptides on an allogeneic cell that
are each
recognized by different Tcell clones; and the presence of many foreign molecule
s per cells,
resulting in activation of T cells with low affinity, which normally woul
d not be
stimulated. The mixed lymphocyte response (MLR) is an in vitro correlate of dire
ct
allorecognition. 5 Indirect allorecognition is the second pathway by which
the immune
system recognizes foreign HLA pro teins. 10 Indirect allorecognition is analogo
us to the normal
mechanism of recognition of foreign antigens, as it involves the uptake, process
ing, and
presentation of foreign HLA proteins by recipient antigenpresenting cells to re
cipient CD8+ T
cell CD8 CD8 CD4 CD4 Graft cell with foreign MHC I antigen CD4+ T cell APC with
digested foreign
MHC antigen = Tcell receptor for antigen = Digested = MHC class I antigen = MHC
class II antigen
= Antigen presenting cell APC Cell death Production of cytokines and antibody A
Direct
Allorecognition B Indirect Allorecognition FIGURE 172. Direct versus indirect al
lorecognition.
(A) In direct allorecognition, cytotoxic T cells bind directly to foreign HLA pr
o teins on graft
cells. (B) In indirect allorecognition, foreign MHC antigens are presented by ph
agocytic cells,
and CD4 T cells respond. 1814_Ch17_278291.qxd 7/10/09 3:51 PM Page 281 282 SEC
TION 3 Immune
Disorders T cells. Indirect allorecognition plays a predominant role in acute an
d chronic
rejection. The effector responses against transplanted allogeneic tissue include
direct
cytotoxicity, delayedtype hypersensi tivity responses, and antibodymediate
d mechanisms.
Antibody may mediate antibodydependent cellular cyto toxicity reactions and ma
y x complement,
resulting in cell death. Rejection episodes vary in the time of occurrence and t
he effector
mechanism that is operative. TRANSPLANT REJECTION Hyperacute Rejection Hyperacut
e rejection
occurs within minutes to hours after the vascular supply to the transplanted org
an is
established. This type of rejection is mediated by preformed antibody that react
s with donor
vascular endothelium. ABO, HLA, and certain endothelial antigens may elicit hype
racute rejec
tion. Binding of preformed antibodies to the alloantigens activates the compleme
nt cascade and
clotting mechanisms and leads to thrombus formation. The result is ischemia and

necrosis of the
transplanted tissue. 11 Hyperacute rejection is seldom encountered in clinical t
ransplantation.
Donorrecipient pairs are chosen to be ABO identical or compatible, and patients a
waiting trans
plantation are screened for the presence of preformed HLA antibodies. In additio
n, the absence of
donor HLA speci c antibodies is con rmed prior to transplant by the perform ance of
a crossmatch
test. These approaches have virtually eliminated hyperacute rejection episodes.
Some individuals
possess very low levels of donor speci c antibody in the pretransplant peri
od. In these
cases, antibodymediated rejection may take place over several days. This has be
en termed
accelerated rejection. 9, 12 Like hyperacute rejection, accelerated rejection in
volves intravas
cular thrombosis and necrosis of donor tissue. Acute Cellular Rejection Days to
weeks after
transplant, individuals may develop acute rejection. This is a cellulartype rej
ection but may
involve antibody as well. 11 Acute rejection is characterized by parenchymal and
vascular injury.
Interstitial cellular in l trates contain a predominance of CD8positive T cells
as well as CD4
T cells and macrophages. CD8 cells likely mediate cytotoxic reactions to foreign
MHCexpressing
cells, while CD4 cells likely produce cytokines and induce delayedtype hypersen
sitivity (DTH)
reactions. Antibody may also be involved in acute graft rejection by binding to
vessel walls,
activating complement, and inducing trans mural necrosis and inflammation a
s opposed to
the thrombosis typical of hyperacute rejection. The develop ment and applicatio
n of potent
immunosuppressive drugs that target multiple pathways in the immune response to
alloantigens has
improved early graft survival of solidorgan transplants by reducing the inciden
ce of acute
rejection and by providing approaches for its effective treatment. However, it i
s chronic
rejection that remains the most sig ni cant cause of graft loss after the rst year
posttransplant, because it is not readily amenable to treatment. Chronic Reject
ion Chronic
rejection results from a process of graft arte riosclerosis characterized by pr
ogressive brosis
and scarring with narrowing of the vessel lumen due to proliferation of smooth m
uscle cells. 13
Several predisposing factors impact the development of chronic rejection, includ
ing prolonged
cold ischemia, reperfusion, acute rejection episodes, and toxicity from immunosu
ppressive drugs.
Chronic rejection is also thought to have an immunologic component, pre sumably
a delayedtype
hypersensitivity reaction to foreign HLA proteins. 9 This is indicated in studie
s employing ani
mal models of graft arteriosclerosis in which mice lacking IFN gamma do not deve
lop graft
arteriosclerosis. In addi tion, similar studies support an important role for C
D4 T cells and B

cells in this process. Cytokines and growth factors secreted by endothelial cell
s, smooth muscle
cells, and macrophages activated by IFN gamma stimulate smooth muscle cell accum
ulation in the
graft vasculature. GRAFTVERSUSHOST DISEASE Stem cell transplants (and less com
monly lung and
liver transplants) are complicated by a unique allogeneic responsegraftver
sushost
disease (GVHD). Recipients of stem cell transplants for hematologic malignancies
typi cally have
depleted bone marrow prior to transplantation as a result of the chemotherapy us
ed to treat the
malignancy. Next, donor bone marrow or, more commonly, peripheral blood stem cel
ls are infused.
The infused products often con tain some mature T cells. These cells have sever
al bene cial
effects, including promotion of engraftment, reconstitution of immunity, and
mediation of a
graftversusleukemia effect. However, these mature T cells may also med
iate GVHD. Acute
GVHD occurs during the rst 100 days postin fusion and targets the skin, gastroin
testinal tract,
and liver. 14 In mismatched allogeneic stem cell transplantation, the tar gets
of GVHD are the
mismatched HLA proteins, while in matched stem cell transplantation, minor histo
compatibil ity
antigens are targeted. The infused T cells can mediate GVHD in several ways,
including a
massive release of cytokines due to largescale activation of the donor cells
by MHC mismatched
proteins and by in ltration and destruc tion of tissue. The incidence and seve
rity of GVHD
is related to the match status of the donor and recipient as well as other fac
tors. 15 In
efforts to reduce the incidence and severity of GVHD, several approaches are tak
en, including
immuno suppressive therapy in the early posttransplant period and 1814_Ch17_27
8291.qxd
7/10/09 3:51 PM Page 282 CHAPTER 17 Transplantation Immunology 283 removal o
f T lymphocytes
from the graft. Tcell reduction is very effective in lowering the incidence of
GVHD, but it can
also reduce the graftversusleukemia (GVL) effect of the infused cells and incr
ease the
incidence of graft failure. Beyond 100 days posttransplant, patients may experi
ence chronic
GVHD. This condition resembles autoimmune disease, with fibrosis affecting the s
kin, eyes, mouth,
and other mucosal surfaces. IMMUNOSUPPRESSIVE AGENTS There is a growing list
of agents
that are employed to suppress antigraft immune responses in solidorgan and s
tem cell
transplantation. Immunosuppressive agents are used in several ways, including in
duction and
maintenance of immune suppression and treatment of rejection. Combinations
of different
agents are frequently used to prevent graft rejection. However, the immunosuppre
ssed state (and
graft survival) induced by these agents comes at a price of increased susceptibi
lity to

infection, malignan cies, and other associated toxic side effects. There
are several
classes of immunosuppressive agents, which are brie y discussed in the following s
ections.
Corticosteroids Corticosteroids are potent antiin ammatory and immuno suppressiv
e agents used
for immunosuppression maintenance. At higher doses, they are used to treat acute
rejection
episodes. Steroids act by blocking production and secretion of cytokines,
in ammatory
mediators, chemoattractants, and adhesion molecules. These activities decrease m
acrophage
function and alter leukocytetraf cking patterns. However, longterm use is associ
ated with
several complications, includ ing hypertension and diabetes mellitus. Antimetab
olic Agents
Antimetabolic agents interfere with the maturation of lym phocytes and kill pr
oliferating
cells. 16 Azathioprine was the rst such agent employed. It has been replaced in l
arge part by
mycophenolate mofetil, which has a more selective effect on lymphocytes compared
to azathioprine
and thus fewer side effects. Calcineurin Inhibitors Cyclosporine and FK506 (tac
rolimus) are
compounds that block signal transduction in T lymphocytes, resulting in impairme
nt of cytokine
syntheses, including IL2, 3, 4, and interferongamma. 16 Inhibition of cytokine
synthesis blocks
the growth and differentiation of T cells, impairing the anti graft response. R
apamycin
(sirolimus) 17 is an agent that inhibits Tcell proliferation by binding to spec
i c intracel
lular proteins, including mammalian target of rapamycin (mTOR). Monoclonal Anti
bodies Several
monoclonal antibodies that bind to cell surface mol ecules on lymphocytes are u
sed as induction
agents and to treat severe rejection episodes. OKT3 is a mouse mono clonal anti
body the binds to
the CD3 receptor on human lymphocytes. 16 Binding of OKT3 to the CD3positi
ve Tcell surface
has several outcomes. Binding may modulate CD3 from the cell surface, rendering
the affected T
cells nonfunctional. Higher doses of antibody deplete T cells from the circula
tion via
complementmediated lysis or opsonization for removal by phagocytic cells. Two
antiCD25
monoclonal antibodies are available for use in transplant patients. 18 Basili
ximab and
dacluzimab both bind the CD25 (IL2 receptor) and thus interfere with IL2mediate
d Tcell
activation. It may also deplete CD25 expressing cells. An additional monoclonal
antibody that
targets the CD52 receptor found on T and B lymphocytes is alemtuzumab, which may
be used for
induction therapy at the time of transplantation. 18 A problem with monoclonal a
ntibody
preparations administered to patients is the devel opment of antimouse antibod
y. Polyclonal
Antibodies Two polyclonal antiTcell antibody preparations are used to treat se
vere rejection.

Thymoglobulin is an antithymocyte antibody prepared in rabbits, and ATGAM is a p

olyclonal
antiserum prepared from immunization of horses. Both are potent immunosuppressiv
e agents that
deplete lymphocytes from the circulation. The development of these antimouse an
tibodies can
interfere with the activity of the monoclonal antibody. A drawback associated
with
administration of polyclonal antibody preparations is the development of
serum sickness
due to antibody responses to the foreign immunoglobulin. CLINICAL HISTOCOM
PATIBILITY
TESTING Appreciation of the bene cial role of HLA matching 9,19 and the detrimenta
l role of
antibody to HLA proteins 12 on graft survival provided the impetus for developme
nt and appli
cation of specialized testing to aid in the selection of the most appropriate do
nors for patients
needing transplanta tion. Histocompatibility laboratories provide specialized t
esting for both
solidorgan and stem cell transplantation programs. Two main activities are carr
ied out by these
lab oratories in support of transplantation, HLA typing, and HLA antibody
screening/identi cation. HLA Typing HLA typing is the phenotypic or genotypic de nit
ion of the
HLA antigens or genes in a transplant candidate or donor. For clinical HLA testi
ng, phenotypes or
genotypes of the clas sical transplant antigens or genes are determined (HLAA,
B,
1814_Ch17_278291.qxd 7/10/09 3:51 PM Page 283 284 SECTION 3 Immune Disorders
Cw, DR, DQ).
This information is used to nd the most suit able donorrecipient combination f
rom an
immunologic standpoint. It must be stressed that other considerations go into th
e choice of a
particular donor for any given patient, be it a solidorgan or stem cell transpl
ant. HLA
Phenotyping The classic procedure for determining the HLA pheno type is the com
plementdependent
cytotoxicity (CDC) test. Panels of antisera or monoclonal antibodies that de ne in
dividual or
groups of immunologically related HLA anti gens are incubated with lymphocytes
from the
individual to be HLA typed. Puri ed T lymphocytes are used for HLA class I typing,
while purified
B lymphocytes are used for HLA class II typing. After incubation with the antise
ra, com plement
is added. In the presence of bound antibody, which occurs only if the lymphocyte
expresses the
HLA antigen targeted by the antisera, complement is activated and cells are kill
ed. A vital dye
is then added that distinguishes live cells from dead when they are viewed micro
scopically. Using
this assay, an extensive array of HLA antigens can be de ned (Table 171). There are
several
limitations to the CDC method for HLA typing. Viable lymphocytes must be u
sed, which
demands timely performance of the assay. Separation of T and B lymphocytes is re
quired for
definition of class I versus class II antigens. The source of antisera for HLA t

yp ing is not
always consistent or reliable. Thus, reagents can vary in quality or quantity ov
er time. Finally,
the level of resolution (i.e., the ability to distinguish two closely related ye
t distinct HLA
antigens) is limited. The limits of resolu tion dont significantly impact the ro
le of this
technology for matching solidorgan donors and recipients. However, for unrelate
d stem cell
transplantation, a higher level of resolution is required. DNAbased (molecu
lar) HLA typing
methods are now commonly employed in histocom patibility laboratories, because
they address the
limitations of CDCbased methods and are amenable to higher throughput fo
rmats. HLA
Genotyping Molecularbased HLA genotyping methods use polymerase chain reaction
(PCR)based
amplification of HLA genes followed by analysis of the ampli ed DNA to identify th
e speci c HLA
allele or group of alleles. 20 The most common approaches for analysis include P
CR ampli cation
of HLA genes with panels of primer pairs, each of which ampli es specific alleles
or related
allele groups. Amplification is detected by agarose gel electrophoresis (Fig. 173
). Only those
primer pairs that bind to the target gene result in detection of an ampli cation p
roduct. The HLA
type is then identi ed by determining which primers resulted in ampli cation. A se
cond common
approach for HLA genotyping is to perform a single PCR reaction that will amplif
y all HLA gene
variants at a speci c locus (referred to as a generic amplification). The amplif
ied gene is
then subjected to hybridization with a panel of DNA probes, each speci c for a u
nique HLA
allele or allele group. Only those probes that speci cally hybridize to the ampli ed
DNA will be
detected. The HLA genotype is determined by assessing which probes hybridi
zed. A third
common method for HLA geno typing is sequencing of PCRampli ed HLA genes. HLA ge
notyping
overcomes the limitation of CDC based HLA phenotyping. Cells do not need to be
viable in order
to obtain DNA for HLA genotyping. Typing reagents are chemically synthesized; th
us, there is no
reliance on human donors of antisera. HLA genotyping can provide varying levels
of resolution
that can be tailored to the spe cific clinical need. DNAbased typing can provi
de results at a
level of resolution comparable to CDCbased typing (antigen equivalent) or ca
n provide
allelelevel results (required for matching of unrelated stem cell donors and r
ecipients).
Allelelevel HLA typing has demonstrated the incredible extent of polymorphism w
ithin the HLA
loci (Table 171). HLA Antibody Screening and Identi cation Antibodies to HLA antige
ns can be
detected in candidates and recipients of solidorgan transplants. These antibodi
es develop in
response to multiple blood transfusions; to prior HLAmismatched transplants; an

d, in women, to
multiple pregnancies. Because of the potential adverse impact HLA antibodies can
have on graft
survival, patients awaiting solid organ transplantation are screened periodical
ly for their
presence. 12 If detected, the speci city (which HLA proteins they bind) of the
antibodies is
then determined so that donors possessing those HLA antigens can be eliminat
ed FIGURE 173.
An example of a PCR with sequencespeci c primers (PCRSSP) analysis of the HLADQ
B1 locus. Each
lane of this ethidium bromide stained agarose gel contains the ampli cation produc
t of an
individual PCR reaction. Each reaction contains primers to a ubiquitous gene and
should
demonstrate amplification (the larger band in each lane). This serves as an inte
rnal control to
document successful ampli cation in each reaction. Each reaction also con tains p
rimers speci c
for various HLADQB1 alleles. An ampli cation product of small size is seen in sev
eral lanes,
indicating the presence of the target HLADQB1 gene for those speci c primers. The
particular
pattern of ampli ed primers is assessed to determine the HLA DQB1 type of the sam
ple. (Color
Plate 13). 1814_Ch17_278291.qxd 7/10/09 3:51 PM Page 284 CHAPTER 17 Transpla
ntation
Immunology 285 from consideration for donation to that patient. Patients are t
ested monthly for
the presence of HLA antibodies while they are waiting for an organ offer. Antibo
dy screening and
identi cation is also performed posttransplantation to aid in the diagnosis of an
tibodymediated
rejection and to assess the effectiveness of therapy for antibodymediated rejec
tion. The methods
used for antibody detection and identifi cation have changed signi cantly in rece
nt years. The
CDC method used for HLA typing is also used for HLA antibody detection and ident
i cation. In this
case, panels of lympho cytes with de ned HLA phenotypes are incubated with the pa
tients serum.
If the serum contains HLA antibodies, they will bind to those lymphocytes in the
panel that
express the cognate HLA antigen. Binding is detected by addition of complement a
nd a vital dye to
assess cell death microscop ically. In some scenarios, the level of antibody in
a patient serum
may be below a level detectable by the CDC assay. In these cases, antihuman glob
ulin (AHG) may be
added to the CDC assay to increase the tests sensitivity. The AHG CDC assay can
detect lower
levels of antibody as well as isotypes of bound antibody that dont activate compl
ement and thus
wouldnt normally be detected in the standard CDC assay. The proportion of lymphoc
ytes in the
panel (usually 30 to 60 unique lymphocyte preparations are included in th
e panel) that are
killed by the patients serum is referred to as the percent panel reactive antibod
y (%PRA). In
addition, the specificity of the antibodies can be deter mined by evaluating th

e phenotype of
the panel cells. Enzymelinked immunosorbent assay (ELISA) has been developed in
recent years as
a substitute for CDCbased HLA antibody testing. 21 ELISA assays utilize pu
rified HLA
antigens bound to the wells of microtiter plates. Patient serum is add
ed to the
wells of the plate, and if HLAspecific antibody is present, it will
bind. Bound
antibody is detected by addition of an enzymelabeled anti immunoglobulin reage
nt. Addition of
substrate results in a color change in the wells that have bound antibody. The w
ells of the ELISA
plate may contain a pool of HLA anti gens, thus serving as a qualitative screen
for the presence
of HLA antibody in a serum. Alternatively, each well may con tain HLA antigens
representing a
single donor and thus can be used in a fashion analogous to a CDCbased analysis
, allowing %PRA
and speci city to be determined. Another approach for antibody detection and ident
i ca tion is
ow cytometry. 22 Antibody in patient serum can be incubated with latex beads that
are coated
with HLA anti gens, either from a single donor or a single puri ed HLA protein. P
atient serum is
incubated with the beads, and bound antibody is detected by adding an FITClabel
ed antiIgG
reagent (Fig. 174). A more recent version of ow cytometrybased antibody detection i
s the
multiplex bead array system that can assess binding of up to 100 different HLA a
ntigens in a
single tube using a dedicated owbased detection system. 23 Flow cytometrybased me
thods are the
most sensitive technology for detecting HLA antibodies. In addition, they can pr
ovide the most
speci c determination of the speci city of HLA antibodies when beads coated with a s
ingle HLA
antigenic type are used. Once a donor has been identi ed for a particular patient,
a
donorrecipient crossmatch test is performed to con rm the absence of donorspeci c an
tibody.
Donor lymphocytes are incubated with recipient serum in a CDC assay to ver ify
a lack of binding
as detected by microscopic analysis after addition of a vital dye. Alternatively
, binding of
antibody can be detected by ow cytometry using an FITClabeled anti IgG reagent.
As for
antibody screening and identi cation, the ow cytometric crossmatch is the most sens
itive method
for detecting donorspeci c antibody. 12 SUMMARY The immune systems ability to reco
gnize and
respond to the myriad of infectious agents that humans encounter has obvious ben
efits. However,
the mechanisms that impart this ability make the transplantation of organs and c
ells between
allogeneic individuals difficult. The target of the response to transplanted tis
sues is HLA
proteins. These proteins play critical roles as antigenpresenting molecules for
CD4 and CD8 T
cells, resulting in the phenomenon of MHC restriction. While an individuals immun

e system
develops to respond to the foreign proteins presented on its own MHC antigens, i
t responds even
more intensely to foreign MHC proteins. Both the humoral and cellular arms of th
e immune system
contribute to this allogeneic immune response and mediate graft rejection. Even
in the face of
this intense response, transplantation has become an effective treatment for a v
ariety of
diseases due to the development of immunosuppressive agents that block the immun
e system from
responding to the allogeneic MHC proteins in the transplanted tissue. Unfortunat
ely, these agents
also block immune response to infectious organ isms and tumors. In concert
with the
development of immunosuppressive drugs, defining the MHC antigens of donors an
d recipients
and monitoring the allospecific immune response are critical components of cl
inical trans
plantation and contribute to the continually improved outcomes of transplantatio
n.
1814_Ch17_278291.qxd 7/10/09 3:51 PM Page 285 286 SECTION 3 Immune Disorders
AntiIgG HLA Ab
A Bead with HLA protein B 80 70 60 50 40 30 20 10 0 0 200 400 600 FL1H C o u n
t s 800 1000 M1 C
1000 800 600 400 200 0 0 200 400 600 AntilgG FITC S p e c i f .
B e a d s
P E 800 1000 FIGURE 174. (A) Flow cytometric detection and identi cation of
HLA antibodies
employs latex beads that are coated with HLA proteins from individual donors or
single HLA
molecules. For qualitative determination of the presence of HLA antibodies, mult
iple beads, each
coated with the product of an individual donor, are pooled together so as to rep
resent the
majority of common HLA antigens. They are then incubated with patient serum. To
determine the
speci city of HLA antibodies in a serum sample, beads coated with a single HLA pro
tein species
can be incubated with patient serum. Once incubated with serum, both bead types
are then
incubated with an FITC labeled antiIgG reagent that will detect the presence o
f antibodies
bound to the bead. (B) Singleparameter histogram display of an HLA class I anti
body screen. A
pool of HLAcoated beads was incubated with a patient serum, washed, and then in
cubated with an
FITClabeled antiIgG reagent. Unbound FITClabeled reagent was washed away and
the beads
analyzed for uorescence on a ow cytometer. The large peak represents beads with no
bound
antibody, while the smaller peak to the right indicates the presence of HLA anti
body bound to
approxi mately 19 percent of the HLA class I coated beads. This represents a po
sitive HLA class
I antibody screen. (C) Nine individual clusters of latex beads, each coated with
a single HLA
class I antigen species, are identi ed in the dual parameter dot plot of the seru

m from (B). The

HLA antigen coated on each bead is indicated to the right of the bead. The bead
coated with
HLAA11 has shifted to the right relative to the other beads, indicating that th
e HLA antibodies
in this patients serum are speci c for the A11 antigen. 1814_Ch17_278291.qxd 7/10
/09 3:51 PM
Page 286 CHAPTER 17 Transplantation Immunology 287 A 40yearold mother of thr
ee needs to have
a second kid ney transplant. Her rst transplant was lost due to chronic rejectio
n. The mothers
HLA typing, HLA antibody, and ABO blood group status was determined. The patient
was found to
have antibodies to HLAB35 by complement dependent cytotoxicity testing with a
panel of
lymphocytes. The HLA type and blood group were also determined for two of her si
blings and two
close friends (who were also siblings) who are interested in donating a kidney t
o the patient.
From the available donors, who would be the most compatible for donation to this
patient? BLOOD
IDENTIFICATION GROUP A B CW DR DQ Recipient O 1,2 8,44 7,5 17,4 2,7 Sibling 1
O 1,11 8,35 7,4
17,1 2,5 Sibling 2 A 3,11 7,35 7,4 15,1 6,5 Friend 1 B 2,24 57,7 6,7 7,15 2,6 Fr
iend 2 O 2,24
57,7 6,7 7,15 2,6 CASE STUDY 1814_Ch17_278291.qxd 7/10/09 3:51 PM Page 287 2
88 SECTION 3
Immune Disorders HLA HAPLOTYPE SEGREGATION ANALYSIS Principle Serological HLA ty
ping provides
information about the HLA protein polymorphisms expressed on the cell surface of
an individual.
HLA phenotype information is limited to the alloantigens that are recognized by
the reagent panel
selected for a particular indication. Each reagent panel con sists of polyclona
l antisera or
monoclonal antibodies selected to recognize private or shared HLA speci cities and
HLA antigens.
An HLA class I panel might detect the products of HLAA, B, C loci; a class II
panel might
recognize the products of the DR and DQ loci. Donorrecipient match ing for renal
(kidney)
transplantation is usually based on the HLAA, B, DR, because these antigens s
eem to be the
most immunogenic. By convention, HLA phenotypes are written as a string of
antigens (e.g.,
HLAA1, 3; B7, 8; DR7, 17). If only one HLA antigen is identi ed for a locus, then
an X or blank
is inserted. To help distinguish these pos sibilities, retyping with a differen
t panel might be
needed, or the entire family might be typed. Family typing is essen tial if the
goal is to nd a
genotypic HLAidentical donor (i.e., a sibling who shares two haplotypes with a
patient). Each
parentchild combination will share only one haplo type. Segregation analysis inv
olves
inspection of phenotype data from parental and offspring generations of a nuclea
r family so that
the four haplotypes can be inferred from the HLA phenotype data. Haplotype segre
gation analysis
is based on the principle that HLA genes are transmitted en bloc and yields the

HLA genotype of
the individual. Sequencebased HLA typing is a method of direct allele typing. S
egregation
analysis using the HLA allele assign ments of family members is still nee
ded to define
the segregating haplotypes. Predicted haplotypes of some indi viduals can be
deduced without
family studies if the particular HLA alleles at closely linked loci exhibit
linkage
disequilibrium. By referring to published allele frequency tables for the approp
riate ethnic
group, the two haplotypes of many individuals can be assigned. Procedure 1. Arra
nge the HLA
phenotypes in the order in which the corresponding genes are arranged on chromos
ome 6, from
centromeric (left) to telomeric (right), to recognize and interpret rare intraH
LA recombination
events that occur in approximately 1 to 2 percent of families. 2. Identify the
antigens at
each locus from one parent that could only have come from the father and des
ignate these as
haplotypes (a) and (b) or could only have been inherited from the mother (haplot
ypes [c] and [d],
where [a, b, c, d] is a shorthand representation of the inferred haplotypes). Fo
r each child,
list the two haplotypes that can account for the phenotype of the child (i.e., a
c, ad, bc, bd).
3. Write out each of the four haplotypes (i.e., HLADQ_, DR_, B_, C_, A_) where
the underline
would be replaced with the number corresponding to the particular anti gen assi
gned to each
locus in the string. 4. If there is no antigen available to assign a given locus
, insert X after
the locus designation. A blank X may re ect homozygosity at the locus. 5. Each ch
ild will
inherit one paternal haplotype and one maternal haplotype. If an extra haplo
type is required
to explain the data, then consider the following pos sibilities: (a) incomplete
typing or missed
antigen, (b) incorrect typing, (c) nonpaternity, or (d) interlocus crossov
er during meiosis
in one of the parents. Jones Family Genotype Analysis Worksheet 1. Using the fol
lowing worksheet,
deduce the haplotypes that segregate in the Jones family. The HLA phenotypes of
the Jones family
are listed in the centromeric telomeric order. Father: DQ2,XDR13,17 B8,X C
w7,XA1,24 (a)
DQ___DR___B___ Cw___A___ (b) DQ___DR___B___ Cw___A___ Mother: DQ1,X DR
1,13 B35,60
Cw3,w4 A2,3 (c) DQ___DR___B___ Cw___A___ (d) DQ___DR___B___ Cw___A___
Child 1:
(patient) DQ1,2 DR1,17 B8,35 Cw4,w7 A3,24 ( ) DQ___DR___B___ Cw___
A___ ( )
DQ___DR___B___ Cw___A___ Child 2: DQ1,2 DR13,17 B8,60 Cw3,w7 A2,24 (
)
DQ___DR___B___ Cw___A___ ( ) DQ___DR___B___ Cw___A___ Child 3: DQ1,2 D
R13,17 B8,60
Cw3,w7 A1,2 ( ) DQ___DR___B___ Cw___A___ ( ) DQ___DR___B___ Cw___A___
Child 4: DQ1,2
DR13,X B8,60 Cw3,w7 A1,2 ( ) DQ___DR___B___ Cw___A___ ( ) DQ___DR__

_B___ Cw___A___
Child 5: DQ1,2 DR1,13 B8,35 Cw4,w7 A1,3 ( ) DQ___DR___B___ Cw___A_
__ ( )
DQ___DR___B___ Cw___A___ 2. Using the (a, b, c, d) as an abbreviation for the
four hap
lotypes, which paternal and maternal haplotypes were present in each of the ve ch
ildren? 3.
Which sibling is the best match for Child 1, who is in need of an organ transpla
nt, and why?
EXERCISE 1814_Ch17_278291.qxd 7/10/09 3:51 PM Page 288 CHAPTER 17 Transplant
ation Immunology
289 Interpretation of Results HLA genotyping is important in histocompatibility
testing,
especially when serotyping data is incomplete and the possi bility of a related
donor is under
consideration. The chance that two siblings will be HLA identical by sharing a p
ater nal and a
maternal haplotype is 1:4, or 25 percent. The chance that two sibling
s share one
haplotype is 1:2, or 50 percent. The chance that two siblings share zero hapl
o types is 1:4,
or 25 percent, following the Mendelian principle. If a child needing a renal tra
nsplant shares
two haplotypes with a sibling (e.g., patient: a, c/sibling: a, c), then it follo
ws that they
probably share the same HLA antigens at closely linked loci that may not have be
en typed, such as
DP, or at a locus where the phenotype assignment is problematic. Allele typing by
DNAbased
methods yields the geno type directly at the coding level. However, DNA typing
results, taken
alone, provide no information about whether the allele is expressed. Although HL
A expression is
codom inant, certain virus infections and cancers interfere with HLA gene expre
ssion and thus
alter the phenotype. 1814_Ch17_278291.qxd 7/10/09 3:51 PM Page 289 290 SECTI
ON 3 Immune
Disorders 1. The type of allograft rejection associated with vascular and parenc
hymal injury with
lymphocyte infiltrates is which of the following? a. Hyperacute rejection b. Acu
te cellular
rejection c. Acute humoral rejection d. Chronic rejection 2. Antigen receptors o
n T lymphocytes
bind HLA class II molecules with the help of which accessory molecule? a. CD2 b.
CD3 c. CD4 d.
CD8 3. Patients at risk for graftversushost disease (GVHD) include each of the
following,
except recipients of a. bone marrow transplants. b. lung transplants. c. liver t
ransplants. d.
irradiated leukocytes. 4. HLA molecules exhibit all of the following properties,
except that they
a. belong to the immunoglobulin superfamily. b. are heterodimeric. c. are integr
al cell membrane
glycoproteins. d. are monomorphic. 5. Kidney allograft loss from intravascular t
hrombosis with
out cellular in ltration 5 days posttransplant raises the suspicion level about w
hich primary
rejection mechanism? a. Hyperacute rejection b. Accelerated humoral rejection c.
Acute humoral
rejection d. Acute cellular rejection 6. Which reagents would be used in a

direct
(forward) donorrecipient crossmatch test? a. Donor serum and recipient lymphocyte
s rabbit
serum complement b. Recipient serum and donor lymphocytes
rabbit serum complemen
t c. Donor
stimulator cells recipient responder cells
complete culture medium d. Recipient
stimulator
cells
donor responder cells
complete culture medium 7. The indirect allorecognit
ion pathway
involves which one of the following mechanisms? a. Processed peptides from polym
orphic donor pro
teins restricted by recipient HLA class II molecules b. Processed peptides from
polymorphic
recipient proteins restricted by donor HLA class I molecules c. Intact polymorph
ic donor protein
molecules recog nized by recipient HLA class I molecules d. Intact polymorphic
donor protein
molecules recog nized by recipient HLA class II molecules 8. Which immunosuppre
ssive agent
selectively inhibits IL2 receptormediated activation of T cells and causes cle
arance of
activated T cells from the circulation? a. Mycophenolate mofetil b. Cyclosporine
mofetil c.
Corticosteroids d. Daclizumab REVIEW QUESTIONS 1814_Ch17_278291.qxd 7/10/09 3
:51 PM Page 290
CHAPTER 17 Transplantation Immunology 291 References 1. www.unog.org. 2. Appel
baum, FR.
Hematopoieticcell transplantation at 50. N Eng J Med 357:14721475, 2007. 3. Simp
son, E, Scott,
D, James, E, et al. Minor H antigens: Genes and peptides. Transpl Immunol 10:1151
23, 2002. 4.
Zwirner, NW, Dole, K, and Stastny, P. Differential surface expression of MICA by
endothelial
cells, fibroblasts, ker atinocytes, and monocytes. Hum Immunol 60:323330, 1999.
5. Zou, Y,
Stastny, P, Susal, C, Dohler, B, and Opelz, G. Anti bodies against MICA antigen
s and
kidneytransplant rejection. N Eng J Med 357:12931300, 2007. 6. Thielke, J, Kapla
n, B, and
Benedetti, E. The role of ABO incompatible living donors in kidney transplantat
ion: State of the
art. Semin Nephrol 27:408413, 2007. 7. Rarag, SS, Fehinger, TA, Ruggeri, L, Velar
di, A, and
Caliguri, MA. Natural killer cell receptors: New biology and insights into the
graftversusleukemia effect. Blood 100:19351947, 2002. 8. Ruggeri, L, Capanni, M
, Urbani, E, et
al. Effectiveness of donor natural killer cell alloreactivity in mismatche
d hematopoietic
transplants. Science 295:20972100, 2002. 9. Abbas, A, Lichtman, AH, and Pillai, S
. Cellular and
molecular immunology, ed. 6. Saunders, Philadelphia, 2007, pp. 375396. 10. Afzali
, B, Lechler,
RI, and HernandezFuentes, MP. Allore cognition and the alloresponse: Clinical
implications.
Tissue Antigens 69:545556, 2007. 11. Leichtman, AB. Primer on Transplantation. Am
erican Society
of Transplant Physicians, Thorofare, 1998, pp. 217222. 12. Gebel, HM, Bray, RA, a
nd Nickerson,
P. Pretransplant assess ment of donorreactive, HLAspecific antibodies i
n renal

transplantation: Contraindication vs. risk. Am J Transplant 3:14881500, 2003. 13.

Colvin, RB.
Chronic allograft nephropathy. N Eng J Med 349:22882290, 2003. 14. Goker, H, Hazn
edaroglu, IC,
and Chao, NJ. Acute graft vs. host disease: pathobiology and management. Experim
ental Hematology.
29(3):259277, 2001. 15. Copelan, EA. Hematopoietic stemcell transplantation. N E
ng J Med
354:18131826, 2006. 16. Halloran, PF, and Lui, SL. Primer on Transplantati
on. American
Society of Transplant Physicians, Thorofare, 1998, pp. 93102. 17. Augustine, JJ,
Bodziak, KA,
and Hricik, DE. Use of sirolimus in solid organ transplantation. Drugs 67:369391,
2007. 18.
Buhaescu, I, Segall, L, Goldsmith, D, and Covic, A. New immunosuppressive thera
pies in renal
transplantation: Monoclonal antibodies. J Nephrol 18:529536, 2005. 19. Lee, SJ, K
lein, J,
Haagenson, M, et al. Highresolution HLA matching contributes to the success of
unrelated donor
mar row transplantation. Blood 110:45764583, 2007. 20. Schmitz, JL. Molecular di
agnostics for
the clinical laborato rian, ed. 2. Humana Press, New York, 2005, pp. 485493. 21.
Lucas, DP,
Paparounis, ML, Myers, L, et al. Detection of HLA class Ispeci c antibodies by th
e QuikScreen
enzyme linked immunosorbent assay. Clin Diag Lab Immunol. 4:252257, 1997.
22. Bray, RA,
Nickerson, PW, Kerman, RH, and Gebel, HM. Evolution of HLA antibody detection: T
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Luminex technology for antiHLA antibody screening: Evaluation of performance an
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7/10/09 3:51
PM Page 291 18 Tumor Immunology Diane L. Davis, PhD, MT, SC, SLS (ASCP), CLS(NC
A) 292 LEARNING
OBJECTIVES After nishing this chapter, the reader will be able to: 1. Compare and
contrast the
normal cell and the tumor cell and describe the process by which tumors develop.
2. Discuss
evidence to support and to refute the theories of immunosurveil lance and immun
oediting. 3.
De ne tumorassociated antigen (TAA) and list the categories of common tumor marke
rs, giving
examples of each category. 4. Describe the principles of how tumor markers may b
e appropriately
used for screening, diagnosis, and disease classi cation/monitoring. 5. Discuss th
e impact of
disease prevalence and test sensitivity/speci city on the clinical value of a test
. 6. Describe
characteristics of an ideal tumor marker. 7. List and describe the laboratory te
chniques used for
tumor marker analy sis, including the advantages and disadvantages of each, wit
h particular
attention to the effects of antigen excess, antibody crossreactivity, and heter
ophile antibodies
in immunoassays. 8. For each of the following tumor markers, describe how it is
best used

clinically: alphafetoprotein (AFP), -2 microglo
ulin, CA-15.3, CA-19.9, CA-125,

calcitonin,
carcinom
ryonic antign (CEA), clustrs of iffrn- tiation (CD) in WBC, stro
gn/progstron
rcptors, fcal occult
loo, HER2/nu, human chorionic gonaotropin (hCG), mon
oclonal
immunoglo
ulins an light chains, parathyroi hormon (PTH), prostat- spci c ant
ign (PSA), an
thyroglo
ulin (TG). 9. Compar an contrast passiv vrsus activ immunothrapy,
scri
ing
common tchniqus us in ach. KEY TERMS Anaplastic Anti
oy conjugats Bnign
Cytogntics
Dissmination Dysplasia Graft vrsus lukmia High os hook ffct Immunoitin
g
Immunohistochmistry Immunosurvillanc Immunothrapy Immunotoxins Inuction In
situ Invasion
Mass spctromtry (MS) Mtastasis Microarray Noplasia Oncoftal antign Oncopp
tiomics
Protomics Proto-oncogns Suscpti
ility gns Tumor-associat antign (TAA) T
umor in ltrating
lymphocyt (TIL) TNM systm Tumor supprssor gns 1814_Ch18_292-310.qx 7/10/0
9 3:00 PM Pag
292 CHAPTER 18 Tumor Immunology 293 INTRODUCTION AND DESCRIPTION OF TUMORS Tumor
immunology is
th stuy of th antigns associat with tumors, th immun rspons to tumors,
th tumors
ffct on th hosts immun status, an th us of th immun systm to
hlp raicat
th tumor. Tumor immunology is
st unrstoo with a
ack- groun on if
frncs
twn
tumor clls an normal clls. 1 Normal cll growth an ivision ar rgulat pr
ocsss sign
to rapily prouc nw clls whn ncssary, inhi
it cll ivision whn su
fficint clls
ar prsnt, an limit cll lif span (programm cll ath, or apoptosis). Rg
ulatory gns
that promot cll ivision, th proto- oncogns, can caus uninhi
it cll
ivision if
thir xprssion is altr or if thy ar mutat into oncogns. Similarly, mu
tations or
malfunctions in tumor supprssor gns that rmov growth-inhi
itory signals
can caus
tumors. Tumors, thrfor, ar compos of clls that pos- sss many of th attr
i
uts of th
normal clls from which thy aros
ut hav acclrat or ysrgulat growth.
Similarity to
normal tissu is on rason why th immun systm os not automatically raica
t all tumors. If
a tumor os not inva surrouning tissu an nor- mal
oy function is lar
gly prsrv,
it is sai to

nign. Malignant tumors can inva surrouning tissus an
gratly isrupt
normal
oy function. Mtastasis is whn th malignant clls travl through th

oy, causing nw
foci of malignancy until
oy function is so isrupt that ath occurs. Malign
ant clls
typically iffr visually from normal clls, ar mta
olically mor activ to su
pport thir
growth, an xprss iffrnt gns or iffrnt lvls of gn proucts as comp
ar to normal

clls. 1 For xam- pl, lukmic clls can hav a
rrant morphology an 
xprss iffrnt
surfac antigns ithr in typ or amount. Th convrsion of a normal cll to a
malignant cll is
typically a procss, not an vnt. 2 During th inuction phas, clls ar xpos
 to a varity
of nvironmntal insults, incluing chmical carcinogns, oncognic viruss, an
rai- ation
(ioniing an ultraviolt). Cancr may only vlop as th rsult of multipl mu
tations caus
y
ths insults, an it mor raily vlops in clls gntically prispos to
ths mutations.
During th inuction phas, which may tak months to yars, clls xhi
it yspla
sia or a
normal
growth that is not yt consir noplasia, or consistnt with a tumor. Th in
situ phas of
cancr is whn noplastic clls hav form
ut ar con n to th tissu of origi
n. If th clls
ar malignant, th cancr procs to th invasion phas an thn to issminati
on throughout th

oy, usually via th
loo an lymphatics. Tratmnt is far mor ffctiv th
arlir th
malignancy is tct,
ut tction is far mor if cult in th arly stags, as
thr ar
fwr clls to tct an thy mor closly rsm
l normal clls if thy ar y
splastic rathr
than noplastic. 2 Th progny of th cll that unrgos transformation ar mon
oclonal in
origin, maning thy ar initially inti- cal phnotypically an gnotypical
ly. This can

 an important fatur in iagnosing malignancy. As rapi uncon- troll proli
fration
continus, mistaks can occur in DNA rplication, causing cllular phnotypic an
 gnotypic htrognity to vlop. 1,2 This a
ility to mutat may hlp cancr clls scap
from
oth th
immun systm an from chmothraputic agnts, as scri

low. Furthr, it
complicats
intification of rlia
l tumor markrs, as markr xprssion may chang ovr t
im. In
pathology, tumors mor similar to ftal or m
ryonic tissu ar classi  as poorl
y
iffrntiat, or anaplastic, whil wll-iffrntiat tumors ar mor similar
to nor- mal
tissu. Gnrally, th poorly iffrntiat tumors ar mor aggrssiv an l
a to poorr
patint prognosis. Tumors ar also classi 
y th TNM systm
y th si of th
primary tumor
(T), th involvmnt of ajacnt lymph nos (N), an th tction of mtastasi
s (M). 1
IMMUNOSURVEILLANCE Immunosurvillanc
y th immun systm to raicat cancr c
lls as thy form
has long
n postulat. Thr is incras incinc of tumors in thos
with  cint
immun systms such as th lrly an in immunosupprss iniviuals,
ut this
is not proof of
th xistnc of immuno- survillanc. Incras lif span implis incras xp
osur to
carcinogns, which caus tumors. In immunosupprss popl, many tumors ar
 ssntially

infctions, as thy ar of CHAPTER OUTLINE INTRODUCTION AND DESCRIPTION OF TUMOR

S
IMMUNOSURVEILLANCE TUMOR-ASSOCIATED ANTIGENS PRINCIPLES OF LAB TESTS FOR SCREENI
NG, DIAGNOSING,
AND MANAGING TUMORS LAB TESTS FOR TUMOR MARKER DETECTION IMMUNOTHERAPY Passiv I
mmunothrapy
Activ Immunothrapy SUMMARY CASE STUDIES EXERCISE: SEMIQUANTITATIVE PSA MEMBRAN
E TEST FOR THE
DETECTION OF ELEVATED SERUM LEVELS OF PSA REVIEW QUESTIONS REFERENCES 1814_Ch18_
292-310.qx
7/10/09 3:00 PM Pag 293 294 SECTION 3 Immun Disorrs viral rathr than spon
tanous origin.
Furthr, th incinc of nonviral tumors in T-cll- cint iniviuals is
not incras,
although this may
 u to aquat NK cll activity. 35 Ths topics ar still
unr stuy.
Thr is, howvr, vinc that supports th hypoth- sis that th immun syst
m provis tumor
survillanc for th
oy: NK clls, T lymphocyts (tumor in ltrating lympho- cyt
s [TIL]),
an macrophag infiltrats hav
n monstrat in som tumors, an thy a
r associat
with a
ttr prognosis. 3 A common charactristic of many tumors is loss of MHC
xprssion an
su
squnt poor antign prsntation, allowing tumor clls to scap from T cll
s. 3 Crtain
thraputic avancs irct towar up- rgulating th immun systm to ght
a particular
can- cr hav shown som succss. 3 Spontanous rgrssion of som tumors h
as
n
o
srv. 4 If immunosurvillanc is in occurring, mchanisms
y which tumor
s scap is
important to trmin. Som tumor antigns may
 poorly immunognic, particula
rly if thy ar
vry similar to normal slf antigns an inuc immun tolranc. Th tumor cll
may lack MHC
class I, class II, or accssory molcul xprssion. Th tumor may
 rsistant
to th immun
rspons, or th tumors growth rat may xc th a
ility of th immun
rspons to
stroy it, particularly in popl with  cint immun sys- tms. Solu
l antign
rlas
y
th tumor may
in to th T-cll rcptor, thr
y prvnting intraction with t
h tumor cll.
In aition, th local nvironmnt of som tumors has
n shown to
 imm
unosupprssiv,
inclu- ing tction of immunosupprssiv cytokins such as transforming
growth factor.
3,57 It is also possi
l that immunosurvillanc itslf is ulti- matly rsponsi

l for tumors
that can scap th immun systm
caus of immunoiting. 5 In th limination p
has of
immunosurvillanc, tumors ar sufficintly immunognic to
 liminat

y th immun
systm. During th quili
rium stag, as mutations occur ovr tim, clls that ar
lss
immunognic hav a growth avantag. Finally, in th scap phas, cancr clls ha
v mutat

yon th immun systms a
ility to control thm. Although rsarch has
n una

l to

immiatly tr- min th xtnt an prcis mchanisms of tumor immuno
survillanc,
rsarch into th manipulation of th immun systm to fight cancrs is ongoing
an iscuss
latr in this chaptr. TUMOR-ASSOCIATED ANTIGENS Tumor-associat antigns (TAA)
ar antigns
prsnt in th tumor tissu in highr amounts than in normal tis- su. 3 Thy ar
 oftn th
proucts of mutat gns an viruss,
ut thy can also aris from a
rrant xp
rssion of normal
gns. For xampl, oncoftal tumor antigns ar most highly xprss in
oth n
ormally
vloping ftal tissu an in crtain kins of cancrs. Tumors also gnrally 
xprss normal
antigns an proucts from thir tissu of origin. Virtually no tumor-associat
antigns ar
tumor- spcific,
caus thy also hav
n foun in som noncancrous
human tissu. 4
Th prfct tumor-associat antign uniqu to a particular tumor coul ai in t
h scrn- ing,
iagnosis, histopathologic valuation, staging, monitoring, localiation, an im
munothrapy of
various malignancis,
ut such a prfct antign has not yt
n foun. PRINCIP
LES OF LAB TESTS
FOR SCREENING, DIAGNOSING, AND MANAGING TUMORS Scrning tsts ar us in ostn
si
ly normal
popl to tct occult cancr. Diagnostic tsts ar thos that hlp trmin 
iffrntial
iagnosis, tumor stag, prognosis, an thrapy slction. Ths ar two vry if
frnt functions, an th a
ility of la
tsts to prform all ths functions wl
l is still
imprfct. Disas prvalnc profounly impacts th tsts usful- nss. Bays t
horm
pro
a
ility calculation shows th following: A goo cancr tst with 99 p
rcnt
snsitivity an 95 prcnt spci city will
 positiv in 99 out of 100 popl wi
th isas;
will
 ngativ in 95 out of 100 popl without isas. If th cancr rat in
th population
is 0.1 prcnt, thn 98 prcnt of positivs woul
 fals positivs. At
a 1 prcnt
cancr rat, th fals-positiv rat is still 83 prcnt. Assuming that a clinic
ian can
intify this cancr
y signs an symptoms 75 prcnt of th tim, if this sam
tst is appli,
th fals-positiv rat is 1.7 prcnt. Tsts for iffrntial iagnosis, thn,
gnrally prform
rlativly wll,
caus th clinical suspicion of cancr trans- lats to a high
r cancr
prvalnc in th population
ing tst. Th prsumptions for scrning tsts
ar that a
rlativly low num
r of popl
ing scrn actually hav cancr an that it
woul
 wors to
miss a cancr than to o furthr tsting on a normal prson to xclu can- cr.
Th concpt of a
normal or rfrnc rang osnt rally apply, as it may
 ifficult to trmin
 with crtainty that a rfrnc population os not hav cancr, an valus from normal
an cancrous

populations may ovr- lap. Cutoff valus for tumor markrs ar typically slct
 a
ov th point
at which furthr tsting will
 on, so cutoff valus for scrning tsts ar
gnrally st
with th xpctation that thr will
 an xtrmly high num
r of fals positi
vs u to low
isas prvalnc. This is not a
nign choic, as aitional tsting can
 in
vasiv, costly,
or anxity-provoking. Thrfor, wispra us of a la
oratory tst to scrn f
or cancr is
justi  if 810
:
1814_Ch18_292-310.qx 7/10/09 3:00 PM Pag 294 CHAPTER 18 Tumor Immun
ology 295 th tumor
is an important halth pro
lm for th popu- lation
ing scrn; thr is a r
cognia
l
arly symptom or markr that can
 us for scrning with rasona
ly high sns
itivity an
spci city; it is a tumor for which tratmnt at an arly stag is mor succssful
than at a
latr stag; th scrning tst is accpta
l to th population; th costs an

n ts of th
scrning tst ar accpta
l to th population. Th
n ts inclu improv surv
ival tim, lss
raical tratmnt n for tumors tct arlir, an rassur- anc for thos
 with ngativ
rsults. Th costs inclu longr mor
iity in patints whos prognosis is
not chang,
ovrtratmnt of qustiona
l iagnoss, mislaing rassur- anc for thos with
fals-ngativ
rsults, anxity an possi
l mor
iity from mor invasiv tsting for thos wit
h fals- positiv
rsults, th actual physical haars of th scrning tsts, an th actual oll
ar costs of th
scrning tst. 810 To improv th cost-to-
n t ratio, slct su
groups, such a
s patints
with a family history of a cancr, shoul
 scrn whn possi
l inst
a of th ntir
population. Diffrntial iagnosis of tumor typ can
 on
y tissu/c
ll morphology
an tction of tumor markrs irctly from tumor tissu. Immunohistochmistry
can tct
xprss antigns using la
l anti
ois, an molcular tchniqus such
as uorscnt in
situ hy
riia- tion (FISH) can tct a
normal gn xprssion using nuclic ac
i pro
s 11 (s
Chaptr 11). Th rquirmnts for using a tumor markr to facilitat iffrntia
l iagnosis
y
th pathologist ar lss stringnt than th rquirmnts for using tsts for wi
spra
scrning. To
 hlpful in pathological iagnosis, th markr must
 iffrnt
ially xprss
in th tumor of origin an othr tumors, which may hav a similar apparanc his
tologically.
Ths markrs must
 com
in with othr clinical rsults,
caus th iffrn
tiation that
occurs with trans- formation somtims can rsult in loss of th markr. This fa
ls-ngativ
situation is rlativly common. In aition, th DNA changs that occur with mal
ignant

transforma- tion somtims can caus xprssion of a markr that is not normally
associat
with th tumor typ in qustion, although this occurrnc is rlativly unc
ommon. Disas
managmnt with la
oratory tsts is typically on with srial trminations of
a tumor markr.
12 A
as- lin lvl at initial iagnosis is sta
lish. As th isas an tr
atmnts
progrss, aitional lvls ar trmin to sta
lish prognosis, monitor th r
sults of
thrapy, an tct rcurrnc. This is an ara in which many tumor markrs ar

st us
clinically,
caus it is not th a
solut valu of th tumor markr that is imp
ortant
ut rathr
th upwar or ownwar trn whn th markrs
iological half-lif is consir.
Srial
trminations on to ai th clinician in making important cisions concrnin
g th thraputic rgimn must
 on
y th sam mthoology so that changs ar u to
actual
altrations in th patint, not iffrncs in mthos. 12 An iali mol of
using tumor
markrs to gui thrapy is shown in Figur 181. Pro
lms with prostat-spci c ant
ign (PSA)
ar a goo illustration of th ilmmas associat with tumor mark- rs. 2,10,12
,13 No othr
tissu in mn is known to prouc PSA, so it is vry spci c for th prostat glan
 an incrass
in T u m o r
M a r k  r
L  v  l 160 140 120 100 80 60 40 20 0 Jan F
Mar Apr May No vinc
of isas Uppr
limit of rfrnc intrval Rlaps Chmothrapy #1 Thrapy sms succssful Ch
mothrapy #2
Using a Tumor Markr to Monitor Cancr Thrapy unsuccssful Surgry Dclin of m
arkr consistnt
with
iological half-lif No vinc of isas Jun Jul Aug Sp Oct Nov Dc Jan
2009 2008 F

Mar FIGURE 181. Tumor markr analysis. A curv showing a sam- pl scnario monito
ring a cancr
patint for tumor rcurrnc an for thrapy f cacy using lvls of a tumor-assoc
iat antign.
1814_Ch18_292-310.qx 7/10/09 3:00 PM Pag 295 296 SECTION 3 Immun Disorrs
almost all
prostat cancrs. Howvr, in a halthy prson, th amount of PSA prouc is i
rctly rlat to
th glans si, an many mn vlop
nign nlarg prostats as thy ag. Fu
rthr, as mn
ag, thy ar mor likly to vlop prostat cancr,
ut thy ar lss li
kly to i from
it. In othr wors, as mn ag, thr ar prostat cancrs that can an shoul

 lft alon. It
is rcommn that PSA scrning cas onc a mans rmaining lif xpctancy is
lss than 10
yars. Grat ffort has
n xpn to is- criminat
twn
nign prostatic
hyprtrophy,
wakly aggrssiv cancrs, an highly aggrssiv cancrs using PSA. If th frto-
oun ratio of

PSA is low or th rat of PSA is incrasing at a rat that xcs 0.5 ng/mL pr
yar 13 (PSA
vlocity), this is mor associat with cancr an is justi - cation for a
iopsy.
Du to th
fact that an incras PSA os not always inicat a cancrous stat or an aggr
ssiv cancr
that must
 trat, th nt
nfit to th wi- spra PSA scrning currntl
y on in th
Unit Stats may
 qustiona
l. 10 LAB TESTS FOR TUMOR MARKER DETECTION Clini
cians scrn for
th prsnc of malignancis
y a varity of mthos. 2 Commonly us tsts incl
u stool occult

loo an colonoscopy for colorctal carcinoma, Papanicolaou smar for crvical
cancr,
slf-xams for
rast an tsticular cancr, x-ray mammography for
rast cancr
, an igital
rctal xam for prostat cancr. La
oratory tsts can provi important ajunct
information to
patint historis an physical xams. Broaly, th thr typs of la
oratory mt
hos for cancr
scrning an iagnosis ar gross an microscopic morphol- ogy of tumors, tct
ion of
antign/protin tumor markrs, an DNA/RNA molcular iagnostics. Ths tchniqu
s ar
complmntary in that many of th DNA changs an su
squnt mRNA xprssion rs
ult in th
altr antigns/ protins tct or morphology visuali, so th choic of m
tho oftn
pns on convninc, cost, snsitivity, an spci city. Pathologists an histol
ogy la
s
procss suspct tumor tissu with gross issction an prparation of slis f
or microscopic
analysis. A varity of spcial stains, nuclic aci pro
s, an tumor markr ant
i
ois can

appli to th slis to nhanc th visi
l faturs. Evn so, valuation of mo
rphology an
staining pattrns can
 vry su
jctiv, an classi cation catgoris can
 rath
r
roa.
Consira
l skill is rquir to accuratly iagnos cancr
y morphology alon
, an nal
iagnosis is oftn ma with supplmntal clinical information an aitional t
sting, which is
scri

low. 14 Som of th molcular iagnostic tchniqus that hav
com
incrasingly
routin inclu th following: Cytogntic stuis: Many cancrs ar associat
with particular
karyotyps. Howvr, as mor prcis knowl- g of th xact gn fcts prs
nt in various
cancrs is gain, tsting for th a
rrant gns is
coming mor prvalnt. 14
Nuclic aci
amplification tchniqus: Polymras chain raction (PCR) an its variants incr
as th inhrnt lvl of DNA or RNA, allowing th tction of small populations of cancr c
lls (incluing
circulating clls in mtastasis) an th tction of mutations, ltions, an
gn
rarrangmnts/translocations. 14 (S Chaptr 11 for a complt iscussion of P
CR.)
Fluorscnt in situ hy
riiation (FISH): Nuclic aci pro
s capa
l of
ining
to squncs of

intrst ar tagg with uorophors an appli to clls. Clls con- taining th s
qunc of
intrst can
 visuali with uorscnt microscops. Similar tchniqus using n
on- fluorscnt
la
ls such as nyms an silvr stains ar also
coming availa
l. Caniat
DNA/RNA squncs
for gntic scrning of cancrs a
oun,
ut most ar still in th rsarch stag
. Th BCR-ABL
translocation associat with chronic mylognous lukmia is a wll-rspct m
arkr for this
isas, 15 an monoclonal xprssion of B-cll DNA rarrangmnt is prs- nt a
lmost xclusivly
in multipl myloma or othr lymphoi malignancy. 16 Most othr phnotypically r
lat cancrs
hav htrognous gntic causs, so univrsal an rlia
l gntic a
normaliti
s ar not yt
scri
. Th futur may li with microarray tsts that ar currntly
in
g vlop with
multipl nuclic aci tsts contain on a singl chip to allow for simu
ltanous tsting
of a sampl for multipl gns. On potntial us of this tchnology is
tction an
smiquantitation of mRNA xprssion in clls to istin- guish pattrns (rathr t
han singl
markrs) consistnt with cancr. 14 Som gntic a
normalitis ar associat
 with an
incras risk of vloping a cancr or with a poorr prog- nosis. Exampls of
suscpti
ility
gns ar th BRCA-1 an BRCA-2 mutations link with an incras risk of
ras
t, ovarian, an
prostat cancrs. 10 An xampl of a prog- nostic markr is ovrxprssion of th
 Hr2/nu
oncogn. Brast cancrs with this oncogn tn to
 mor aggrs- siv
ut w
ill mor likly
rspon to crtain thrapis (trastuuma
). 10 Antign/protin tumor markrs
ar su
stancs
xprss
y cancr clls or
y th
oy in rspons to th prsnc of cancr.
Th ial
tumor markr has th following charactristics: It must
 prouc
y th t
umor or as a
rsult of th tumor an must
 scrt into som
iological flui that can

analy asily
an inxpnsivly for lvls. Its circulating half-lif must
 long nough to p
rmit its
concntration to ris with incrasing tumor loa. It must incras to clinically
signi cant
lvls (a
ov
ack- groun control lvls) whil th isas is still trata
l
an with fw
fals ngativs (suf cint snsitivity). Th antign must
 a
snt from or
at

ackgroun lvls in all iniviuals without th malignant isas in qustion t
o minimi
fals-positiv tst rsults (suf cint spci city). 2,10,12 1814_Ch18_292-310.qx 7
/10/09 3:00
PM Pag 296 CHAPTER 18 Tumor Immunology 297 Non-nuclic aci tumor markrs
gnrally fall
into svn catgoris: cll surfac markrs, protins, oncoftal antigns, car

ohyrat
antigns,
loo group antigns, nyms/isonyms, an hormons. 12 Exampls
ar shown in

Ta
l 181. Most ar tct
y immunologic mthos with anti
ois to istinc
t pitops on
th molculs. Prostat-spci c antign (PSA), for xampl, is an nym,
ut it
is typically
tct as an antign. Tumor markrs ar not always irctly associat with th
 malignant
transformation. Oftn, thy ar th normal proucts of th tissu of origin
in
g xprss, an
this is mor likly if th tumor is wll iffrntiat. For xampl, nocrin
glan tumors
oftn prouc gnrous amounts of hormon that th tissu of origin proucs. Al
though thr ar
scors of possi
l tumor markrs in th litratur, lss than a on hav
FDA approval
as such. 10,12 Howvr, many non-FDA-approv markrs ar availa
l to clinician
s, with a
notation on th la
rport stat- ing that rsults ar for rsarch us only. Th
National Acamy
of Clinical Biochmistry has vlop a st of vry usful consnsus guili
ns rgaring
th clinical us of tumor markrs. 10 Thy list mthos an markrs for a vari
ty of purposs
that hav accpta
l vinc of valiity. Ths guilins rcommn vry fw m
arkrs for
scrning/arly tction an still rcommn using ajunct tsts or scrning h
igh-risk
populations. Ths markrs ar list in Ta
l 182. All othr rcommn markrs
hav various
uss in iagnosis an isas monitoring. Som of th most common an usful mar
krs ar list
in Ta
l 183, along with important noncancrous conitions that caus lvations.
Th nw l
of protomics mploys mass spctrom- try (MS) to intify an quantify an array
of protins
simultanously prsnt in a sampl. This has givn
irth to a nw l call onco
pptiomics. 17
Protin pro ling in cancr patints will ai in th iscovry of nw tumor mark- 
rs or pattrns
of protin xprssion that ar consistnt with cancr. Oncopptiomics may allow
mor su
tl
incrass of tumor markrs to hav iagnostic signi canc, sinc mul- tipl mark
rs can

masur an th ovrall pattrn assss,
ut this is currntly only at th
rsarch stag.
Thr ar som important aspcts to la
oratory tsting for tumor markrs. Most t
umor markrs ar
tct using anti
ois
caus of th spcificity of anti
ois an th gnr
al rlia
ility of
immunoassays. Howvr, thr ar som important limitations to using anti
ois
as ragnts.
Anti
ois ar irct at spcific pitops, an th anti-
ois from iffrn
t manufacturrs
may vary gratly in trms of what is masur, particularly if monoclonal anti
ois ar us.
This maks it important to us th sam mtho for monitoring patints ovr tim
, an clinicians
shoul
 awar of this if patints chang clinics or la
ora- toris. It also m
ans that if
la
oratoris switch mthos, thy must provi a transition prio uring which
spcimns ar

masur
y
oth mthos an spcimns ar archiv until nw ata is sta
lish
 for ach
patint. 10 Ta
l 18-1. Catgoris of Protin/Antign Tumor Markrs 2,12 TUMOR M
ARKER CLASS
EXAMPLES DISEASE ASSOCIATIONS Cll surfac markrs Estrogn/progstron rcp
tors Prognosis
for hormon thrapy in
rast cancr CD markrs on whit
loo clls Clonality a
n linag of
whit
loo cll noplasms Protins Thyroglo
ulin (TG) Wll-iffrntiat papil
lary or
follicular thyroi carcinoma Immunoglo
ulins (Ig) an Ig light chains Multipl
myloma an
lymphoi malignancis (Bnc Jons protins) Oncoftal antigns Alpha-1-ftoprot
in (AFP) Grm
cll carcinoma, hpatocllular carcinoma Carcinom
ryonic antign (CEA) Colorct
al carcinoma an
som othrs Car
ohyrat antigns CA 125 Ovarian cancr CA 15-3 Brast cancr Bl
oo group
antigns CA 19-9 (rlat to Lwis antigns) Pancratic an gastrointstinal can
crs
Enyms/isonyms Prostat-spci c antign (PSA) Prostat cancr Alkalin phospha
tas (ALKP)
Bon an livr cancr Nuron spci c nolas Nural tissu noplasms Hormons Huma
n chorionic
gonaotropin (hCG) Grm cll carcinoma, tropho
lastic tumors Calcitonin Mullar
y thyroi cancr
Gastrin Pancratic gastrinoma 1814_Ch18_292-310.qx 7/10/09 3:00 PM Pag 297
298 SECTION 3
Immun Disorrs Ta
l 18-2. Tumor Markrs Usful for Cancr Scrning Profssio
nal Consnsus
Rcommnations from th National Acamy for Clinical Biochmistry La
Micin
Practic
Guilins 10 CANCER TYPE MARKER ADJUNCT TEST POPULATION RECOMMENDED Prost
at
Prostat-spci c antign (PSA, Digital rctal xam Mn ovr 50 an with at last
10 yars of
lif total an fr) xpctancy Colorctal Fcal occult
loo Gntic tsting
Su
jcts ovr 50
yars ol for occult
loo; gntic tsting in high-risk su
jcts Livr Alpha-1
-ftoprotin
(AFP) Ultrasoun High-risk su
jcts Ovarian Car
ohyrat antign 125 (CA 125) U
ltrasoun
Su
jcts with family history of ovarian cancr Ta
l 18-3. Common Tumor Markrs
2,10,12,27
NONCANCEROUS NORMAL CONDITIONS WITH MARKER CANCER(S) USES* SOURCES ELE
VATIONS
COMMENTS AFP Nonsminomatous 1, 2, 3, 4 Ftal livr an Prgnancy, non- Scr
ning for
high-risk tsticular yolk sac, ault noplastic livr isas populations for
livr Grm cll
livr cancr such as thos Livr with livr cirrhosis an chronic hpatitis In g
rm cll tumors,

oth AFP an hCG ar lvat. 2 Lymphocyt 2 MHC class I In ammatory an high Hig
hr lvls
imply microglo
ulin malignancis cll turnovr conitions poor prognosis in mult
ipl myloma.
Calcitonin an Familial mullary N/A Thyroi In hyprcalcmia, incras Can

lvat in Ca
thyroi carcinoma calcitonin is xpct. othr forms of cancr. Srum Ca
may

low whn

calcitonin is lvat in mullary carcinoma. CD markrs Whit
loo cll N/A

All WBCs WBC
incras such as An array of CD markrs (WBC) infction ar associat with WBC
malignancis.
CEA Colorctal 2, 3, 4 Tissus of Rnal failur, non-noplastic Valus incras
 with Brast
ntormal livr an intstinal isas, ag ag an in smokrs. origin CA125 O
varian 1, 2, 3,
4 Various Enomtriosis, plvic Dont collct spcimn anocarcinoma in ammatory 
isas,
uring mnstruation utrin
rois, an (fals incras). prgnancy CA15-3 Bras
t 4 Mammary
Bnign
rast isas Epitop of pisialin. Can
 incras in tissu Bnign l
ivr isas 15-3
is monoclonal pancratic, lung, anti
oy. Othrs ar colorctal, ovarian, 27.29
an 549. an
livr cancrs CA 19-9 Pancratic 1, 2, 4 Sialyat Lwis a Bnign hpato
iliary
an Can

lvat in som
loo group pancratic conitions nonpancratic antign maligna
ncis. Su
jcts
who ar Lwis a an
ngativ cannot synthsi CA 19-9. 1814_Ch18_292-310.qx
7/10/09 3:00 PM
Pag 298 CHAPTER 18 Tumor Immunology 299 Ta
l 18-3. Common Tumor Markrs 2,10,
12,27 Cont
NONCANCEROUS NORMAL CONDITIONS WITH MARKER CANCER(S) USES* SOURCES ELE
VATIONS
COMMENTS ER/PR Brast 2 Brast N/A ER/PR
rast cancrs anocarcinoma
n t fro
m strogn/
progstron ruction thrapy. Fcal occult Colorctal cancr N/A N/A Sourcs o
f proxias in
Hm functions as a
loo fcs othr than
loo proxias an can
 (crtai
n foos) tct
chmically. Bttr spci city with anti
ois against glo
in chains of hmoglo
in.
hCG
Nonsminomatous 1, 2, 3, 4 Placnta Prgnancy hCG high homology with tsticular
cancr
lutiniing hormon (LH). Grm cll Malignancis can prouc tropho
lastic fr
alpha an
ta
chains (hyatiiform as wll as intact alpha/ mol, choriocar-
ta imr. Immun
oassays cinoma)
that tct only intact hCG shoul not
 us for tumor markr tction. In g
rm cll tumors,

oth AFP an hCG ar lvat. HER2/nu Brast 2 Growth factor N/A Cancr assoc
iat with gn
in all clls ovrxprssion of HER2/ nu an goo rspons to monoclonal anti
o
y thrapy
(trastuuma
) Monoclonal Plasma cll, B 1, 2, 3, 4 Normal Igs ar Monoclonal ga
mmopathy of Bnc
Jons protins ar fr Ig light lymphocyts polyclonal. Fw untrmin signi c
anc fr Ig
light chains in chains fr light chains (MGUS), amyloiosis, urin tct

y hating. xist.
nonscrtory myloma This mtho is largly historic. Monoclonal Plasma cll, B
1, 2, 3, 4
Normal Igs ar Monoclonal gammopathy Monoclonal IgG/IgA Igs lymphocyts polyclona
l. of
untrmin signi canc multipl myloma Monoclonal IgM Walnstrms macroglo
ulinmi
a PSA
Prostat 1, 2, 3, 4 No tissus othr UTI/prostatitis,
nign Lvls irctly pr
oportional than

prostat prostatic hyprtrophy to prostat si. Many lvations ar
nign or n

ot clinically
signi cant. Dcras prcnt of fr PSA an 0.5 mg/mL/year increase (PSA velocity
) more
associated with malignancy. Collect specimen be
ore ejaculation, digital rectal
exam, or prostate
manipulation. Continued 1814_Ch18_292-310.qxd 7/10/09 3:00 PM Page 299 300 SE
CTION 3 Immune
Disorders While antibodies are employed
or their speci
icity, it is not a
bsolute.
Antibodies will cross-react with similar structures, and this is particularly pr
oblematic when
the cross- reacting substances are in excessive amounts, as can occur in cancer.
For example, hCG
is made o
an alpha subunit and a beta subunit. The alpha subunit is virtually i
dentical to the
alpha subunit o
luteinizing hormone (LH), and the beta sub- units are 80 percen
t homologous, so
epitope choice is quite important. Further, assay con guration in uences what is mea
sured.
Cancers may produce
ree alpha and beta chains in addition to intact hCG, so an
immunochemical
method that relies only on beta chain epitopes to minimize LH inter-
erence wil
l measure
something completely di

erent than a method that sandwiches intact hCG between

an antialpha
capture antibody and an antibeta labeled antibody. 10 By virtue o
unchecked gro
wth and
aggressive metab- olism, some tumors may produce massive amounts o
tumor
marker
molecules. The prozone e

ect is a well- known limitation o

antibody-based
assays in which
antigen saturation o
antibodies inhibits the cross-linkage required to visualiz
e the reaction.
In immunoassays, a sim- ilar phenomenon has been called the high-dose hook e

ec
t, 18 and the
result is a
alsely decreased measurement, as shown in Figure 182. It is critical
that criteria
be developed to identi
y situations in which the hook e

ect may be present so t

hat specimens can
be diluted and accu- rate results obtained. A related problem o
antigen excess
in automated
systems is specimen carryover, so in addi- tion to diluting the specimen with ex
cessive antigen,
the specimen being tested immediately a
ter it may need to be repeated. Finally,
because many o

the antibodies used in immu- noassays are animal in origin, heterophile an

tibodies in
specimens can inter
ere pro
oundly with results. 18 In a tragic case involving

alse-positive
hCG results
rom an automated analyzer, several women had unnecessary chemothe
rapy or
hysterectomies
or presumed undetected cancer. 19 Although heterophile antibodie
s are mostly
asso- ciated with
alse increases by mechanisms similar to that shown in Figure
183,
alse
decreases are also possible. Antibody-blocking reagents are commercially availab
le (e.g.,
Scantibodies) to block heterophile antibodies in suspicious specimens, and many
manu
acturers are

employing block- ing agents within the routine reagents. Specimens with Table 18
-3. Common Tumor
Markers 2,10,12,27 Contd NONCANCEROUS NORMAL CONDITIONS WITH MARKER CANCER(S)
USES*
SOURCES ELEVATIONS COMMENTS PTH and Parathyroid 1, 2, 3, 4 Parathyroid In
hypocalcemia,
increased PTH has a short hal
-li
e, Ca carcinoma glands PTH is expected. Serum
so levels are
done Ca
may be high when intraoperatively to ensure PTH is elevated in complete p
arathyroid
parathyroid carcinoma. tumor removal. TG Thyroid 3, 4 Thyroid TG re ects thyroid m
ass, Assays
must simultaneously injury, and TSH levels. test
or thyroglobulin Thyroid mark
ers (T4,
antibodies (
alse decrease). TSH) generally normal O
ten tested a
ter TSH in th
yroid cancer.
stimulation to see i
TG increases. Also can withhold thyroid medication to incr
ease TSH in
patients with thyroid gland removed. Abbreviations: AFP
alpha-1-
etoprotein ; CA
carbohydrate
antigen; Ca
serum calcium; CD
clusters o
di

erentiation in WBC; CEA carcinoembry

onic
antigen; ER/PR
estrogen/progesterone receptors; hCG
human chorionic gonadotropin
; Ig
immunoglobulins; PTH
parathyroid hormone; PSA
prostate-speci c antigen; TG thyrogl
obulin;
UTI urinary tract in
ection * NACB recommendations: 1
diagnosis/case nding; 2
staging/prognosis; 3
detecting recurrence; 4
monitoring therapy Antigen concentr
ation Same
signal, di

erent concentrations Area o

antigen excess Optimum antigen area S i
g n a l
The Hook E

ect FIGURE 182. The high-dose hook e

ect. Antigen excess can

satu- rate
antibodies, and the intended sandwich con gurations cannot
orm, leading to a
alse
decrease in
signal. 1814_Ch18_292-310.qxd 7/10/09 3:00 PM Page 300 CHAPTER 18 Tumor Immun
ology 301
nonlinear behavior on dilution or with discrepant results using di

erent method
s or a
ter
applying antibody-blocking reagents may have heterophile antibodies and should n
ot be reported
until the issue is resolved. IMMUNOTHERAPY Immunotherapy is the nal aspect o
tum
or immunology
to be discussed. The possibility o
stimulating the patients own immune system to
respond to
tumor-associated anti- gens has long intrigued scientists. This chapter cannot c
over all the
di

erent protocols, but increasing knowledge con- cerning tumors and the im
mune system has
led to new research and recent optimism in this area. Immunotherapeutic meth
ods used can
be separated into two types: passive or active immunotherapy. Passive immunothe
rapy involves
trans
er o
antibody, cytokines, or cells to patients who may not be able to mou
nt an immune
response. With active immunotherapy, patients are treated in a manner that s
timulates them
to mount immune responses to their tumors. Passive Immunotherapy Passive trans

er o
allogeneic

cellular immunity
rom one person to another to ght cancer has many barriers beca
use o
possible
recipient rejection o

oreign cells, gra
t-versus- host disease (GVHD), and the

ragility o

live cells, although research models are being studied. Inducing a patients own c
ellular
immunity is
ar more likely to be success
ul, as dis- cussed in the next section
. However, a
orm
o
GVHD called gra
t versus leukemia has been demonstrated with trans-
er o
al
logeneic T cells
and is associated with improved patient prognosis. 20 There
ore, success
ul pass
ive trans
er o

anticancer T cells is theoretically possible. Adoptive T-cell therapy has be

en attempted
using several models. 21 For example, T cells
rom allogeneic donors can b
e immunized
against tumors. A
ter recipients are immunosuppressed to prevent rejection and t
o elimi- nate T
suppressor mechanisms, they receive the T cells. One strategy in this model to t
reat GVHD is to
genetically engi- neer the allogeneic T cells with a suicide switch. This gene mak
es the cells
vulnerable to a drug that will immediately eliminate them in the event o
GVHD.
Other strategies

or adoptive T-cell therapy use autolo- gous T cells. 21 Tumor-in ltrating lymphoc
ytes (TILs) can
be harvested and expanded in vitro using IL-2. The patient is then lymphodeplete
d to remove T
suppressor cells, and high concentrations o
TILs are trans
used. Similarly, aut
ologous T cells
can be harvested, exposed to cancer antigens, expanded with IL-2, and then retur
ned to the
patient. Attempts have also been made to insert genetically engineered T-cell re
cep- tors into
autologous T cells. 20 Passive trans
er o
antibody to treat cancer almost alway
s employs
monoclonal antibodies. Naked monoclonal anti- bodies against cancer could induce
antibody-dependent cell-mediated cytolysis (ADCC), complement-mediated lysis, or
opsonization. I

the antibodies are directed toward particular receptors, they could trigger a de
sirable action in
the cell such as inducing apoptosis or inhibiting growth signals. Antibody conju
gates, or
immunotoxins, are anti- bodies conjugated to toxins or radioisotopes on the prem
ise that they can
kill cancer cells while leaving adjacent cells intact. In order
or these techni
ques to work, the

ollowing are required: 22 Antibodies must be directed at a cell sur

ace antigen
with high
density. The antigen must be present on the primary tumor and on all metastatic

oci. Normal
tissues must be
ree
rom the antigen or not sus- ceptible to the toxin. The ant
ibodies must
have su
cient access to the tumor tissue (i.e., the tumor burden is not such that
certain
portions escape exposure to the antibodies). The obstacles involved in immunothe
rapy with
antibod- ies are: tumor heterogeneity with regard to antigenic expression; antig
enic
modulation or the loss o
antigen
rom the tumor cells.
ailure o
antibo

dies to
penetrate tumor tissue.
ailure o
antibodytoxin conjugates to internalize into t
he cell a
ter
binding and release toxin. toxic e

ects on other tissues, particularly the hema

topoi- etic
organs. the limited amount o
toxin that can be linked to the anti- body without
destruction o

binding activity. host immune response to the injected antibody. circulating ant
igen
orming
immune complexes with the antibodies, removing them
rom circulation. Table 184 s
hows the cancer
immunotherapy antibod- ies currently available in the United States. 22 Most ant
ibodies are
arti cially engineered, as the development o
heterophile Anti-tumor marker captur
e antibody
rom
sheep Labeled anti-tumor marker antibody
rom sheep Heterophile Ab against sheep
in specimen
bridges two antibodies as a tumor marker would Mechanism o

alse-positives due
to heterophile
antibodies FIGURE 183. Heterophile antibody inter
erence. Heterophile anti- bodie
s can cause
both
alse decreases and
alse increases, depending on their reactivity against
the antibody
species used in an assay. However, as shown,
alse increases are most likely.
1814_Ch18_292-310.qxd 7/10/09 3:00 PM Page 301 302 SECTION 3 Immune Disorders
antibodies in
patients receiving therapy is a signi cant inter-
erence. The chimeric antibod
ies splice
animal (usually murine) variable Fab onto human FC
or a
inal composi- tion o

about 25 percent
animal and 75 percent human. Humanized antibodies splice the portion o
animal ant
i- body
required
or epitope binding to human antibody
or a nal composition o
about 5 p
ercent animal
and 95 percent human. This improves antibody hal
-li
e, which is naturally long
in the absence o

heterophile antibodies. Long anti- body hal

-li
e is desirable
or therapeutic e

ects, but it
also prolongs negative e

ects. This is particularly a problem

or antibodies co
njugated to
radioisotopes, which cause bone marrow toxicity and myelosuppression. 22,23 Curr
ently,
immunotherapeutic antibodies are most e

ec- tive against hematologic malignanci

es, small tumors,
and micro metastases, not bulky tumors. 22,23 Antibodies poorly penetrate tumor
mass, because
they are large molecules, and they bind to the rst available antigen encountered
on the outside
o
the tumor. Surgical removal or debulking o
a tumor may ideally precede the u
se o
antibodies.
Further, since there is usually more than one path
or cell growth, antibodies
are rarely, i

ever, used alone. Combination Table 18-4. Currently Available FDA-Approved An

tibodies
or
Cancer Therapy 22 ANTIBODY BRAND GENERIC ANTIGEN THERAPEUTIC NAME NAME C
OMPOSITION
TARGET USE Avastin Bevacizumab Humanized IgG1 monoclonal Vascular endothelial
Prevent blood
vessel
ormation with murine binding region growth
actor (VEGF) in tumors. Era
dicate tumors
or

VEGF through lack o

blood supply, especially colorectal cancers and nonsmall ce
ll lung cancers
Bexxar Tositumomab Iodine-131 ( 131 I) murine IgG2a CD 20 on B cells Radioactiv
e immunotherapy
lambda monoclonal plus non-Hodgkins lymphoma unlabeled tositumomab Campath Alem
tuzumab Humanized
IgG1 monoclonal CD52 on almost T and Antibody-dependent lysis o
with murine bi
nding region

or B cells as well as some lymphocytes in chronic CD 52 other WBC lymphocytic

leukemia Erbitux
Cetuximab Chimeric monoclonal-murine Epidermal growth
actor Reduced proli
erat
ion o
Fab
variable portion and receptor (EGFR) cancers expressing EGFR, human IgG1 kappa
FC portion
especially colorectal cancers and their metastases, head and neck cancers Hercep
tin Trastuzumab
Humanized IgG1 kappa HER2/neu Reduced proli
eration o
breast monoclonal with m
urine cancers
overexpressing HER2/ binding region
or HER2 neu through ADCC, anti- angiogenesi
s, reduced cell
signaling
or growth Mylotarg Gemtuzumab Humanized IgG4 kappa CD33 on leukemic
Toxin
immunoconjugate ozogamicin monoclonal with murine blasts and immature therapyant
ibody is
binding region
or CD33 myelomonocytic cells internalized and toxic linked to c
alicheamicin
calicheamicin kills cells in acute myelogenous leukemia ProstaScint Capromab In
dium-111 ( 111
In) murine Prostate-speci c Radioactive immunotherapy pendetide IgG1 kappa monocl
onal membrane
antigen o
prostate cancer Rituxan Rituximab Chimeric monoclonal-murine CD 20 on
B cells ADCC,
complement lysis, and Fab variable portion and induced apoptosis when two human
IgG1 kappa FC
portion CD20 molecules bind in non-Hodgkins lymphoma Zevalin Ibritumomab Yttrium
-90 ( 90 Y)
labeled CD 20 on B cells Radioactive immunotherapy chimeric monoclonal- o
non-H
odgkins
lymphoma murine Fab variable portion and human IgG1 kappa FC portion plus unlabe
led rituximab
1814_Ch18_292-310.qxd 7/10/09 3:00 PM Page 302 CHAPTER 18 Tumor Immunology 30
3 therapy with
chemotherapeutic agents or other treatments is usually recommended. 22 Radiolabe
led antibodies
are particularly e

ective because o
the bystander or cross
ire e

ect seen
with
high- energy isotopes that can kill up to several hundred cells. This is particu
larly
advantageous
or tumors with hetero- geneous antigen expression and could allow
antibodies to
kill cells that are not expressing antigen at all. An additional use
or radiola
beled antibodies
is imaging studies to locate
oci o
cancer. However, their use must be managed
to min- imize
bone marrow toxicity, although this toxicity is generally reversible. 22,2
3 Currently,
there are no FDA-approved antibodytoxin conjugates, although denileukin di
tit
ox, a
recombinant IL-2 coupled with modi
ied diphtheria toxin, has been approve
d
or T-cell

lymphoma. Only one antibodydrug conjugate (gemtuzumab ozogamicin) has been approv
ed. 22
Conjugation can theoretically allow the use o
ultratoxic drugs and some extreme c
ytotoxins,
because the agents deliv- ery is limited and
ocused by the antibody. Several cyt
otoxins are
under study, including diphtheria toxin, Pseudomonas exo- toxin, abrin, ricin, a
nd saporin. Since
toxins have their own binding mechanisms, antibodies must bypass the normal mech
anism or the
toxin must be modi
ied. Further, some cytotoxins must be internalized to be e

e
ctive, so the
anti- body must target an epitope that would be internalized. 22 Active Immunoth
erapy The goal o

active immunotherapy is to have the patient develop an immune response that will
help eliminate
the tumor. Nonspeci c stimulation by adjuvants such as Bacillus Calmette Guerin (B
CG) was
irst
attempted, and super
i- cial bladder cancer is still treated with BCG. 24 Improv
ed technology has
allowed the production o
novel adjuvants and selective use o
stimulatory cytok
ines (TNF-,
IFN-, IL-1, IL-2, and so on) in immunocompetent patients to enhance the natural a
ntitumor
response and the artificial vaccine-induced response. 6 Other attempts at stimul
atin host immune
systems have involved transfection of normal cells or isolated tumor cells with
enes for
cytokine production and injection of the modified cells into or around the tumor
. 25 This has

een done with many cytokines, includin TNF-, interferons, IL-2, nd gr nulocyte
monocytecolony stimul ting f ctor (GM-CSF). Of the cytokines tr nsfected, GM-CSF
h s shown the
most promise. 25 C ncer v ccines h ve been of gre t interest to rese rchers. Whe
n speci c viruses
re ssoci ted with
c ncer, v ccine construction is rel tively str ightforw rd
, since vir l
ntigens re obviously foreign. The v ccine for hum n p pillom virus (HPV) to pr
event cervic l
c ncer is n excellent ex mple. It is import nt to note th t m ny viruses h ve s
ever l serotypes,
not ll of which m y be ssoci ted with c ncer, so v ccines must be protective
g inst the
ppropri te epitopes. HPV v ccines, for ex mple, re directed epitopes th t prev
ent initi l
infection with c rcinogenic serotypes but do not help tre t est blished cervic l
c ncer, s these
epitopes re down- regul ted in c ncer cells. 7 Therefore, distinction exists
between
prophyl ctic v ccines nd ther peutic ones. In the bsence of vir l c use of
c ncer,
prophyl ctic v ccin tion is more difficult, since m ny of the ntigens expressed
on tumors, s
st ted previously, ppe r to some degree on norm l tissue. High- f nity cytotoxic
T lympho- cytes
g inst tumor- ssoci ted ntigens (TAA) re often deleted by toler nce p thw ys.
20 Further, just
the expres- sion of n ntigen on tumor does not utom tic lly m ke it suit
ble v ccine
t rget, since coexpression with MHC I nd oblig tory ccessory molecules is requ

ired for dequ te

T-cell response. V ccin tion with tumor lys tes nd peptide ntigen v ccines h v
e h d limited
success. This is t le st p rtly due to the heterogeneity of ntigens presented
on tumors nd the
few public ntigens common to ll tumors nd the m ny priv te ntigens unique to n
individu ls tumor. Designing custom v ccines for e ch individu l is obviously mo
re difficult
th n designing more univers l v ccines. V rious str tegies h ve been ttempte
d to identify
suf - ciently immunogenic ntigens nd how to present them to T cells. They re to
o numerous to
outline here. However, n ex mple is the str tegy to prime dendritic cells (DCs)
, which re
p rticul rly potent ntigen-presenting cells th t c n stimul te both humor l
nd cellul r
immunity nd immune toler nce re ctions. The gener l principle of this techniqu
e is th t DC
line is cultured from the p tient nd then exposed to tumor ntigen. Antigen exp
osure m y be done
using coculture with puri ed tumor ntigens or tumor lys te, fusion of DCs with tu
mor cells, DC
ph gocytosis of de d tumor cells, or tr nsfection of RNA. 26 The DCs re reintro
duced in the
p tient by v rious me sures (intr - venously, subcut neously, nd so on) s
v ccine.
Prelimin ry rese rch h s demonstr ted some success with these v ccines, but the
det ils of
optim l ntigens, DC priming, nd modes of DC delivery re still being ev lu ted
. It rem ins to
be seen which, if ny, of the numerous immuniz tion protocols currently under st
udy will work.
These protocols will be import nt djuncts to tr dition l ther pies in which tum
ors will first be
debulked nd then the immune system will er dic te residu l tumor nd mic
romet st ses. 25
The incre sed underst nding of tumor immunology in recent ye rs h s m de this
e
ld of ctive
study nd incre sed optimism. SUMMARY Tumor immunology is the study of the ntig
ens ssoci ted
with tumors, the immune response to tumors, the tumors effect on the hosts imm
une st tus,
nd the use of the immune system to help er dic te the tumor. C ncer begins
with ch nges in
one norm l cell, nd these ch nges llow 1814_Ch18_292-310.qxd 7/10/09 3:00 PM
P ge 303 304
SECTION 3 Immune Disorders the cell to grow nd prolifer te without reg rd to no
rm l growth
sign ls or controls. As these bnorm l cells grow nd continue to mut te, they m
y show
differences in gene expres- sion both in type nd mount. Antigens expressed in
gre ter mounts
in tumor tissue re c lled tumor- ssoci ted ntigens. There is evidence th t imm
unosurveill nce
m y protect g inst growth of some tumors. T cells nd NK cells c n recognize tu
mor cells s
foreign nd destroy them. Ultim te development of c ncer m y be due to immunoedi
ting by cellul r
immunity. Very immunogenic tumor cells re elim- in ted, cre ting n environment
th t llows

tumor cells to ev de the immune system. The clinic l l bor torys role in detectin
g nd monitoring c ncer is b sed on the presence of v rious tumor m rkers. These include lte
red
ntigen/receptor expression, bnor- m l genes, overproduced cellul r products,
nd subst nces
produced in response to the tumor. Assessment of tumor m rkers h s been pplied
for tumor
screening, di gnosis, histop thologic ev lu tion, st ging, dise se monitoring, l
oc l- iz tion of
met st sis, nd ther py selection. Dise se prev lence in popul tion profoundly
ffects the
clinic l utility of tumor m rker testing. Existence of dise se in symptom tic p
opul tions will
be low, so the m jority of positive tumor m rker tests will be f lse positives.
Few tumor m rkers
re recommended for screening, nd most should be used in conjunction with other
tests or in
high-risk pop- ul tions. Dise se prev lence is higher in popul tions being diffe
renti lly
di gnosed or monitored for known c ncer, so the utility of tumor m rker ss ys f
or these purposes
is f r superior. Tumor m rker tests re gener lly best utilized to follow the co
urse of known
c ncer, since the m rkers trend up or down is more import nt th n its bsolute v
lue. Both
p ssive nd ctive immunother py directed t ppropri te tumor m rkers h ve
begun to show
some signs of success. While most immunother py is still t the rese rch st ge,
sever l
monoclon l
ntibodies h ve g ined FDA pprov l for c ncer tre tment. The hum
n p pillom virus
v ccine is FDA pproved for c ncer prevention, so the con- tinued identific tion
of tumor m rkers
nd elucid tion of the immune systems response g inst c ncer is worthwhile.
1814_Ch18_292-310.qxd 7/10/09 3:00 PM P ge 304 CHAPTER 18 Tumor Immunology 30
5 1. A
45-ye r-old wom n went to her physici ns of ce fter noticing lump during her bre
st
self-ex m. She h d strong f mily history of bre st c ncer. The lump w s detect
ed on
m mmogr phy
nd w s found to be
0.5 cm m ss th t w s dherent to her ski
n. An lysis found
her CA-15.3 levels to be 60 IU/mL, which is double the upper limit of the refere
nce interv l.
After surgery, the levels of CA-15.3 dropped, but t r te th t w s slower th n
the biologic l
h lf-life. They rem ined bove 30 IU/mL. The tumor morphology indic ted m lign n
cy, so it w s
tested for Her2/neu expression, which w s elev ted, nd estrogen/progesterone re
cep- tors, which
were neg tive. Questions . Do you think th t there is residu l tumor, nd why?
b. In ddition to
chemother py, wh t other ther py would you recommend, nd why? c. Wh t type of t
her py is
unlikely to be successful, nd why? 2. Sever l weeks go, 25-ye r-old wom n h
d thyroidectomy to remove
well-differenti ted follicul r c rcinom . After surgery,
her thyroglobulin
(TG) level decre sed to undetect ble. She rem ins symptom tic but w s sked

to discontinue
her thyroid hormone repl cement ther py for 2 weeks nd return for the l b- or
tory
determin tions indic ted in the t ble below: Questions . Wh t test bove is don
e to ensure
ccur cy of the TG test? Is this TG me surement likely to be ccu- r te or not?
b. Wh t test
bove is done to verify th t residu l thyroid c ncer is likely to be
producing TG?
Is rem ining c ncer being stimul ted or not? c. Wh t does this TG level indic te
in the p tient?
3. A 66-ye r-old bl ck m le went to
urologist compl in- ing of frequent urin t
ion with only
sm ll volumes of urine, cre ting gre t urgency. During the digit l rect l ex m,
the urologist
felt n enl rged prost te with no distinct nodules or bnorm l re s. The p tien
ts serum level
of prost te-speci c ntigen (PSA) w s determined nd comp red to the level from th
e previous
ye r. The physici n lso sked th t the bound-to-tot l PSA r tio be determined.
The test results
re shown below. Questions . Do ny tissues other th n the prost te produce PSA
? Could there
be nother source of the PSA in this c se? b. Wh t is PSA velocity? c. Sho
uld this m n h ve
biopsy? Do you think this m n h s c ncer? Why? d. At wh t point is it unethic
l to test for
PSA? CASE STUDIES PATIENT REFERENCE TEST RESULTS INTERVAL PSA October 2008
3.9 ng/mL 03.5
ng/mL PSA October 2007 3.5 ng/mL 03.5 ng/mL Bound/free PSA 25.8% 23.4% Laborator
y Results
PATIENT REFERENCE RESULTS RANGE TG 7 ug/L
5 ug/L in athyroidic patients Anti
-TG antibodies
None detected
1:10 Thyroid-stimulating 10.7 mU/L 0.44.2 mU/L hormone (TSH)
1814_Ch18_292-310.qxd 7/10/09 3:00 PM Page 305 306 SECTION 3 Immune Disorders
SEMIQUANTITATIVE
PSA MEMBRANE TEST FOR THE DETECTION OF ELEVATED SERUM LEVELS OF PSA Principle* T
his is a
solid-phase chromatographic capture membrane enzyme immunoassay
or the detectio
n o
PSA in
patient serum. Elevated PSA is
ound in men with prostate cancer and in men with
benign prostatic
hypertrophy. O
ten, age-adjusted cuto

s are used or compared to previous measur

ements (PSA
increase velocity). This assay uses an internal standard o
4 ng/mL to allow a v
isual comparison
o
the specimen reac- tion to determine i
it exceeds 4. Patients without tumors
rarely have
levels above 10 ng/mL, but many patients with tumors and those with benign prost
atic hypertrophy
will be in the 4 to 10 ng/mL range. A positive above 4 will warrant
urther inve
stigation with
a quantitative test and a digital rectal examination. This rapid test
or
PSA per
orms
according to the
ol- lowing principle: The membrane o
the test cassette
is impregnated
with murine monoclonal antihuman PSA as the immobilized antibody in the test reg
ion and with
immo- bilized antimurine antibody in the internal standard and control regions.
Antibodies in the

internal standard region are adjusted to re

lect a positive reaction consistent
with 4 ng/mL o

PSA. Antihuman PSA monoclonal conjugate labeled with red-colored gold is impregn
ated in the
cassette but not immobilized. When serum is applied, PSA in the patients serum re
acts with the
red-colored monoclonal mouse anti-PSA. Through the capillary e

ect in the membrane o

the test
cassette, the reaction mixture is carried to each o
the areas with immobilized
antibodies. I

PSA is present, it will be captured by anti-PSA in the test region, and the redlabeled antibody
can be visualized. Mobilized red-gold antimurine antibodies are bound in the int
ernal standard
and control regions by the murine antiglobulin, independent o
the existence o

PSA. A
ter the
device is incubated at room temperature
or 10 minutes, a visual interpretation
is per
ormed. No
line in the test region or one lighter in color than the 4 ng/mL line is a negat
ive result, and
a line as dark or darker than the 4 ng/mL standard is a positive result
(Fig. 184). In
this test system, the high-dose hook e

ect (prozone) has been observed at conce

ntrations
exceeding 500 ug/mL. Further,
ree PSA reacts slightly better than complexed PSA
, reducing the
tests speci city
or cancer as compared to other quantitative methods. EXERCISE Sam
ple well Test
result Control result Internal standard equivalent to 4 ng/mL PSA SERATEC PSA Te
st Cassette C
T
FIGURE 184. A schematic showing the Seratec PSA test cassette. In this ex
ample, the patient
line T has more PSA than internal stan- dard, representing 4 ng/mL control, and
the control line
C indicates a valid reaction. (This test is available as seratec PSA semi-quant
[re
psm400t]

rom Serological Research Institute, Joan Matthew, 3053 Research Drive, Richmond
, CA 94806, USA
[Telephone: 1-510-223-737; Fax: 1-510-222-8887; e-mail: contact@seratec.com or
jmatthew@serological.com]. Above in
ormation taken
rom seratec PSA semi-quant [
re
psm400t]
literature rev. May 2005.) * This test is available
rom Seratec (Telephone:
49
551 50480-0;
Fax: 49 551 50480-80; e-mail: seratec@t-online.de). 1814_Ch18_292-310.qxd 7/10/
09 3:00 PM
Page 306 6. Both AFP and hCG exhibit serum elevations in a. pregnancy. b. ovaria
n germ cell
carcinoma. c. nonseminomatous testicular cancer. d. all o
the above. 7. Calcito
nin and
parathyroid hormone levels should not be evaluated as indicating possible tumors
without also
measuring serum a. CEA. b. calcium. c. thyroglobulin antibodies. d. thyroid horm
ones (TSH, T4).
8. Consensus guidelines indicate enough evidence to use all o
the
ollowing
or
cancer screening
in the groups indicated except a. CA-125/women o
reproductive age. b. AFP/subje
cts at high risk


or liver cancer. c.
ecal occult blood/people over 50 years o
age. d. PSA/men
over 50 with at
least 10 years o
li
e expectancy. 9. A 57-year-old man had a massive tumor remo
ved
rom his
colon. His serum specimen had the
ollowing results using an automated ELISA ant
ibody sandwich
assay
or CEA. The stated linearity o
the test is 100 ug/L. What should be done
? FINAL RESULT
TEST USING DILUTION ABSORBANCE RESULT FACTOR Undiluted 1.026 107.1 ug/L 10
7.1 ug/L specimen
Specimen 1.269 125.7 ug/L 1257.0 ug/L
10 Specimen 0.995 95.8 ug/L 9580.0 ug/L
100 Specimen
0.101 9.6 ug/L 9600.0 ug/L 1000 a. Retest all specimens using a di

erent kit lo
t. b. Retest
the specimen at 1:10,000 and 1:100,000 dilutions. c. Retest the specimen using h
eterophile
antibody blocking reagent. d. Retest any specimen pipetted a
ter the undiluted s
pecimen and
report 9580.0 ug/L
or this specimen. CHAPTER 18 Tumor Immunology 307 1. How can
normal cells
become malignant? a. Overexpression o
oncogenes b. Underexpression o
tumor-sup
pressing genes c.
Viral in
ection d. All o
the above 2. Which o
the
ollowing best summarizes th
e concept o

tumor development via immunoediting? a. Cytokines produced by tumor cells are to

xic to T cells
expressing receptors
or tumor-associated antigens. b. Cells that can escape the
immune system
have a growth advantage over more immunogenic tumor cells that are destroyed by
T cells during
immunosurveillance. c. T-cell activity causes an up-regulation o
MHC expression
on tumor cells
that allows them to escape the immune system. d. Expression o
secreted tumor-as
sociated antigen
saturates T-cell receptors and renders them incapable o
binding to the actual t
umor cells. 3. A
woman goes 3 days per week to a tanning bed
or 20 minutes o
UV expos
ure per visit.
Which stage o
cancer is this? a. Induction b. In situ c. Invasion d. Disseminat
ion 4. I
a
disease is present in 1 in every 10,000 individuals, and a marker can detect thi
s disease with
100 percent sensitivity and 95 percent speci city, what would be the number o

al
se positives

or each cancer
ound? a. 5 b. 50 c. 500 d. 5000 5. Each o
these markers ma
y be elevated
in multiple myeloma except a. CA-125. b. -2 microglo
ulin. c. monoclonal intact
immunoglo
ulin
molculs. . fr monoclonal light chains from th immunoglo
- ulin molcul. R
EVIEW QUESTIONS
1814_Ch18_292-310.qx 7/10/09 3:00 PM Pag 307 308 SECTION 3 Immun Disorrs
10. A tumor
foun in th prostat os not stain with anti-
oy to PSA. Is this proof that
th tumor cam
from a iffrnt organ? a. Ys
. No 11. In orr to us a tumor markr to monit
or th cours of
th isas, which of th following must
 tru? a. Th la
oratory masurs th
markr with th
sam mtho ovr th ntir cours of th patints tratmnt.
. Th markr must

 rlas

from th tumor or
caus of th tumor into a
oy ui that can
 o
tain an t
st. c. Th
markrs half-lif is such that th markr prsists long nough to r ct tumor
ur
n
ut clars
fast nough to intify succssful thrapy. . All of th a
ov. 12. Which of th
 following
markrs coul
 lvat in
nign livr isas? a. AFP
. CEA c. CA 15-3 . C
A 19-9 . All of
th a
ov 13. Choos th incorrct statmnt. a. Srum CA 19-9 lvls shoul not

 collct
from smokrs.
. Srum CA-125 spcimn shoul not
 collct from womn who ar
 mnstruating.
c. Fcs for occult
loo shoul not
 collct from su
jcts who rcntly at
proxiascontaining foos. . Srum PSA spcimns shoul
 collct
for any manipula
tion of th
prostat, incluing igital rctal xam. 14. Immunotoxin anti
ois us for can
cr thrapy ar
humani to prvnt su
jcts from vloping a. graft-vrsus-host isas.
. ht
rophil
anti
ois. c. mylosupprssion. . srum sicknss. 15. Each markr
low is cor
rctly pair
with a isas in which it can
 us for conitional monitoring xcpt a. CEA/
choriocarcinoma.

. CA-15.3/
rast anocarcinoma. c. CA 125/ovarian anocarcinoma. . CA-19.9/p
ancratic
anocarcinoma. 1814_Ch18_292-310.qx 7/10/09 3:00 PM Pag 308 CHAPTER 18 Tum
or Immunology 309
Rfrncs 1. Caull, K. Altrations in cll growth an noplasia. In Porth, C
(): Essntials
of Pathophysiology. Lippincott Williams & Wilkins, Philalphia, PA, 2004. 2. El
kins, B.
Noplasia. In Kaplan, L, Psc, A, Kamircak, S (s): Clinical Chmistry: Th
ory, Analysis an
Corrlation. Mos
y, St. Louis, MO, 2003. 3. A

as, A, an Lichtman, A. Cll
ular an
Molcular Immunology, . 5. WB Saunrs, Philalphia, PA, 2003. 4. Roitt, I, B
rostoff, J, an
Mal, D. Immunology, . 6. Mos
y, Lonon, Englan, 2001. 5. Kim, R, Emi, M, an
Tana
, K.
Cancr immunoiting from immun survillanc to immun scap. Immunology 121:
114, 2007. 6.
Rao, V, Dyr, C, Jaml, J, Drw, P, an Grman, J. Potntial prognostic an
thraputic
rols for cytokins in
rast cancr. Oncol Rp 15(1):179185, 2006. 7. Lggat
t, G, an
Frar, I. HPV vaccins: Th
ginning of th n for crvical cancr. Curr Op I
mmunology
19:232238, 2007. 8. Sturgon, C. Practic guilins for tumor markr us in th
clinic. Clin
Chm 48(8):11511159, 2002. 9. Pritkr, K. Cancr
iomarkrs: Easir sai than o
n. Clin Chm
48(8):11471150, 2002. 10. National Acamy of Clinical Biochmistry. La
orat
ory Micin
Practic Guilins: Tumor Markrs. Draft Guilins 2005. www.nac
.org. Acc
ss Sptm
r
2007. 11. Tuma, R. Inconsistncy of HER2 raiss qustions. J Natl Cancr
Inst
99(14):10641065, 2007. 12. Sokoll, L, an Chan, D. Tumor markrs. In Clark, W, a
n Dufour, D

(s): Contmporary Practic in Clinical Chmistry. AACC Prss, Washington, DC,

2006. 13. Lo
,
S, an Catalona, W. Prostat-spcific antign in clinical practic. Cancr
Lttrs
249:3039, 2007. 14. Cross, D, an Burmstr, J. Th promis of molcular pro l- ing
for cancr
inti cation an tratmnt. Clin M & Rs, 2(3):147150, 2004. 15. Mlo, J, an Ba
rns, D.
Chronic myloi lukamia as a mol of isas volution in human cancr.
Nat Rv Cancr
7(6):441453. 2007. 16. Ros, N, Hamilton, R, an Dtrick, B. Manual of Clinical L
a
oratory
Immunology. ASM Prss, Washington, DC, 2002. 17. Tammn, H, Zucht, H, an Bu,
P.
Oncopptiomicsa commntary on opportunitis an limitations. Cancr Lttrs 249:
8086, 2007.
18. Clark, W. Immunoassays. In Clark, W, an Dufour, D (s): Contmporary Pra
ctic in Clinical
Chmistry. AACC Prss, Washington, DC, 2006. 19. Col, LA, Rinn, KM, Shaha

i, S, an
Omrani, A. Fals positiv hCG lvls laing to unncssary surgry an
chmothrapy,
an nlss occurrncs of ia
ts an coma. Clin Chm 45:313314, 1999. 20. Xu
, S, an
Stauss, H. Enhancing immun rsponss for can- cr thrapy. Cll Mol Immunol 4(3
):173184, 2007.
21. Jun, C. Aoptiv T cll thrapy for cancr in th clinic. J Clin Invst. 11
7(6):14661476,
2007. 22. Sharky, R, an Goln
rg, D. Targt thrapy of cancr: Nw prospc
ts for anti
ois
an immunoconjugats. CA Cancr J Clin 56(4):226243, 2006. 23. Goln
rg, D, an
Sharky, R.
Avancs in cancr thrapy with raiola
l monoclonal anti
ois. Q J Nucl M
Mol Imaging
50(4):248264, 2006. 24. Oostrlinck, W. Guilins on iagnosis an tratmnt of
supr cial

lar cancr. Minrva Urol Nfrol 56(1):6572, 2004. 25. Li, C, Huang, Q, an Ku
ng, H. Cytokin
an immuno-gn thrapy for soli tumors. Cll Mol Immunol 2(2):8191, 2005. 26. S
chott, M.
Immun survillanc
y nritic clls: Potntial implication for immunothrapy
of nocrin
cancrs. Enocr Rlat Cancr 13(3):779795, 2006. 27. Wu, A. Tit Clinical Gu
i to
La
oratory Tsts, . 4. Saunrs, Philalphia, PA, 2006. 1814_Ch18_292-310.
qx 7/10/09
3:00 PM Pag 309 1814_Ch18_292-310.qx 7/10/09 3:00 PM Pag 310 IV SECTION S
rological
Diagnosis of Infctious Disas 311 1814_Ch19_311-327.qx 7/10/09 3:53 PM Pag
 311 19
Srological an Molcular Dtction of Bactrial Infctions Dorothy Fik MS, MT(
ASCP) SBB an
Christin Stvns LEARNING OBJECTIVES Aftr nishing this chaptr, th rar will

 a
l to: 1.
Discuss mchanisms of host fns against
actria. 2. Discuss how crtain
act
ria va th
immun systm. 3. Dscri
 how th M protin contri
uts to th virulnc of Str
ptococcus
pyogns. 4. Dscri
 symptoms of acut rhumatic fvr. 5. Discuss how pathogn
sis occurs in

glomrulonphritis. 6. List an scri
 v xoantigns prouc
y group A strp

tococci. 7.
Discuss rasons for prforming anti
oy rathr than antign tsting for squla
of strptococcal
infctions. 8. Stat th principl of th antistrptolysin O (ASO) titr tst. 9
. Explain how th
prsnc of anti-oxyri
onuclas B (anti-DNas B) is tct. 10. Compar th
snsitivity of
th strptoym tst to othr tsts for strptococcal anti
ois. 11. Intrprt
la
oratory ata
to iagnos squla of strptococcal infctions. 12. Discuss how Hlico
actr p
ylori may caus
ulcrs. 13. Discuss th various typs of tsting that may
 on to tct H. p
ylori infction.
14. Explain th tsting that may
 on to tct anti
ois to H. pylori. 15.
Discuss how
Mycoplasma pnumonia may caus infctions. 16. Explain th anti
oy tsting tha
t is prform to
tct infctions to Mycoplasma pnumonia. 17. Intify srological an molcul
ar tchniqus to
tct rickttsial infctions. KEY TERMS Acut rhumatic fvr Anti-DNas B ASO
titr Exoantign
Group A Strptococci Hlico
actr pylori Lanc l group Mycoplasma pnumonia Pos
tstrptococcal
glomrulonphritis Rickttsia Strptolysin O Strptoym Spott Fvr Typhus U
ras 312
1814_Ch19_311-327.qx 7/10/09 3:53 PM Pag 312 CHAPTER 19 Srological an Mol
cular Dtction
of Bactrial Infctions 313 IMMUNE DEFENSES AGAINST BACTERIAL INFECTIONS AND MEC
HANISMS OF
EVASION Prvious chaptrs in this txt hav iscuss
oth innat an spci c immu
n rsponss
that srv as protction from for- ign antigns, incluing
actria. Th rst lin
 of fns
against pathogns is intact skin an mucosal surfacs that srv as structural

arrirs.
Lysoym, an nym foun in many scrtions such as tars an saliva, is capa
l
 of stroy- ing
many
actria. Othr solu
l innat componnts that protct us from pathogni
c
actria ar
intrlukins, prostaglanins, an lukotrins, which caus fvr, an acut pha
s ractants such
as C-ractiv protin, haptoglo
in, an cruloplasmin, which ithr coat
actri
a or rmov su
stancs that might promot
actrial growth. Phagocytosis
y nutrophils, monocy
ts, an
macrophags is anothr mch- anism of innat immunity that will protct us again
st
actria. Th
phagocytic procss is nhanc
y th activation of th altrnativ complmnt c
asca, which is
triggr
y micro-
ial cll walls or othr proucts of micro
ial mta
olism. T
h spci c immun
rspons inclus formation of anti-
ois that ar prouc to xotoxins or ot
hr scrt

actrial proucts. Anti
oy formation is th main fns against xtracllular

actria.
Anti-xotoxin anti
ois
in to th xotoxin an form immun complxs that ar
lim- inat.
Through this procss,
actrial toxins ar nutrali an ffctivly prvnt
from causing

pathology. In ai- tion, aftr anti
oy formation, th classical pathway

of complmnt is
activat, laing to lysis of
actrial clls an nhancmnt of in ammatory ff
cts.
Opsoniation an phagocytosis can also occur whn th FC rcptor on th phagocy
tic cll
ins to
th FC rgion of th IgG anti
oy molcul. Cll-miat immunity, th othr
r
anch of th
spci c immun rspons, is hlpful in attacking intracl- lular
actria, such as
Myco
actrium
tu
rculosis, Lgionlla pnumophila, Listria monocytogns, an Rickttsia sp.
Bactria hav
vlop svral ways to inhi
it th immun rspons or mak it mor if cul
t for th
immun rspons to occur. Thr main mchanisms ar avoiing anti-
oy,
lockin
g phagocytosis,
an inactivating th complmnt casca (Fig. 191). On mchanism of vaing th
ffcts of
anti
oy occurs through altring
actrial antigns CHAPTER OUTLINE IMMUNE
DEFENSES AGAINST
BACTERIAL INFECTIONS AND MECHANISMS OF EVASION GROUP A STREPTOCOCCI Clinical Man
ifstations of
Group A Strptococcal Infction Dtction of Group A Strptococcal Antigns Dt
ction of
Strptococcal Anti
ois HELICOBACTER PYLORI Charactristics of Hlico
actr Pyl
ori Infctions
Antign-Dtction Procurs Dtction of Hlico
actr Pylori Anti
ois MYCOPLA
SMA PNEUMONIAE
Charactristics of Mycoplasma Infction Dtction of Mycoplasma Pnumonia
y Cu
ltur Dtction
of Anti
ois to Mycoplasma Pnumonia Molcular Tchniqus RICKETTSIAL INFECTIO
NS
Charactristics of Rickttsial Infctions Srological Diagnosis Molcular Tchni
qus SUMMARY CASE
STUDIES EXERCISE: STREPTOZYME TEST REVIEW QUESTIONS REFERENCES Digstion Cll 

ris Ahrnc
Diapsis R
loo clls Enothlium Nutrophil IgA C3
C3
Encapsulat
act
ria Chmotaxis C
E D A B FIGURE 191. Ways
actria ar a
l to va host fnss. (A) Inhi
iting
chmotaxis.
(B) Blocking ahrnc of phagocyts to th
actrial clls. (C) Blocking igst
ion
y toxin
prouction, which kills th phagocyt. (D) Inhi
iting
ining of complmnt comp
onnt C3
. (E)
Clvag of IgA. 1814_Ch19_311-327.qx 7/10/09 3:53 PM Pag 313 314 SECTION 4
Srological
Diagnosis of Infctious Disas (pitops) as a rsult of gntic mutations. Som
 strptococci
hav this capa
ility. In this situation, th spci c anti
ois vlop
y th h
ost
com
inffctiv, an rsponss to th nw antigns must vlop. Bactria can also 
va th spci c
immun rspons through own-rgulation of MHC molculs an prouction of prot
ass that gra
IgA. 1 Nissria gonorrhoa, Hamophilus in una, an Strptococcus sanguis ar
all xampls
of
actria that can clav IgA. 2,3 Howvr, most of th vasion mchanisms tar
gt th procss
of phagocytosis. Bactria can mount a fns at svral stags in th procss,
incluing

chmotaxis, ah- sion, an igstion. 2,3 Som pathogns such as Nissria gono
rrhoa, for
xampl, inhi
it th rlas of chmotactic factors that woul
ring phagocytic
clls to th
ara. Th cll walls of S. pyogns prouc an M protin that intrfrs with a
hsion to th
phagocytic cll. Aitionally, th prs- nc of a polysacchari capsul foun
in such organisms
as Nissria mningitiis, Strptococcus pnumonia, Yrsinia pstis, an Hamop
hilus in una
inhi
its th a
ility of nu- trophils an macrophags to
in to initiat phagoc
ytosis. 3
Microorganisms us svral iffrnt mchanisms to rsist igstion. Som

actria can
lock
fusion of lysoso- mal granuls with phagosoms aftr
ing ngulf
y th phago
cyt. Salmonlla
spcis ar a
l to o this, as can Myco
actrium tu
rculosis an Myco
actri
um lpra. In M.
tu
rculosis an M. lpra infction, ach
acillus is con- tain in a mm
ran
-nclos ui
compartmnt call a pristiophorus vacuol (PV), which os not fus with th ly
so- soms. This
is u to th complxity of th aci-fast cll walls. An aitional mchanism of
rsisting
igstion involvs organisms proucing toxins that triggr th rlas of lysosomal contnts
into th cytoplasm of th phagocytic clls, su
squntly killing ths clls. Ex
ampls of ths
toxins ar lukociin, prouc
y Staphylococcus; listriolysin O, pro- uc

y Listria
monocytogns; an strptolysin, prouc
y Strptococcus. 2 Th last major f
ns som

actria us is to
lock th action of complmnt. Organisms mntion prviousl
y that prouc a
capsul o not
in th complmnt com- ponnt C3
, which is important in nhanc
ing phagocytosis.
3 Such organisms cannot
 phagocyti unlss coat
y opsonins (s Chaptr 1
). Aitionally,
som organisms xprss molculs that isrupt on or mor of th compl- mnt pa
thways. Protin
H, prouc
y S. pyogns,
ins to C1
ut os not allow th complmnt
casca to
proc furthr. 3 Anothr xampl is group B strptococci, which hav sialic ac
i in thir cll
walls. This inhi
its th complmnt pathway
y graing C3
. Th rst of thi
s chaptr
arsss th immunologic rspons to svral important
actria that caus in
vasiv isas
an for which srological tsting plays a major rol. Molcular mthos of tc
tion ar also
iscuss an com- par to srological tsting. GROUP A STREPTOCOCCI Strptococ
ci ar
gram-positiv sphrical, ovoi, or lanct- shap organisms that ar catalas n
gativ an ar
oftn sn in pairs or chains. 4 Thy ar ivi into groups or srotyps on th

asis of
crtain cll wall componnts. Th outrmost cll wall componnt contains two maj
or protins known
as M an T, an ths trmin th srogroup or srotyp (Fig 19-2). T
h srotyp is


as on minor variations in th protins that can
 inti  srologically. Int
rior to th
protin layr is th group-spci c car
ohy- rat that ivis strptococci in
to 20 fin
groups, signat A through H an K through V. Ths ar known as th Lanc l
groups,
as
on th pionring work of Dr. R
cca Lanc l. Som strains possss a hyaluronic
aci capsul
outsi th cll wall that contri
uts to th
actriums antiphagocytic proprti
s. Capsul M
an T protins DNas
NADas
Strptokinas
Hyaluronias
Strptolysin O
Cytoplasm
ic
mm
ran Pptioglycan layr Group car
ohyrat Exoantigns: FIGURE 192. Diagram
of antignic
compo- nnts of Strptococcus pyogns. 1814_Ch19_311-327.qx 7/10/09 3:53 PM
Pag 314 CHAPTER
19 Srological an Molcular Dtction of Bactrial Infctions 315 Strptococcus
pyogns, which

longs to Lanc l group A, is on of th most common an u
iquitous pathognic

actria an
causs a varity of infctions. Th M protin is th major virulnc factor of t
h group. It is a
filamn- tous molcul consisting of two alpha-hlical chains twist into a rop
lik structur
that xtns out from th cll sur- fac. Thr is a nt-ngativ charg at th
amino-trminal
n that hlps to inhi
it phagocytosis. In aition, prsnc of th M protin l
imits position
of C3 on th
actrial surfac, thr
y iminishing complmnt activation. 5
,6 Immunity to
group A strptococci appars to
 associ- at with anti
ois to th M protin
. Thr ar mor
than 100 srotyps of this protin, an immunity is srotyp- spcific. 6,7 Thr
for, infctions
with on strain will not provi protction against anothr strain. Aitional v
irulnc factors
inclu xoantigns or xo- toxins, protins xcrt
y th
actrial cl
ls as thy
mta
oli uring th cours of strptococcal infctions. Pyrognic xotoxins A,
B, an C ar
rsponsi
l for th rash sn in scarlt fvr an also appar to contri
ut to
patho- gnicity.
7,8 Anti
ois ar prouc to th following xoantigns: nyms strptoly
sin O,
oxyri
onuclas B (DNas B), hyaluronias, nicotinami anin inucloti- 
as (NADas), an
strptokinas. Cultur an rapi scrning mthos ar routinly us for iagno
stic tsting
arly in th infction. Howvr, iag- nosis of th squla such as glomrulon
phritis an acut
rhumatic fvr is
st achiv
y tction of anti
ois. For this rason, la

oratory mthos
for tcting anti
ois to ths strptococcal proucts ar prsnt in this c
haptr. Clinical
Manifstations of Group A Strptococcal Infction Strptococcus pyogns caus i
nfctions ranging
from pharyn- gitis to lif-thratning illnsss such as ncrotiing fasciitis a
n strptococcal
toxic shock synrom. Th two major sits of infctions in humans ar th uppr
rspiratory tract

an th skin, with pharyngitis an imptigo
ing th most com- mon clinical m
anifstations. 9
Symptoms of pharyngitis inclu fvr, chills, svr sor throat, haach, t
onsillar
xuats, ptchia on th soft palat, an antrior crvical lymphanopathy. 9
Th most common
skin infction is strptococcal pyorma, or imptigo, charactri
y vsic- u
lar lsions on
th xtrmitis that
com pustular an crust. Such infctions tn to occur
in young
chilrn. 7 Othr acut or suppurativ complications inclu otitis mia, scarl
t fvr,
rysiplas, cllulits, purpral spsis, an sinusitis. Sptic arthritis, acut

actrial
nocaritis, an mningitis also can rsult from a pharyngal infction. 5,7 S
vr invasiv
infctions with group A strptococci hav also
n associat with toxic shock
synrom an
ncro- tiing fasciitis. 9,10 Toxic shock synrom is a lif-thratning multisy
stm isas that
initiats as a skin or soft tissu infc- tion an may proc to shock an rna
l failur u to
ovrprouction of cytokins. 4,9 Ncrotiing fasciitis rsults from a skin infc
tion that invas
th muscls of th xtrmitis or th trunk. Th onst is quit acut an is a m
ical mrgncy.
Exotoxins prouc
y S. pyogns caus a rapily spraing infction p in th
 fascia,
rsulting in ischmia, tissu ncrosis, an spticmia if not trat promptly.
Th isas may

 asso- ciat with prisposing conitions, such as chronic illnss in th l
rly or variclla
in chilrn,
ut halthy prsons can
 affct as wll. 10,11 Rporting of nc
rotiing fasciitis an toxic shock synrom is part of a survillanc program conuct

y th Cntrs
for Disas Control an Prvntion. Whil th incinc of this synrom
has clin in
th Unit Stats, a signi cant num
r of cass ar still rport ach yar. 12 T
h two main
amaging squla to group A strptococ- cal infctions ar acut rhumatic fv
r an
poststrptococcal glomrulonphritis. 4 Ths conitions rsult from th host r
spons to
infction. Srological tsting plays a major rol in th iagnosis of ths two
isass,
caus
th organism itslf may no longr
 prsnt. Acut rhumatic fvr vlops as
a squla to
pharyn- gitis or tonsillitis in 2 to 3 prcnt of infct iniviuals. It os
not occur as a
rsult of skin infction. Th latncy prio is typically 1 to 3 wks aftr ons
t of th sor
throat. Charactristic faturs of acut rhumatic fvr inclu fvr, pain cau
s
y
in ammation in th joints, an in ammation of th hart. Th isas is most likly
u to
anti
ois or cll- miat immunity originally prouc against strptococcal
antigns
ut that
cross-ract with human hart tissu. 4,13 Chif among th anti
ois ar thos 
irct towar

th M protins, which hav at last thr pitops that rsm-
l antigns in h
art tissu,
prmitting cross-ractivity to occur. Titrs of som anti
ois may rmain
high for
svral yars following infction. Th scon main complication following a str
ptococ- cal
infction is glomrulonphritis, a conition charactri
y amag to th glom
ruli in th
kiny. This conition may follow infction of ithr th skin or th pharynx, w
hil rhumatic
fvr follows only uppr rspiratory infctions. 9 It is most common in chil
rn
twn th
ags of 2 an 12 an is spcially prvalnt in th wintr months. 14 Symptoms o
f
glomrulonphritis may inclu hma- turia, protinuria, ma, an hyprtnsion
. Patints may
also xprinc malais,
ackach, an a
ominal iscom- fort. 15 Rnal functio
n is usually
impair
caus th glomrular filtration rat is ruc,
ut rnal failur
is not typical.
Th most wily accpt thory for th pathognsis of poststrptococcal
glomrulonphritis is that it rsults from position of anti
oy-strptococcal
antign immun
complxs in th glomruli. Ths immun complxs stimulat an inflammatory
rspons that
amags th kiny an impairs function u to rlas of th lysosomal c
ontnts of
lukocyts an activa- tion of complmnt. 4,14 1814_Ch19_311-327.qx 7/10/09
3:53 PM Pag 315
316 SECTION 4 Srological Diagnosis of Infctious Disas Dtction of Group A S
trptococcal
Antigns Diagnosis of acut strptococcal infctions typically is ma
y cultur
 of th organism
from th infct sit. Th spci- mn is plat on shp
loo agar an incu
at
. If group A
strptococcus is prsnt, small translucnt colonis with a clar on of
ta h
molysis aroun
thm will
 visi
l. Prsumptiv inti cation is ma on th
asis of suscpti
i
l- ity to

acitracin, tsting for L-pyrrolionyl- -naphthylami (PYR) activity, or growth i
n th prsnc
of trimthoprim- sulfamthoxaol. 9 As an altrnativ to cultur, commrcial ki
ts hav
n
vlop to tct group A strptococcal antign using throat swa
s. Antign is
xtract
y
ithr nymatic or chmical mans, an th procss taks anywhr from 2 to 30
minuts,
pning on th particular tchniqu. Eithr nym immunoassay (EIA) or
latx
agglutination is thn us to in- tify th antign. Many of ths tsts rqui
r no mor than 2
to 5 minuts of hans-on tim, proviing a istinct avantag ovr cultur. How
vr, whil th
spci city is high, th snsitivity of th tst varis with th typ of procur
on. Rcnt
vl- opmnts using nym immunoassays hav improv th tsts snsitivity,
ut
culturs
shoul
 prform whn rapi tst rsults ar ngativ. 7,16 Molcular mthos
, incluing
hy
riia- tion of spci c rRNA squncs an ral-tim PCR (polymras chain rac

tion), hav
also
n vlop as a mans to rapily tct group A strptococcal infction
s. 17 Srotyping
an molcular tchniqus can
 us to in- tify a particular strain of group
A strptococcus
associat with an pimic. Srotyping involvs intification of M-prot
in antigns
y
prcipitation with typ-spci c anti- sra. Mor than 80 iffrnt srotyps hav

n inti 

y this mtho. Howvr, srotyping has limitations, inclu- ing limit availa

ility of typing
sra, nw M typs that o not ract with th antisra, an if culty in intrprti
ng th rsults.
16 Gnotyping tchniqus involving PCR ampli ca- tion of a portion of th m
m gn, which
cos for th M protin, follow
y squnc analysis, circumvnts ths pro

lms. 17 Puls
l gl lctrophorsis (PFGE) has also
n us for pimiological stuis. DN
A from group A
strptococcus is sparat
y using an altrnating currnt to o
tain a uniqu pa
ttrn, or
ngrprint. Th pattrns from multipl sourcs may
 compar whn thr is a group
A
strptococcal out
rak. 16 Dtction of Strptococcal Anti
ois Cultur or rapi
 scrning
mthos ar xtrmly usful for iagnosis of acut pharyngitis. Howvr, srolo
gical iag- nosis
must
 us to intify rhumatic fvr an glomrulonphritis,
caus
th organism is
unlikly to
 prsnt in th pharynx or on th skin at th tim symptoms appar
. 13 Group A
strptococci la
orat mor than 20 xo- toxins, an it is th anti
oy rspons
to on or mor
of ths that is us as ocumntation of nonsuppurativ is- as. Som of th
xotoxin proucts
inclu strptolysins O an S; oxyri
onuclass A, B, C, an D; strptokinas;
NADas;
hyaluronias; iphosphopyriin nuclotias; an pyrognic xotoxins 4 (s Fi
g. 192). Th
anti
oy rspons to ths strptococcal proucts is varia
l u to svral fac
tors, such as ag
of onst, sit of infction, an timlinss of anti
iotic tratmnt. Th most i
agnostically
important anti
ois ar th following: anti-strptolysin O (ASO), anti-DNas
 B, anti-NADas,
an anti-hyaluronias. Assays for tction of ths anti-
ois can
 prfor
m iniviually
or through us of th Strptoym kit which tcts anti
ois to all ths pro
- ucts (s th
xrcis at th n of th chaptr). During group A strptococcal infctions, ot
hr anti
ois
ar ma to cllular antigns, such as th group A car
ohyrat an th M proti
n. 16 Gnrally,
tction of ths anti
ois is on in rsarch or rfrnc la
oratoris,
sinc
commrcial ragnts ar not availa
l. Srological vinc of isas is
as
on an lvat or
rising titr of strptococcal anti
ois. Th onst of clinical symptoms of rhu
matic fvr or
glomrulonphritis typi- cally coincis with th pak of anti
oy rspons, so

a srum spcimn
tst at th tim shoul monstrat an lvation of th concntration of ths
anti
ois. If
acut an conva- lscnt phas sra ar tst in paralll, a fourfol ris in t
itr (a two-tu

iffrnc in th ou
ling srial ilutions) is con- sir signi cant. Th us o
f at last two
tsts for anti
ois to iffrnt xotoxins is rcommn,
caus prouction o
f tcta
l ASO
os not occur in all patints. Th most commonly us tsts ar thos for ASO a
n anti-DNas B.
9 Anti-hyaluronias tsts ar not availa
l commrcially, an tsting for ths
anti
ois is
prform only in rsarch or rfrnc la
oratoris. Antistrptolysin O (ASO) T
sting ASO tsts
tct anti
ois to th strptolysin O nym prouc
y group A strptococcus
, which is a
l
to lys r
loo clls. Prsnc of anti
ois to strptolysin O inicats rc
nt strptococcal
infction in patints suspct of having acut rhumatic fvr or poststr
ptococcal
glomrulonphritis following a throat infction. Th classic hmolytic mtho fo
r trmining th
ASO titr was th rst tst vlop to masur strptococcal anti
ois. This t
st was
as on
th a
ility of anti
ois in th patints srum to nutrali th hmolytic activ
ity of
strptolysin O. Th traitional ASO titr involvs ilution of patint srum, to
which a masur
amount of strptolysin O ragnt is a. Ths ar allow to com
in uring a
n incu
ation
prio aftr which ragnt r
loo clls ar a as an inicator. If nough
anti
oy is
prsnt, th strptolysin O is nutrali an no hmolysis occurs. Th titr is
rport as th
rciprocal of th highst ilu- tion monstrating no hmolysis. This titr
may

xprss in ithr To units (whn th strptolysin ragnt stanar is
us) or in
intrnational units (whn th Worl Halth Organiation intrnational stanar i
s us). 16
1814_Ch19_311-327.qx 7/10/09 3:53 PM Pag 316 CHAPTER 19 Srological an Mol
cular Dtction
of Bactrial Infctions 317 Th rang of xpct normal valus is varia

l an pns
on th patints ag, th gographic location, an th sason of th yar. ASO tit
rs tn to

highst in school-ag chilrn an young aults. Thus, th uppr lim- its of nor
mal must

sta
lish for spci c populations. 16 Typically, howvr, a singl ASO titr is
consir to

 moratly lvat if th titr is at last 240 To units in an ault an 3
20 To units in
a chil. 16 Bcaus of th la
or-intnsiv natur of th traitional ASO titr t
st, an
caus
th strptolysin O ragnt an th r clls us ar not sta
l, ASO tsting is
currntly
prform
y nphlomtric mthos in th routin clinical la
oratory. Nphlom
try has th
avantag of
ing an automat pro- cur that provis rapi, quantitativ m

asurmnt of ASO
titrs. 16 Th antign us in this tchniqu is puri  rcom-
inant strptolysi
n. Whn
anti
oy-positiv patint srum com
ins with th antign ragnt, immun compl
xs ar form,
rsulting in an incras light scattr that th instru- mnt convrts to a pak
rat signal. All
rsults ar rport in intrnational units, which ar xtrapolat from th cla
ssic hmolytic
mtho scri
 prviously. Whn using th nph- lomtric mtho, th uppr li
mits of normal
for iffrnt populations hav not
n sta
lish, so ach la
oratory must t
rmin its own
uppr limit of normal. ASO titrs typically incras within 1 to 2 wks aftr i
nfction an
pak
twn 3 to 6 wks following th initial symptoms (.g., sor thro
at). 13 Howvr,
an anti
oy rspons occurs in only a
out 85 prcnt of acut rhumatic fvr pa
tints within
this prio. Aitionally, ASO titrs usu- ally o not incras in iniviuals w
ith skin
infctions. 5 Anti-DNas B Tsting Tsting for th prsnc of anti-DNas B is c
linically usful
in patints suspct of having glomrulonphritis prc
y strptococcal ski
n infctions, as
ASO anti
ois oftn ar not stimulat
y this typ of isas. 4 In aition,
anti-
ois to
DNas B may
 tct in patints with acut rhumatic fvr who hav a ngati
v ASO tst
rsult. DNas B is mainly prouc
y group A strptococci, so tsting for antiDNas B is highly
spcific for group A strptococcal squla. 6 Macrotitr, microtitr, EIA, an
nplomtric
mthos hav
n vlop for anti-DNas tsting. Th classic tst for th ma
surmnt of
anti-DNas B activity is
as on a nutraliation mthoology. If anti- DNas B
anti
ois ar
prsnt, thy will nutrali ragnt DNas B, prvnting it from polymr
iing DNA.
Prsnc of DNas is masur
y its ffct on a DNA- mthyl-grn conjugat. Th
is complx is
grn in its intact form,
ut whn hyroly
y DNas, th mthyl grn is ru
c an
coms
colorlss. An ovrnight incu
ation at 37C is rquir in som tsting mthoologi
s to prmit
anti
ois to inactivat th nym. Tu
s ar gra for color, with a 4
inicating that
th intnsity of color is unchang, an a 0 inicating a total loss of color. T
h rsult is
rport as th rciprocal of th highst ilution monstrating a color intnsi
ty of
twn 2
an 4 . Normal titrs for chilrn
twn th ags of 2 an 12 yars ran
g from 240 to
640 units. 13 Nphlomtry provis an automat mans of tsting that can
 us
 for rapi
quantitation of anti-DNas B. In this mtho, immun complxs form
twn an
ti
ois in
patint srum an DNas B ragnt gnrat an incras in light scattr. Rsults
ar xtrapolat
from valus from th clas- sic mtho an ar rport in intrnational units p

r mL. 16
Strptoym Tsting Th Strptoym tst is a sli agglutination scrning tst
for th
tction of anti
ois to svral strptococcal anti- gns. Shp r
loo cl
ls ar coat
with strptolysin, strptokinas, hyaluronias, DNas, an NADas so that anti

ois to any
of th strptococcal antigns can
 tct. Ragnt r
loo clls ar
mix with a
1:100 ilution of patint srum. Hmagglutination rprsnts a positiv tst, in
icating that
anti
ois to on or mor of ths antigns ar prsnt. Th tst is rapi an s
impl to prform,

ut it appars to
 lss rprouci
l than othr anti
oy tsts. In aition, m
or fals
positivs an fals ngativs hav
n rport for this tst than for th ASO
an anti-DNas B
assays. 9 Bcaus a largr varity of anti
ois ar inclu in this tst, th
potntial is
highr for tction of strptococcal anti
ois. Howvr, singl-titr trmin
ations ar not as
significant as svral titrations prform at wkly or
iwkly intrva
ls following th
onst of symptoms. 16 Th Strptoym tst is an xcllnt scrning tool,
ut i
t shoul
 us
in conjunction with th ASO or DNas whn squ- la of group A strptococcal in
fction ar
suspct. HELICOBACTER PYLORI Charactristics of Hlico
actr Pylori Infctions
Hlico
actr
pylori has
com an important organism within th past 20 yars. This gram-nga
tiv,
microarophilic spiral
actrium is th major caus of
oth gastric an uonal
ulcrs. 18,19 H.
pylori is foun worlwi, affcting 30 prcnt of th populations in vlop
countris an
mor than 90 prcnt of th populations in vloping countris. 18 Sinc 1994,
th National
Instituts of Halth has rcommn that iniviuals with gastric or uonal u
lcrs caus
y
H. pylori
 givn anti
iotic tratmnt along with th anti-ulcr m- ications.
If untrat,
H. pylori infction will last for th patints lif an may la to gastric
carcinoma. 18 A
varity of tchniqus hav
n vlop to iagnos H. pylori infc- tion u
to th prvalnc
of th organism an th signi canc of th infction. Th gnomic squncs of two
strains of th
organism hav
n trmin using molcular tchniqus. Thr is a larg amoun
t of gntic
htrognity u to th fr- qunt xchang of gntic matrial
twn strains
. On of th
protins of H. pylori, CagA, is highly immunognic an 1814_Ch19_311-327.qx 7/
10/09 3:53 PM
Pag 317 318 SECTION 4 Srological Diagnosis of Infctious Disas is on of th
virulnc
factors of th
actrium. Th svr- ity of th isas is rlat to injction
of th CagA
protin into th gastric pithlial clls. Onc th CagA protin is in th pith
lial clls,
changs occur in th function of th clls signal transuction pathways an in th

 structur of
th cytosklton. 20,21 A scon virulnc factor is vacuolating cytotoxin
, or VacA. Th
VacA gn cos for a toxin prcursor. Epimiological stuis hav shown
that if th CagA
an VacA gns ar prsnt in th strain of
actria infcting th iniviual, t
hr is a highr
risk of vloping gastric or pptic ulcrs or gastric carcinoma. 21,22 AntignDtction
Procurs Sinc H. pylori is foun in th stomach, som of th mthos to tr
min if th
organism is prsnt rquir an noscopy. This inclus culturing for H. pylori,
histologically
xamining gastric
iopsy tissu, an prforming a uras
iopsy tst. Th most s
pci c tst to
tct H. pylori infction is cultur,
ut th snsitivity is usually lowr
than othr
mthos, sinc th organism is not vnly istri
ut throughout th tissu. 23
H. pylori
proucs a larg amount of uras, so a
iopsy ur- as tst may
 on to t
ct th prsnc
of
actria. Th organism is prsnt if thr is a color chang u to
th incras of
pH from th
rakown of ura to ammonia an
icar
onat. Th
iopsy uras tst
is asy to us,
an rsults can
 tct within 2 hours in som tst procurs, mak- ing it
ial for rapi
iagnosis of H. pylori infctions. 24 Procurs for tcting H. pylori that o
not rquir th
us of noscopy inclu ura
rath tsting, nym immunoassays for
ac
trial antigns
in th fcs, molcular tsts for H. pylori DNA, an anti
oy tsts. In th ura

rath tst, th
patint ingsts ura la
l with raioactiv car
on ( 14 C) or a nonraioactiv
13 C. Ura is
mta
oli to ammonia an
icar
onat. Th
icar
onat is xcrt in th
rat
h, an th
la
l car
on ioxi is masur
y tction of raioactivity for ( 14 C) or
mass spctromtry
analysis for 13 C. This
rath tchniqu has xcllnt snsi- tivity an spci cit
y, an it is
hlpful in trmining if th
actria hav
n raicat u to antimicro
ial
thrapy;
howvr, it involvs th us of raioactivity. 4,23 Analysis of stool sampls

for an aftr
antimicro
ial thrapy for H. pylori antigns is mainly on to trmin if th

actria hav

n liminat. 23 Th tst currntly uss an nym-la
l monoclonal anti
o
y. Th snsitivity an spcificity of this monoclonal anti
oy tst ar 84 to 95 prcn
t an 97 prcnt,
rspctivly, as compar to th ura
rath tst. Continu improvmnt ns t
o
 ma in this
tst procur to achiv th lvl of accu- racy of th ura
rath tst. Nonin
vasiv molcular
tsts hav also
n vlop to tct H. pylori DNA. 23,24 Howvr, PCR-
as
mthos, which
tct th prsnc of th organism in fcal sampls, cannot istinguish
twn
living an a
H. pylori. Car must
 takn whn purifying th sampl an prforming th proc

ur so that
contamination or amplification of xognous DNA os not occur. 23 Thr may

 rsiual DNA
lft aftr th
actria hav
n raicat. Ral-tim PCR using TaqMan tchnol
ogy has
n
vlop to trmin th patints
actrial loa an has shown goo corrlation
with th ura

rath tst. Molcular mthos ar quick an prcis an can trmin anti
ioti
c rsistanc as
wll. 23,24 Dtction of Hlico
actr Pylori Anti
ois Srological tsting is a
primary
scrning mtho of tr- mining infction with H. pylori. Infctions from this
organism rsult
in anti
oy prouction of IgG, IgA, an IgM. Th prs- nc of anti
oy in th

loo of an
untrat patint inicats an activ infction, sinc th
actrium os not sp
ontanously
clar. Anti
oy lvls in untrat iniviuals rmain lvat for yars. In tr
at
iniviuals, th anti
oy concntrations cras aftr a
out 6 months to a
out
50 prcnt of th
lvl th patint ha uring th activ infction. This mans that if an anti
o
y tst is us to
tct raication of H. pylori, th prtratmnt sampl shoul
 stor for a
yar so that
paralll tsting can
 prform. 25 A cras in anti
oy concntration of mo
r than 25
prcnt must occur for trat- mnt to
 consir succssful. 25 Most srologi
cal tsts in
clinical us tct H. pylori spci c anti
ois of th IgG class. Although IgM ant
i
oy is
prouc in H. pylori infctions, tsting for its prsnc lacks clinical valu
, sinc most
infctions hav
com chronic
for iagnosis. Thus, IgG is th primary anti

oy foun. IgA
tsting has a lowr snsitivity an spci city than IgG tsting,
ut it may incra
s snsitivity
of tction whn us in conjunction with IgG tsting. 23 Masurmnt of th an
ti
ois may

on
y svral tchniqus, incluing nym-link immunosor
nt assays (ELI
SA), immuno
lot,
an rapi tsts using latx agglutination or flow-through mm
ran-
as nym
immunoassay. Th
tst mthoology of choic for th tction of H. pylori anti
ois is th
 ELISA
tchniqu, which is rlia
l an accurat. 25 Tsts mploying antigns from a po
ol xtract from
multipl an gntically ivrs strains yil th
st snsitivity, sinc H. py
lori is so htrognous. Vry fw, if any, patints prouc anti
ois to all of th H. pylor
i antigns; most
patints prouc anti-
ois against th CagA an VacA protins. Anti
ois to
ths two
protins inicat a mor svr cas of gastritis or gastric carcinoma. 23 Thr
ar also a
num
r of rapi tsts on th markt. It is rcommn that sampls with positiv
 rapi tst
rsults
 tst
y an ELISA tst for corrlation. Whn compar to othr tc
hniqus for
anti
oy tction of H. pylori, ELISA tsts ar snsitiv, spci c, an cost-

ffctiv for
tr- mining th organisms prsnc in untrat iniviuals. 23 Howvr, sinc
anti
ois ar
not rapily clar aftr trat- mnt, anti
oy tsting is not as wll suit fo
r trmining
raication of infction as ar othr mthos. Aitionally, iniviuals who ar
immunocompromis (th lrly or immunosupprss iniviuals) may hav a fals
-ngativ rsult
with anti
oy tsting. 1814_Ch19_311-327.qx 7/10/09 3:53 PM Pag 318 CHAPTER
19 Srological
an Molcular Dtction of Bactrial Infctions 319 MYCOPLASMA PNEUMONIAE Charac
tristics of
Mycoplasma Infction Mycoplasma is a mm
r of a uniqu group of organisms that

long to th
class Mollicuts. Ths organisms lack cll walls an hav a small gnom, stro
ls in thir cll
mm
ran, an complx growth rquirmnts. This maks cultur of ths organisms
ifficult an
tim-consuming. Th
st- known organism of this class is Mycoplasma pnumonia,
which is a
laing caus of uppr rspiratory infctions worlwi. 26 Mycoplasma pnumonia
 infctions ar
foun in all ag groups. Th incu
ation prio is 1 to 3 wks, an th infctio
n can oftn
spra through houshols. Symptoms of infction inclu sor throat, chills, ho
arsnss,
tracho-
ronchitis, an haach. Twnty prcnt of all community- acquir pn
umonias an 20
prcnt of all hospitaliations for pnumonia in th Unit Stats ar thought t
o
 u to
Mycoplasma infctions. 26,27 M. pnumonia may rmain in th rspiratory tract f
or svral
months. It can attach to clls in th rspiratory tract an caus chronic in ammat
ion. 26 Othr
complications may
 xtra-pulmonary, incluing hmolytic anmia, skin rashs, a
rthritis,
mningoncphalitis, pricari- tis, an priphral nuropathy. 27 An autoimmun
rspons may
play a rol in ths xtrapulmonary complications. Dtction of Mycoplasma Pnum
onia
y Cultur
Bcaus M. pnumonia os not hav a cll wall, th organ- ism is snsitiv to
rying, so
transport is xtrmly important to o
taining a cultur. 28 Th transport mia
us can

trypticas soy
roth with 0.5 prcnt al
umin, SP4 mium, or a viral transport
mium. If th
sampl cannot
 plat immiatly, it shoul
 fron at 70C. Culturing of M. p
numonia is
not routinly prform from rspiratory sampls,
caus th culturs ar if cul
t to grow an
rquir spciali agars. Growth may tak svral wks, an srological tstin
g may inicat
th prs- nc of M. pnumonia infction
for th cultur. Dtction of Anti

ois to
Mycoplasma pnumonia For many yars, prior to th vlopmnt of antign-spci c
srological
tsts, la
oratory iagnosis of Mycoplasma pnumo- nia involv tsting for col
agglutinins,

caus ths ar prsnt in a
out 50 prcnt of patints with th infction. Co
l agglutinins

ar autoanti
ois that ract with th I/i antigns on r
loo clls an
caus thir
agglutination at tmpraturs
low 37C. Dvlopmnt of ths anti
o- is is tho
ught to rsult
from cross-raction of th I antign on human r
loo clls or from altration
of th r
loo
clls
y th organism. 29 Tsting for col agglutinins is no longr rcommn
for th tction
of M. pnumonia infctions, sinc som viral infctions an collagn vascular 
isass also
caus prouc- tion of col agglutinins. 29 Mor rcntly, M. pnumonia spci c sr
ological
tsts hav
n vlop. Ths inclu nym immunoassay, particl agglutinat
ion, inirct
immuno- uorscnc, an complmnt xation. Dpning on th tchniqu us, IgM, I
gG, an IgA
classs of anti
oy to M. pnumonia may
 tct. 29 Enym immunoassays (EI
A) ar th most
wily us mtho of tction,
caus thy rquir a small sampl volum an
can tst larg
num-
rs of spcimns in an automat format. 26 Th EIA procur may

 as snsitiv as
PCR if nough tim has laps for anti
ois to
 form. It is
ttr to tst
for th prsnc
of
oth th IgG an IgM classs of anti
oy for gratr accuracy,
ut ach class
of anti
oy must

 tst for sparatly. 29 Th inirct uorscnt assay (IFA) can also tct I
gM or IgG
classs of anti
oy to M. pnumonia. In this tst, Mycoplasma antign is attach
 to a glass
sli. Patint srum is plac on th sli an incu
at. Th sli is
thn wash,
an fluorscnt-la
l anti-IgM or anti-IgG is a to th sli an allow t
o incu
at. Aftr
incu
a- tion, th sli is wash an o
srv unr a uorscnt microscop. If t
h scrning
tst is positiv, thn a srial ilution may
 on to trmin th concntrat
ion of anti-
oy
prsnt. 29 Molcular Tchniqus Molcular tchniqus provi a mor rapi mans
of tct- ing
Mycoplasma infction than cultur or srological tsting. PCR tsting for Mycopl
asma may

prform using throat swa
s, nasophayngal swa
s, sputum, x tissu, an cul- t
urs. In
aition to o
taining rapi rsults, PCR tsts ar also avantagous in that onl
y on spcimn is
rquir, tissus that hav alray
n procss for histology can
 us,
an liv
organisms ar not rquir. Howvr, inhi
itors prsnt, spcially in nasop
haryngal
aspirats, may caus a fals-ngativ raction. 29 Thrfor, a ngativ PCR ts
t may inicat
th n for aitional tsting
y othr mans. RNA amplification tchniqus
hav also
n
us. RNA-
as ampli cation mthos ar highly snsitiv u to th larg num
r
of rRNA
(ri
osomal RNA) copis in th M. pnumonia cll, which also inicats that th
organ- ism is
aliv. Nuclic aci ampli cation mthos hav
n vlop that tct svral 
iffrnt gn

targts, inclu- ing th ATPas opron gn, th PI ahsion gn, th 16S RNA g
n, an th tuf
gn. Molcular tsting will likly rplac srological tsting in th futur. R
ICKETTSIAL
INFECTIONS Charactristics of Rickttsial Infctions Th rickttsia ar short r
os, or
cocco
acilli, that ar o
li- gat, intracllular, gram-ngativ
actria. Th
 gnus
1814_Ch19_311-327.qx 7/10/09 3:53 PM Pag 319 320 SECTION 4 Srological Diag
nosis of
Infctious Disas Rickttsia is ma up of two istinct groups: th spott fv
r group (SFG)
an th typhus group (TG). Each is rsponsi
l for a iffrnt st of isass.
Ta
l 191 contains a partial list of spcis foun in ach group. Rickttsial isass ar as
sociat with
arthropos, as thy spn at last part of thir lif cycl in an arthropo host

for
ing
transmitt to humans. 30 Arthropo hosts inclu ticks, mits, lic, or as. Hum
ans ar
accintal hosts for rick- ttsia xcpt for Rickttsia prowakii, which caus
s pimic
typhus. 31 Som of th rickttsia ar foun only in crtain aras of th worl a
n ar
gntically iffrnt nough to
 consir iffrnt spcis. R. japonica is
foun only in
Japan, whil R. rickttsii is foun in th Wstrn Hmisphr. Som spcis, lik
 R. typhi, ar
foun vrywhr in th worl. 32 In th Unit Stats, th main rickttsial is
ass ar two
typs of typhusnmic an pimiccaus
y R. typhi an R. flis, rspctivly,
an Rocky
Mountain spott fvr, caus
y R. rickttsii (s Ta
l 191). Ths isass a
r most
prvalnt
twn May an Sptm
r. 31 In Rocky Mountain spott fvr, sy
mptoms occur
approximatly 7 ays aftr a tick
it. Ths inclu fvr, svr haach, ma
lais, an
myalgia, accompani
y nau- sa, vomiting, a
ominal pain, an somtims a coug
h. A rash, which
starts on th hans an ft an procs to th trunk, appars in 3 to 5 ays a
ftr th

ginning of othr symptoms. 30 Th organism infcts nothlial clls, cau
sing an incras
in vascular prma
ility an focal hmorrhags. Murin typhus, caus
y R.
typhi (which is
carri
y flas), is charactri
y a cough an chst infiltrat suggstiv
of pnumonia. A
rash appars in only a
out on-half of patints. 30 This isas can caus svr
 manifstations such as siurs, coma, an rspiratory failur. Symptoms of isa
s caus
y
R. flis ar not as wll fin. Srological Diagnosis Sroiagnosis is cur
rntly th mtho
of choic for tct- ing rickttsial infctions. Mthos that may
 us ar p
ro
-
as
immunoassays an agglutination assays. 32 For many yars, anti
ois prouc in
patints with
rickttsial infctions wr tct
y an agglutination tst known as th WilFlix tst, which

was
as on cross-ractivity of th patints anti
ois with polysacchari anti

gns prs- nt
on th OX-19 an OX-2 strains of Protus vulgaris an th OX-K strain of Protus
mira
ilis.
Currnt srological assays for rickttsial anti
ois ar organism-spcifi
c an inclu
inirct fluorscnt assays (IFA), microimmunofluorscnt assays (micro-IF), im
munoproxias
assays (IPA), ELISA, an immuno
lot assays (IBA). IFA an IPA rquir th whol

actrium as th
ragnt, whil ELISA an IBA us rickttsial antigns asor
 onto a soli phas
 or
nitrocllulos mm
ran. Th IFA tst an th micro-IF ar currntly consir
th gol stanar
for tcting rickttsial anti
ois. 32 Th IPA, which uss a light microscop
insta of a
uorscnt micro- scop to ra th slis, has a snsitivity an spcificity simi
lar to that of
IFA. 32 Tsting
y
oth of ths mthos can tct signi cant titrs of anti
oi
s in Rocky
Mountain spott fvr
y th scon wk of infction. 30 Howvr, antiricktts
ial tratmnt
ns to
 start
y th fth ay of illnss in orr to
 succssful. Thus, s
rological
iagno- sis tns to
 mor rtrospctiv, aftr tratmnt has
gun. Molcular
Tchniqus
Molcular tction of rickttsial infctions can
 accom- plish
y prformin
g a PCR using
primrs that targt gns of th TG an SFG groups of organisms. To tct
oth
SFG an TG
rickttsia, th citrat synthas gn an th Ta
l 191. Etiology an Ecology of
Rickttsial
Disass GEOGRAPHICAL DISEASE MODE OF ORGANISM LOCATION ASSOCIATION TRANSMISS
ION Spott Fvr
Group (SFG) R. rickttsii Wstrn Hmisphr Rocky Mountain spott fvr Tick

it R. japonica
Japan Japans spott fvr Tick
it R. flis North an South Amrica, Fla-

orn spott
fvr Unknown Europ, Africa R. akari Unit Stats, Ukrain, Rickttsialpox Mi
t
it Croatia,
Kora Typhus Group (TG) R. typhi Worlwi Enmic typhus Fla fcs R. prowak
ii Worlwi
Epimic typhus Lous fcs Worlwi Rcruscnt typhus Yars aftr pimic t
yphus
1814_Ch19_311-327.qx 7/10/09 3:53 PM Pag 320 CHAPTER 19 Srological an Mol
cular Dtction
of Bactrial Infctions 321 17 kDa antign gn ar us, whil th 190 kDa anti
gn (Omp A)
gn an th 120 kDa antign (OmpB) gn ar us only to tct th SFG
group. 32 PCR can
thn
 follow
y rstriction igst or nucloti squnc analysis of th am
plicon, or
ral-tim PCR can
 us to finitivly intify th rickttsial spcis. Th
continu
vlopmnt of ral-tim PCR hols much promis for th futur. Ta
l 192 summari
s an
compars th major typs of tsting availa
l for all organisms list in th ch
aptr. Ta
l
192. Comparison of Tsting Mthos for Bactrial Inti cation ORGANISM TEST METHOD
TESTING

PRINCIPLE COMMENTS Group A Strptococcus Cultur Plat on
loo agar Taks 2448 h
ours Rapi EIA
Extract antign from Rapi
ut not as snsitiv as throat swa
cultur PCR Ampl
ify portions of
th N for spciali quipmnt mm gn Anti
oy tsting Nutraliation of
xotoxins Us
to iagnos strp squla (ASO, anti-DNas, tc.) Sli agglutination Agglutina
tion of r clls
coat Rapi
ut rsults not as rlia
l with xotoxins as othr tsting; goo
initial
scrning tool Hlico
actr pylori Uras tst Plac
iopsy matrial on ura a
gar. Rsults in
2 hours. Invasiv Color chang as ura is
rokn own procur, an organism m
ay
 miss u
to unvn istri
ution in tissu Ura
rath tst Patint ingsts raioactiv ur
a; Rapi an
snsitiv
ut involvs
icar
onat prouc, which is raioactivity
rath ou
t as CO 2 Rapi
EIA To intify antign in stool spcimns Rapi
ut not as snsitiv as ura
r
ath tst. Us
to trmin succss of thrapy Anti
oy tsting EIA tst to trmin prsnc
of Bst for
initial scrning IgG or IgM mtho. Not as rlia
l as antign tsting to valu
at thrapy PCR
Amplify spci c DNA Rapi an snsitiv
ut su
jct to contamination Mycoplasma pn
umonia
Cultur Look for growth on spciali agars Dif cult to grow an taks svral w
ks Col
agglutinin tsting Anti
ois cross-ract with I antign Easy to o
ut not sp
ci c for on group
O r clls; look for Mycoplasma agglutination EIA Tst for prsnc of IgG an
IgM Snsitiv
an automat with puri  antigns IFA Antign attach to sli an thn N t
o hav a
uorscnt ract with anti
oy. An microscop, an sli must
 anti-immunoglo

ulin with a
intrprt carfully uorscnt tag is a DNA or RNA Amplify ithr DNA or RN
A spci c Vry
spci c
ut rquirs spcial ampli cation to th organism quipmnt Rickttsia sp.
Wil-Flix
raction Agglutination of Protus antigns Easy an inxpnsiv,
ut not u to
cross-ractivity
spci c IFA Antign attach to sli an thn N to hav a uorscnt ract wi
th anti
oy.
An anti- microscop, an sli must
 immunoglo
ulin with a uorscnt intrprt
 carfully.
Taks tim tag is a. for anti
oy to
 prouc. Continu 1814_Ch19_311-32
7.qx 7/10/09
3:53 PM Pag 321 322 SECTION 4 Srological Diagnosis of Infctious Disas t
cting ths
anti
ois inclu nphlomtry, latx agglu- tination, an nym nutraliatio
n. H. pylori
causs gastric ulcrs, an if th
actria ar not liminat using anti
iotics,
patints ar at
incras risk of vloping gastric carcinoma. H. pylori infction may
 tc
t
y invasiv
or noninvasiv tsts. Th uras nym prouc
y th organism can
 tct
from gastric

iopsy tissu o
tain
y noscopy, or th
rath of patints can
 an
aly aftr thy

ingst ura la
l with a car
on isotop. Srological procurs, such as ELISA
, immuno
lotting,
an rapi latx agglutination or EIA tsts, may
 us to tct H. pylori anti

ois in patint
srum. PCR mthos can
 us to tct th prsnc of H. pylori DNA in fcal
spcimns.
Anti
oy tsting for Mycoplasma pnumonia an rickttsia has
n prform fo
r many yars. At
on tim, srological iagnosis of ths infctions us th principl of
crossractivity of anti
ois prouc
y patints to nonorganism- spci c antigns. T
sting for col
agglutinins was commonly inclu in th tsting protocol for M. pnumonia,
c
aus a larg
prcntag of ths patints prouc anti
ois to th I/i antigns on r
loo
 clls. Tsting
for rickttsial infc- tions incorporat th Wil-Flix tst, an agglutin
ation mtho that
us strains of Protus
actria known to cross- ract with rickttsial anti
oi
s. Currnt
srological mthos us to tct th anti
ois in ths isass ar mor sn
- sitiv an
organism-spci c. Molcular tchniqus such as PCR hav
n vlop to tct n
uclic aci
from M. pnu- monia an rickttsia. Ths tsts hol much promis for th futu
r. Ta
l 192.
Comparison of Tsting Mthos for Bactrial Inti cationCont ORGANISM TEST METHOD
TESTING
PRINCIPLE COMMENTS IPA Antign attach to sli an thn Can
 ra with an o
rinary ract
with anti
oy. An anti- light microscop
ut taks tim immunoglo
ulin with an 
nym for
anti
oy to
 prouc tag is a. PCR Ampli cation of spci c gns for Fast an
 snsitiv

ut rquirs ach groupTG an SFG spciali quipmnt SUMMARY Whil many
actr
ia ar
stroy or inactivat
y th immun systm, som ar a
l to va th hosts 
fns
mchanisms an caus isas. Som of th mchanisms us to va th immun sy
stm inclu
avoiing anti
oy
y vloping nw antigns,
locking phagocytosis
y mans of
structural
componnts such as capsuls, an inactivating th complmnt casca
y proucin
g crtain
protins. Whn
actria ar a
l to ovrcom host fnss an infc- tion occu
rs, procurs
hav
n vlop that us culturs or molcular tchniqus to tct th
organism itslf
or that us srological tchniqus to tct th prs- nc of anti
ois prouc
 uring
infction. Th
actria iscuss in this chaptr (group A strptococcus, H. pyl
ori, M.
pnumonia, an rickttsia) can all
 tct using ths typs of procurs
. Although group
A strptococcus can
 cultur, an rapi tction of th organism is possi
l
, complications
such as acut rhumatic fvr an poststrptococcal glomru- lonphritis can occ
ur, an th
organism is no longr prsnt in th
oy. Tsting for strptococcal anti
ois
is impor- tant

to tct ths squla. In group A strptococcal infctions, svral an

ti
ois ar
form to th xoantigns (toxins) that ar prouc
y th
actria. Anti-strp
tolysin O an
anti-DNas B ar th two most common anti
ois that ar tct in th clinica
l la
oratory.
Othr anti
ois that ar prouc ar anti-hyaluronias an anti-NADas. Som
of th tst
procurs such as th Strptoym tst tct th prsnc of mor than on ant
i
oy. Mthos
for 1814_Ch19_311-327.qx 7/10/09 3:53 PM Pag 322 CHAPTER 19 Srological an
Molcular
Dtction of Bactrial Infctions 323 1. A 6-yar-ol
oy was
rought to th p
iatric clinic.
His mothr inicat h ha
n ill for svral ays with fvr an gnral lt
hargy. Th
morning of th visit, th
oy tol his mothr that his
ack hurt, an sh ha o

srv what
appar to

loo in his urin. History an physical xamination inicat a
wll-nourish
chil with an unrmarka
l halth history othr than a svr sor throat with f
vr 3 wks
prior that was m- icat with aspirin an throat longs. This chils tmprat
ur was
101.5F, an th physician not ma in th chils hans an ft. Bloo an urin
 spcimns
wr collct for a Strptoym tst, complt
loo cll count, an urinalysis
. La
oratory tst
rsults wr as follows: Complt Bloo Count R cll count Normal Platlt cou
nt Normal Whit

loo cll count 12.7 10 9
/
L (Normal = 4.810.8 10
9
/
L)
Urinalysis Color r (Normal = straw) Clarity Clouy (Normal = clar) Pr
otin 2 (Normal =
ngativ/trac) Bloo Larg (Normal = non) Strptoym Positiv 1:600 Qustions
a. What isorr
is inicat
y th chils history, physical xam, an la
oratory tst rsults?

. What was th
most likly causativ agnt of th sor throat prcing th currnt symptoms? c
. Discuss th
most wily accpt thory xplaining th physiological
asis for this isas.
. Why int
th physician orr throat an/or urin culturs? 2. A 36-yar-ol fmal was s
n
y hr
physician for pnumonia-lik symptoms along with a sor throat an chills. Sh w
as also having
ifficulty
rathing. Th patint ha a tmpratur of 100.2F an was prouc- ing
a morat
amount of sputum. Hr physician ci that th pro
a
l iagnosis was som
typ of
pnumonia an orr th following la
oratory tsts to
 prform: complt

loo count,
sputum cul- tur, tsts for in una virus, an tsts for Mycoplasma pnumonia. T
h rsults of
th tsts wr as follows: Complt Bloo Count R cll count Normal Whit cll
count Somwhat
lvat Sputum Cultur Ngativ Mycoplasma Titr Positiv 1:64 Col Agglutinin
Titr Positiv

1:128 Qustions a. What is th most pro
a
l caus of th pnumonia?
. Why was
th sputum
cultur ngativ? c. Why was th col agglutinin titr highr than th Mycoplasm
a titr? CASE
STUDIES 1814_Ch19_311-327.qx 7/10/09 3:53 PM Pag 323 324 SECTION 4 Srologi
cal Diagnosis of
Infctious Disas STREPTOZYME TEST Procur* 1. Dilut srum or plasma sampl
1:100 with
isotonic salin. If th sampl is priphral
loo from th arlo
 or n- grtip,
ll th
capillary tu
 (suppli with th kit) to th lin. Without allowing th
loo t
o clot an using
th
ul
suppli, xpl th sampl into a tu
 containing 2.4 mL of isotonic
salin. This
tchniqu yils an approximat 1:100 ilution. 2. Fill th capillary tu
 to
th mark (0.05
mL) with th ilut spcimn. Using th
ul
suppli, xpl th spcimn onto
a sction of th
glass sli provi with th kit. 3. Rpat stp 2 for th positiv an ngativ
 controls, placing ach on a sction of th sli that
orrs th patint spcimn. 4. A 1
rop of
ragnt to ach sction. B sur th ragnt is mix wll
for it is us
. 5. Thoroughly
mix all sampls using a isposa
l stirrr. Us a clan stirrr for ach sampl.
6. Rock th
mirror sli
ack an forth gntly for 2 min- uts at th rat of 8 to 10 tim
s pr minut. 7.
At th n of 2 minuts, plac th sli on a at surfac an o
srv for agglutin
ation. 8.
Agglutination must
 ra within 10 scons. Raing is facilitat
y a irct
light sourc
a
ov th sli. NOTE: If th tst is positiv, a titr can
 o
tain
y prpa
ring a 1:100
primary ilution of patint srum, making furthr ilutions (as inicat
low)
an tst- ing
ach ilution
y th a
ov mtho. PREPARATION OF SAMPLE RESULTING DILUTION 1.0
mL of primary
1.0 mL ilution (1:100) salin1:200 0.5 mL of primary ilution
1.5 mL salin 1
:400 0.5 mL of
primary ilution
2.5 mL salin 1:600 0.5 mL of primary ilution
3.5 mL salin
 1:800
EXERCISE Principl Th Strptoym rapi sli tst is an agglutination tst tha
t tcts
anti
ois to v Group A strptococcal xoantigns, incluing strptolysin O, str
ptokinas,
hyaluronias, DNas B, an NADas. Ths antigns ar attach to alhy- x s
hp r
loo
clls. Whn patint srum is a to ths ragnt r
loo clls, agglutinati
on of th r

loo clls occurs if th patint has anti
ois to on or mor of ths antign
s. Sampl
Prparation Bloo is collct asptically. Eithr srum or plasma may
 us,
as wll as
priphral
loo from a ngrtip or ar- lo
. If
loo is collct from a ngr or
ar
punctur, a hparini capillary tu
 or th tu
 suppli with th Strptoym
kit may
 us.
If srum or plasma cannot
 tst within 24 hours of collction, it shoul

stor fron.

Ragnts, Matrials, an Equipmnt Strptoym rapi sli tst kit (Wampol La

oratoris,
Cran
ury, NJ), which contains th following: Strptoym ragnt Positiv contro
l srum Ngativ
control srum Cali
rat capillary tu
s an
ul
s Mirror glass sli Aition
al matrials
n: Stirrrs Tst tu
s Isotonic salin (0.85 prcnt) Piptts WARNING: Bc
aus no tst
mtho can offr complt assuranc that human immuno cincy virus, hpati- tis
B virus, or
othr infctious agnts ar a
snt, controls shoul
 hanl as though capa
l
of transmitting
an infctious isas. Th Foo an Drug Aministration rcommns that such mat
rial
 hanl
at a
iosafty lvl 2. CAUTION: Th ragnts in this kit contain soium ai,
which may ract
with la an coppr plum
ing to form highly xplosiv mtal ais. Upon ispos
al, flush with a
larg volum of watr to prvnt ai
uilup. *Aapt from th packag insrt
for th
Strptoym rapi sli tst
y Wampol La
oratoris, Division of Invrnss Mi
cal Profssional
Diagnostics, Princton, NJ. 1814_Ch19_311-327.qx 7/10/09 3:53 PM Pag 324 CH
APTER 19
Srological an Molcular Dtction of Bactrial Infctions 325 INTREPRETATION A
singl
Strptoym trmination is not signi cant, an srial titrations shoul
 prfo
rm on a
wkly or
iwkly
asis for up to 6 wks following a strptococcal infction.
Any positiv
srum shoul
 furthr ilut to trmin th titr. Positiv rsults wit
h Strptoym
ar oftn o
tain whn th ASO titr is ngativ,
caus multipl anti
ois
can
 tct.
Titrs consir to
 within normal limits may vary with th sason of th ya
r, th gographic location, an th ag group of th patints. 1814_Ch19_311-327.qx 7/10
/09 3:53 PM
Pag 325 326 SECTION 4 Srological Diagnosis of Infctious Disas 1. All of th
following ar
protctiv mchanisms against
actria xcpt a. prouction of fvr.
. phagocy
tosis. c.
activation of complmnt. . altration of surfac antigns. 2. All of th follo
wing ar
charactristics of strptococcal M protins xcpt a. it is th chif virulnc
factor of group A
strptococci.
. it provoks an immun rspons. c. anti
ois to on srotyp p
rotct against
othr srotyps. . it limits phagocytosis of th organism. 3. An ASO titr an
a Strptoym
tst ar prform on a patints srum. Th ASO titr was ngativ, showing hmol
ysis in all
patint tu
s. Th Strptoym tst is positiv, an
oth th positiv an ngat
iv controls
ract appropriatly. What can you conclu from ths tst rsults? a. Th pati
nt has a high
titr of ASO.
. Th patint has an anti
oy to a strptococcal xonym othr
than strptolysin
O. c. Th patint has not ha a prvious strptococcal infction. . Th patint
has scarlt

fvr. 4. Which of th following applis to acut rhumatic fvr? a. Symptoms

gin aftr ithr
a throat or a skin infction.
. Anti
ois to group A strptococci cross-ract
with hart
tissu. c. Diagnosis is usually ma
y cultur of th organism. . All patints
suffr prmannt
isa
ility. 5. Which of th following inicats th prsnc of anti-DNas
 B activity in
srum? a. Ruction of mthyl grn from grn to colorlss
. Clot formation wh
n actic aci is
a c. Inhi
ition of r
loo cll hmolysis . Lack of chang in th color i
nicator 6. Which
of th following is consir to
 a nonsuppu- rativ complication of strptoc
occal infction?
a. Acut rhumatic fvr
. Scarlt fvr c. Imptigo . Pharyngitis 7. All of t
h following ar
ways that
actria can va host fnss xcpt a. prsnc of a capsul.
. s
timulation of
chmotaxis. c. prouction of toxins. . lack of ahsion to phagocytic clls. 8.
Anti
oy tsting
for Rocky Mountain spott fvr may not
 hlpful for which rason? a. It is n
ot spci c.
. It
is too complicat to prform. c. It is if cult to o
tain a
loo spcimn. . An
ti
oy
prouction taks at last a wk
for tction. 9. Which of th following n
yms is us to
tct th prsnc of H. pylori infctions? a. DNas
. Hyaluronias c. Uras
. Pptias 10.
Which of th following rasons mak srological in- ti cation of a currnt infc
tion with
Hlico
actr pylori if cult? a. No anti
ois appar in th
loo.
. Only IgM is
prouc. c.
Anti
ois rmain aftr initial tratmnt. . No ELISA tsts hav
n vlop
. 11. M.
pnumonia infctions ar associat with which anti
ois? a. Col agglutinins

. Anti
ois to
ATPas c. Anti
ois to DNas . Anti
ois to Protus
actria 12. Which of th
following
st
scri
s th principl of th IFA tst for tction of anti
ois prouc in
Rocky Mountain
spott fvr? a. Protus antigns ar us to trmin cross- ractivity.
. A
light microscop
is us to tct antign anti
oy com
ination. c. Whol
actria ar us to t
ct anti
ois.
. Anti
ois ar tct
y irct uorscnc. REVIEW QUESTIONS 1814_Ch19_311-3
27.qx 7/10/09
3:53 PM Pag 326 CHAPTER 19 Srological an Molcular Dtction of Bactrial I
nfctions 327
Rfrncs 1. Finly, BB, an McFan, G. Anti-immunology: Evasion of th host
immun systm
y

actrial an viral pathogns. Cll 124:767782, 2006. 2. Black, JG. Micro
iology
Principls an
Explorations, . 6. Wily, Ho
okn, NJ, 2005, pp. 446469. 3. Mak, TW, an Saun
rs, M. Th
Immun Rspons: Basic an Clinical Principls. Elsvir Acamic Prss, Burling
ton, MA, 2006,
pp. 641694. 4. For
s, BA, Sahm, DF, an Wissfl, A. Baily an Scotts Diagnosti
c
Micro
iology, . 12. Mos
y Elsvir, St. Louis, 2007, pp. 265280. 5. Fischtti,
VA.

Strptococcal M protin. Sci Am 264:58, 1991. 6. Larson, HS. Strptococcacca. I

n Mahan, CR, an
Manuslis, G (s): Txt
ook of Diagnostic Micro
iology, . 2. WB Saunrs, Phi
lalphia, 2000,
pp. 345371. 7. Wssls, MR. Strptococcal an ntrococcal infctions. In Braunwa
l, E, Fauci,
AS, Kaspr, EL, t al. (s): Harrisons Principls of Intrnal Micin, . 15.
McGraw-Hill,
Nw York, 2001, pp. 901909. 8. Saua, M, Wu, W, Conran, P, t al. Strptococcal p
yrognic
xotoxin B nhancs tissu amag initiat
y othr Strptococcus pyogn
s proucts. J
Infct Dis 184:723731, 2001. 9. Spllr
rg, B, an Brant, C. Strptococcus. In
Murray, PR,
Baron, EJ, Jorgnsn, JH, t al. (s): Manual of Clinical Micro
iology
, . 9. ASM
Prss, Washington, DC, 2007, pp. 412429. 10. Moss, AE, Gol
rg, S, Kornman, Z,
t al.
Invasiv group A strptococcal infctions. Isral Emrg Infct Dis 8:421426, 2002
. 11. Davis,
HD, McGr, A, Schwart, B, t al. Invasiv group A strptococcal infctions in
Ontario, Canaa,
Ontario Group A Strptococcal Stuy Group. N Eng J M 335:547554, 2001. 12. Cnt
rs for Disas
Control an Prvntion. Summary of noti- a
l isassMarch 21, 2008. MMWR 55(53):
194, 2008.
13. Activ Bactrial Cor Survillanc. Activ Bactrial Cor Survillanc
(ABCs) Rport,
Emrging Infctions Program Ntwork: Group A strptococcus 2006. Availa
l at ht
tp://
www.cc.gov/ncio/
m/a
cs/survrports/gas06.pf. Accss March 2008. 14. Bra
y, HR, an
Brnnr, BM. Pathognsis of glomrular injury. In Braunwal, E, Fauci, AS, Kasp
r, DL, t al.
(s): Harrisons Principls of Intrnal Micin, . 15. McGraw- Hill, Nw York,
2001, pp.
15721580. 15. Lang, MM, an Towrs, C. Intifying poststrptococcal glomr
ulonphritis.
Nurs Pract 26:34, 2001. 16. Sht, A, an Kaplan, E. Diagnostic mthos for grou
p A strptococcal infctions. In Dtrick, B, Hamilton, RG, an Fols, JD, (s): Manual
of Molcular
an Clinical La
oratory Immunology, . 7. Amrican Socity for Micro
io
logy, Washington,
DC, 2006, pp. 428433. 17. CDC, Biotchnology Cor Branch Facility. Introuction t
o mm typing:
M protin gn (mm) typing Strptococcus pyogns. Availa
l at:
http://www.cc.gov/ncio/
iotch/ strp/M-ProtinGn_typing.htm. Accss Marc
h 2008. 18.
National Instituts of Halth Consnsus Confrnc. Hlico
actr pylori in
pptic ulcrs.
JAMA 272:6569, 1994. 19. Malfrthinr, P, Mgrau, F, OMoran, C, an th Europan
Hlico
actr
pylori Stuy Group (EHPSG). Currnt concpts in th managmnt of Hlico
act
r pylori
infctionth Maastricht 2-2000 Consnsus Rport. Alim Pharmacol Thr 16:167180, 20
0, 2002. 20.
Sgal, ED, Cha, J, Lo, J, Falkow, S, an Tompkins, LS. Altr stats: Involvm
nt of
phosphorylat CagA in th inuction of host cllular growth changs
y Hlico
a

ctr pylori. Proc

Natl Aca Sci USA 96:1455914564, 1999. 21. Mgrau, F. Impact of Hlico
actr pyl
ori virulnc
in th outcom of gastrouonal isas: Lssons from th micro-
iologist. Dig
st Dis
19:99103, 2001. 22. Sokic-Milutinovic, T, Wx, T, Toorovic, V, Milosavljvic, T,
an
Malfrthinr, P. Anti-CagA an anti-VacA anti
ois in Hlico
actr pyloriinfct
 patints
with an without pptic ulcr isas in Sr
ia an Montngro. Scan J Gastron
trol 3:222226,
2004. 23. Dunn, BE, an Phanis, SH. Srologic an molcular iag- nosis of H
lico
actr
pylori infction an raication. In Dtrick, B, Hamilton, RG, an Fols, JE
(s): Manual of
Molcular an Clinical La
oratory Immunology, . 7. Amrican Socity for
Micro
iology,
Washington, DC, 2006, pp. 462467. 24. Mgrau, F, an Lhours, P. Hlico
actr py
lori tction
an antimicro
ial suscpti
ility tsting. Clin Micro
iol Rv 20(2):280322, 2
007. 25.
Mgrau, F, an Fox, JG. Hlico
actr. In Murray, PR, Baron, EJ, Jorgnsn, JH
, t al.
(s): Manual of Clinical Micro
iology, . 9. ASM Prss, Washington, DC, 200
7, pp. 947962.
26. Waits, KB, an Taylor-Ro
inson, DT. Mycoplasma an ur- oplasma. In Murray,
PR, Baron, EJ,
Jorgnsn, JH, t al. (s): Manual of Clinical Micro
iology, . 9. ASM
Prss,
Washington, DC, 2007, pp. 10041020. 27. Foy, HM. Infctions caus
y Mycoplasma
pnumonia an
possi
l carrir stat in iffrnt populations of patints. Clin Infct Dis 17(
suppl.
1):S37S46, 1993. 28. For
s, BA, Sahm, DF, an Wissfl, A. Baily an Scotts Dia
gnostic
Micro
iology, . 12. Mos
y Elsvir, St. Louis, 2007, pp. 525532. 29. Waits, K
B, Brown, MB,
an Simcka, JW. Mycoplasma: Immunologic an molcular iagnostic mthos. I
n Dtrick, B,
Hamilton, RG, an Fols, JE (s): Manual of Molcular an Clinical La
oratory I
mmunology, . 7.
Amrican Socity for Micro
iology, Washington, DC, 2006, pp. 510517. 30. Walkr,
DH, an Bouyr,
DH. Rickttsia an orintia. In Murray, PR, Baron, EJ, Jorgnsn, JH, t al. (
s): Manual of
Clinical Micro
iology, . 9. ASM Prss, Washington, DC, 2007, pp. 10361045. 31.
Anrson, B,
Friman, H, an Bninlli, M. Rickttsial Infction an Immunity. Plnum Prss
, Nw York, 1997.
32. Hchmy, KE, Rikihisa, Y, Macaluso, K, Burgss, AWO, Angrson, BE,
an Thompson,
HA. Th Rickttsiaca, Anaplasmataca, Bartonllaca, an Coxillaca. In
Dtrick, B,
Hamilton, RG, an Fols, JE (s): Manual of Molcular an Clinical La
oratory I
mmunology . 7.
Amrican Socity for Micro
iology, Washington, DC, 2006, pp. 527539. 1814_Ch19_31
1-327.qx
7/10/09 3:53 PM Pag 327 20 Srological Rspons to Parasitic an Fungal Infc
tions Patsy C.
Jarrau, MHS, CLS(NCA), MT(ASCP) LEARNING OBJECTIVES Aftr nishing this chaptr,

th rar will

 a
l to: 1. Explain why a host has mor if culty ovrcoming parasitic isass
than thos
caus
y
actria or viruss. 2. Discuss outcoms that may follow invasion
y a
parasit,
incluing a rason for ach outcom. 3. List ways parasits va host fnss.
4. Stat
xampls that illustrat how host rsponss to parasits caus othr pathology f
or th host. 5.
Discuss th rol of IgE in parasitic infctions. 6. Stat important iagnostic i
nformation
provi
y srological tsting for Toxoplasma gonii. 7. Explain how srologica
l tsts for
Entamo
a histolytica, E. histolytica/ispar, Giaria lam
lia, Trichomonas vagin
alis, an
Cryptosporiium parvum iffr from srological tsts for othr parasits. 8. Lis
t possi
l
limitations associat with parasitic srology. 9. List factors that hav l to
a nota
l
incras in fungal infctions in th last 25 yars. 10. Dscri
 th tiological
an
physiological factors to
 xamin whn a mycosis is suspct. 11. Dscri
 t
h typs of
immun fnss mount
y th host in rspons to fungal infctions. 12. Discus
s th importanc
of sroiagnosis in making a rapi an prsumptiv iagnosis of fungal infction
s. 13. List thr
aras of tsting in which immunologic procurs hav major application for myco
tic iagnosis.
14. Rcogni th clinical isas an pimiology of asprgillosis, caniiasi
s,
cryptococcosis, an cocciioiomycosis. 15. List at last thr srological tst
s currntly us
for th sroiagnosis of asprgillosis, caniiasis, cryptococcosis, an coccii
oiomycosis. KEY
TERMS Antign switching Antignic variation Asprgillosis Caniiasis Cocciioi
omycosis Conia
Cryptococcosis Cyst Eosinophil chmotactic factor Fungi Hypha Mol Myclium Myc
oss Sphrul
Thrmal imorphism Toxoplasmosis Yast 328 1814_Ch20_328-346.qx 7/13/09 5:11
PM Pag 328
CHAPTER 20 Srological Rspons to Parasitic an Fungal Infctions 329 Convnt
ional la
oratory
iagnosis of parasitic an fungal isass is trmin
y o
srving morphologi
cal faturs of
parasits an cultur (in th cas of fungi). Though sro- logical tsting for
parasitic an
fungal infctions has incras, it is still not wily us. Srological tst
ing for parasits
an fungi is limit, an th antigns for
oth ar oftn crur than thir coun
trparts for
viral an
actrial iagnosis. This rsults in cras spci city in many iagnostic
procurs. A furthr similarity
twn th two groups of organisms is that
ot
h parasitic an
fungal is- ass hav rciv mor attntion in rcnt yars
caus of thir
rol as
opportunistic infctions in immunocompro- mis patints. Ths iniviuals ruc
 a
ility to
prouc anti
ois limits th utility of srological tsts. Thrfor, it sms

appropriat to
iscuss immunologic factors for ths two typs of infctions in on chaptr. Th
is chaptr provis information on th immunologic aspcts of ths infctions along wi
th a iscussion
of availa
l srological tsting. PARASITIC IMMUNOLOGY Until th mrgnc of
HIV, parasitic
infctions wr uncommon in th Unit Stats. With th spra of AIDS, svra
l infctions such
as toxoplasmosis, giariasis, an cryp- tosporiiosis hav
com mor important
pro
lms. Othr
parasitic isass such as malaria, schistosomiasis, an lish- maniasis ar amo
ng th most
harmful infctiv isass afflicting humans worlwi. 1 A furthr unrstanin
g of parasitic
immunology will la to
ttr iagnosis an con- trol of ths isass an
will a to
th funamntal knowlg of th immun rspons. This sction covrs th vario
us immunologic
stratgis us
y a host as it com
ats parasitic infctions. Spci c tails a
ou
t various
parasits ar consir latr in th chaptr, as is th iagnostic rol
 of srological
tsting mthos as thy rlat to ach parasit. Whn a parasit ntrs a host,
thr ar svral
possi
l rsults. First, thr may
 no infction at all,
caus th hosts inna
t immunity
prvnts th parasit from sta
lishing an infction. In this cas, th host fai
ls to provi
ithr th nc- ssary physical nvironmnt or som important nutritional factor
(s) n for
th parasits survival. Th host may vn prouc proucts that ar toxic to th
parasit. In
th scon possi
l outcom, th parasit may inva th host,
com sta
lish
, an thn

kill an limi- nat
y host fns mchanisms. Ths vnts inicat that a
n ffctiv
immun rspons has occurr. Typically, th host rmains immun to rinfction
for som tim.
This rspons rquirs anti
oy formation an cll-miat immunity. Th thir
possi
l outcom
is th rvrs of th scon th parasit may ovrwhlm an kill th host. Rason
s for this
inclu an organism that multiplis so rapily that th host os not hav tim
to mo
ili its
fnss, th parasit invas a vital organ(s), or th host has an ina
quat immun
systm. A fourth possi
l outcom is a long-lasting infction in which th host

gins to
liminat th parasit
ut cannot rmov it compltly. In th
st cas, th ho
st controls th
isas for an xtn prio an vntually ovrcoms an liminats th paras
it. In th worst
cas, th host ulti- matly succum
s to th infction an is. An, nally, a fth
possi
l
outcom is th host mounts a rspons that attacks not only th parasit
ut als
o host tissus.
Th ffcts of this inappropriat rspons (hypr- snsitivity or immun isas
) may
 mil or
svr; oftn th consquncs for th parasit ar minimal. In fact, most of th

 pathology
associat with a parasitic infction rsults from th immunologic rsponss to
th offning
organ- ism an may
 mor angrous for th host than th invar. Ev
asion of Host
Dfnss Any parasits survival pns on its a
ility to liv in a paca
l man
nr with its
host. If th host is, thn so os th parasit. Howvr, whn th host rcogn
is a parasit
as a forign ntity, th rlationship
coms com
ativ. As a host fns itsl
f, th parasit
attmpts to va ths CHAPTER OUTLINE PARASITIC IMMUNOLOGY Evasion of Host Df
nss Immunologic
Rspons to Parasits Srology in Protooan Disass Hlminth Infctions Limitat
ions of Parasitic
Srology FUNGAL IMMUNOLOGY Charactristics of Fungi Mycotic Infctions (Mycoss)
Immun Rspons
to Fungi Srology in Mycotic Disass Molcular Tsting SUMMARY CASE STUDY EXERC
ISE: GIARDIA
LAMBLIA ANTIGEN TESTING BY A SANDWICH ELISA METHOD REVIEW QUESTIONS REFERENCES
1814_Ch20_328-346.qx 7/13/09 5:11 PM Pag 329 330 SECTION 4 Srological Diag
nosis of
Infctious Disas fns mchanisms. Parasits ar larg organisms that ar v
ry antignically
complx an hav complx lif cycls. Diffrnt stags of th organism may
 fo
un in various

oy locations. Th nt rsult las to a situation in which th host may partia
lly control th
infction
ut not limi- nat th parasit. Th host continus to fight
with incrasingly
complx rsponss until ithr th host or th parasit prvails. Frquntly, th
r is only an
unasy com- promis that can
 tipp in favor of ithr si. Many factors, so
m known an many
othrs not yt known, work togthr to prsrv th parasits xistnc. If a par
asit
coms
squstr within host clls, th parasit is protct. For xampl, whn para
sits such as th
tissu protooans inva macrophags or livr clls, thy ar tmporarily hin
from th immun
systm. Th host cannot rcogni th parasits whil thy rsi insi ths c
lls. 2 Howvr,
vntually thy must xit ths clls an inva nw ons. Whn thy mov
tw
n clls, thy ar
vulnra
l to th hosts fnss. Som parasits isguis thmslvs
y acquiring
host antigns.
Schistosoms surviv in unprotct
loo vs- sls,
caus th invaing form (
schistosomula)
acquirs r
loo cll antigns that latr hlp protct th ault from attack

y th immun
systm. 3 Anothr procss of vasion som parasits mploy is call anti
gnic variation.
African trypanosoms va th immun rspons
y prioically changing thir su
rfac antigns. A
variant surfac glycoprotin proucs an unlim- it group of varia
l antig
n typs. 4,5
Th host
uils anti
oy, mainly IgM, to th on antign, thr
y rucing th
infction. Th
parasit rspons
y changing its anti- gn, making th currnt anti
oy inffc
tiv. This

switching may occur vry rapily, within 5 to 6 ays. Th host must thn prouc
 a nw
anti
oy. This procss of antign switching can continu for long prios of
tim. Anothr
mchanism of scap involvs antign sh from th parasit. For xampl, Entamo

a histolytica
can sh antigns. 2 Anti
oy is form an attachs to th antign. Sinc th a
ntign is not
attach to th parasit, th immun rspons is una
l to harm th offning or
ganism. Molcular
mimicry may rsult from parasitic antigns that ar similar to antigns normally
foun on host
tissu. This may la to autoimmun pro
lms such as th cariac an intstinal
consquncs that
occur in th lat stags of Chagas isas. 2 Along with ths spcific mchanis
ms parasits us
to va th hosts immun systm, thr ar numrous non- spci c factors that int
rfr with
th normal opration of th systm as a whol. Whn a host liminats a
actria
l or viral
infction, th immun systm rsts until a nw ncountr occurs. Parasiti
c infctions,
howvr, ar chronic,
caus th host is rarly a
l to totally liminat th s
ourc of
infction. Th immun systm is forc to rmain activ. Oftn th rsult is imm
unosupprssion,
isruption of nor- mal B-cll an T-cll functions, an hyprsnsitivity r
actions. For
xampl, schistosoms hav mchanisms that can own-rgulat th hosts immun sys
tm. This
immun moulation promots th parasits survival
ut also limits svr amag t
o th host. As
a rsult, ault schistoms can liv in th human host for up to 40 yars
for n
ally
ing
liminat. 6,7 All of ths complicat mchanisms along with a multitu of ot
hr vasiv
mchanisms ithr con- tri
ut to or irctly caus th pathology sn in parasi
tic isass.
Immunologic Rspons to Parasits Innat an aaptiv immun rsponss ar impor
tant in protcting th host from parasitic infction. Working in concrt, svral cll typ
sincluing B an
T lymphocyts, natural killr clls, macrophags, othr antign-prsnting clls
, granulocyts,
an mast cllsactivly participat in th rcognition an limination of para
sits from
th host. Solu
l su
stancs, incluing immunoglo
ulins, compl- mnt, an cyto
kins, also
participat in th hosts a
ility to ovrcom parasitic infctions. Innat immun
rsponss ar
critical in th arly rcogni- tion of an infction. As part of th innat immun
 fns,
nutrophils act as phagocyts to stroy
oth intracllular an xtracllular pa
rasits. Hyrogn
proxi kills xtracl- lular organisms whil nutrophilic granuls stroy ing
st organisms.
Bcaus natural killr (NK) clls can
 activat without prior xposur to th
pathogn, thy
ar important in th arly fns against parasitic infction. NK clls pro- u
c gamma

intrfron (IFN-), which inhi
its the replication of intracellular parasites. 8 C

omplement
activation provides protection throuh the production of chemotactic compo- nent
s, opsonization
for enhanced phaocytosis, and lysis of extracellular parasites. Cellular immuni
ty is responsi
le
for the production of cytokines and chemokines, which enhance the cytotoxic func
- tion of
effector cells, increase the num
ers of immune cells, and attract immune cells t
o the site of
infection. Many proto- zoan parasites are effectively reduced or eliminate
d
y macrophaes
that are activated
y sensitized T cellenerated cytokines such as interferon- and
the
miration inhi
itory factor. Other cytokines such as interleukin-3 and ranulocy
te- monocyte
colony-stimulatin factor act on cells of the myeloid line to cause an incre
ase in
neutrophils, eosinophils, and macrophaes; thus, additional effector cells are
recruited to
com
at the infection. The immunolo
ulin response to parasites includes for- mat
ion of IM,
IG, IA, and IE anti
odies. Specific anti
ody can damae protozoa, neutra
lize parasites
y

lock- in attachment to the host cell, prevent the spread of the parasite, prom
ote complement
lysis, and enhance phaocy- tosis and destruction throuh anti
ody-dependent cel
lular cytotoxity.
Helminth infections in particular are character- ized
y eosinophilia and hih l
evels of IE. IE

inds to mast cells and
asophils. When specific antienanti
ody com
inations oc
cur, mast
cells deranulate and release chemotactic factors, histamine, prostalandins,
and other
1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 330 CHAPTER 20 Seroloical Respons
e to Parasitic
and Funal Infections 331 mediators. One of the most important mediators relea
sed is eosinophil
chemotactic factor, which attracts eosinophils to the infected area. Eosinophils
can destroy
helminths
y deranulation or throuh anti
ody-dependent cellular cyto- toxicity
. It is
elieved
that the a
ility to produce IE evolved mainly for the purpose of dealin with p
arasitic
infections. 3 Althouh anti
odies are detecta
le in serum and may
e useful uid
es when
dianosin diseases caused
y parasites, they have little or no correlation with
the course or
pro- nosis of the disease. Later protection from reinfection
y the parasite ca
nnot
e predicted
on the
asis of circulatin anti
ody levels, nor can any other useful informatio
n
e ained
y
measurin cellular responses. However, seroloy can
e used to identify parasite
s that are
present in orans or other deep tissues such as the
rain or muscle and that are
not recovera
le
in
lood, urine, or feces. If a person travels to an endemic area,
ecomes infec
ted with a
parasite, and then returns to a nonendemic area, a pos- itive seroloy test can
indicate a recent

infection. Conversely, a positive test result in a person who lives in an endemi

c area may only
re ect a previous parasitic infection. Because anti-
ody levels may remain elevat
ed for years,
the positive result may not relate to the patients current condition. Seroloy in
Protozoan
Diseases Falciparum malaria and African trypanosomiasis continue to produce hih
mortality rates
despite intense efforts to control the parasites responsi
le for these diseases.
Relatively mild
diseases such as ame
iasis, iardiasis, and toxoplasmo- sis are found worldwi
de
ut have

ecome sinificant pro
lems for patients who have suppressed immune sys- tems.
Immunosuppressive
drus used to treat cancer, prevent rejection of transplanted orans, and, more
importantly,
treat diseases such as AIDS have caused increased suscepti-
ility to these infe
ctions.
Infections with Cryptosporidium parvum, Cyclospora cayetanensis, and several Mic
rosporidium
species have
ecome more prevalent, especially in immuno- compromised hosts. Bec
ause protozoa can
live in the
lood, the astrointestinal tract, oran tissues, and macrophaes, c
linical
manifestations and host responses are quite varied. Toxoplasmosis and other repr
esentative
protozoan diseases for which seroloical testin is useful are presented here. T
oxoplasmosis
Toxoplasmosis results from infection with Toxoplasma ondii, a u
iquitous pr
otozoan parasite
that infects humans
y inestion of infective cysts (oocysts). It is thouht tha
t cysts are
transferred
y hand-to-mouth contact from con- taminated soil or cat litter or

y inestin
oocysts in raw or partially cooked pork, mutton, or
eef. Transmission may also
occur throuh

lood transfusions or oran transplan- tation. Cats are known to
e de nitive host
s for this
parasite,
ut other definitive hosts must also exist, since nearly 24 pe
rcent of the U.S.
population over ae 12 is infected with this parasite. 9 This disease is nearly
always
asymptomatic or may present with a mild lymphadenopathy. Rarely, the parasite re
aches the central
nervous system (CNS) or the eye. Invasion of the CNS can
e fatal
ut typically
occurs only in
immunosuppressed patients. 10 This is one of the more common opportunistic infec
tions seen in
individuals with AIDS. Encysted oranisms can
e found in any tissue (Fi. 201).
These cysts may
remain via
le for years, which explains why immunosuppressed patients can exhi
i
t symptoms lon
after initial infection. The reatest concern, however, is that Toxoplasm
a species can
cross the placenta. If the fetus is exposed durin the rst trimester, death
nearly always
occurs as a result of CNS damae. Infection durin the second trimester
may result in
hydrocephaly,
lindness, or other nerv- ous system damae. Later infection may r
esult in


lindness or mild CNS defects. However, women who are exposed to this parasite

efore prenancy
do not transmit the infection to the fetus. T. ondii is capa
le of replicatin
inside human
macro- phaes. The parasite can survive inde nitely here,
ecause the oranism c
an prevent
the fusion of lysosomes with phaosomes. Normally a phaocytic cell diests
the mate- rial
within the phaolysosome,
ut in this case, diestion does not occur. The macrop
haes a
ility to
kill the para- site is reatly increased if the parasite is rst opsonized
y anti

ody.
Anti
ody-coated Toxoplasma oranisms trier phaocytosis, which ultimately d
estroys the
oranism. Immunocompromised patients often have severe pro
lems with this infect
ion, a fact that
further confirms the role played
y anti
ody in controllin this disease. Serolo
ical testin
plays an important role in dianosin toxoplasmosis,
ecause isolation of the or
anism requires
tis- sue
iopsy. Generally, seroconversion to an anti
ody-positive state, a four
fold increase in
the titer of IG, or hih levels of IM and IG anti
odies are considered diano
stic. However,
detecta
le elevations of IM may persist for months to years, 50 m FIGURE 201. Pse
udocyst of
Toxoplasma ondii seen in a
rain section. (From Leventhal, R, and Cheadle, RF.
Medical
Parasitoloy: A Self-Instructional Text, ed. 3. FA Davis, Philadelphia, color pl
ate 123, 1989,
with permission.) See Color Plate 15. 1814_Ch20_328-346.qxd 7/13/09 5:11 PM P
ae 331 332
SECTION 4 Seroloical Dianosis of Infectious Disease makin determination of ac
ute infection
dif cult. IA levels can also
e detected in early infection and can persist for 3
to 9 months.
Concurrent elevations of IM and IA indicate early infection and are used to sc
reen prenant
women. 2 Enzyme immunoassays (EIA) for IM, IG, or IA and indirect uorescent an
ti
ody (IFA)
assays for IG are avail- a
le and should
e performed when conenital toxoplasm
osis is
suspected. IFA testin has
een widely used,
ut EIA is the method of choice, 11
as it is more
sensitive, less difficult to perform, and easier to interpret. Elevated titers o
f IG, IM, or
IA anti
ody classes suest that infection has occurred within the previous 3 t
o 9 months.
Elevated IG titers with- out IM anti
ody suest an older infection. Paired sa
mples whose
collection is separated
y 3 weeks are tested to con- rm the presence of recent i
nfection. In a
recent infection, titers of
oth of these anti
ody classes will rise. New
orns w
ith conenital
toxoplasmosis usually have detecta
le IM anti
ody. Presence of elevated IM tit
ers in the
mothers serum further supports the dianosis (Ta
le 201). Speci c IA anti
ody assay
s provide
even more sensitive methods for early detection and con rmation of conenital toxo
plasmo- sis

and should
e performed on new
orns suspected of har
orin the parasite.

Early dianosis
and maternofetal treatment can reduce fetal infection to mild or su
clinical. Pr
enatal conenital
toxoplasmosis can
e dianosed
y per- formin polymerase chain reaction (PCR) t
echnoloy on
amniotic uid to detect T. ondii DNA. 12 Currently, there are no useful seroloic
al procedures
for dianosin CNS infection in immunocompromised patients. 9,11 These indi
viduals often
do not produce detecta
le levels of specific anti
ody aainst the parasite, an
d procedures to
detect circulatin antien appear to lack sensitivity. PCR is, therefore, the me
thod of choice to
detect T. ondii DNA in CSF. Other Protozoa Serum anti
ody studies are not used
to identify
several proto- zoa,
ecause conventional methods of identi cation are easier to pe
rform and
provide more de nitive results. Entamoe
a histolytica, Entamoe
a histolytica/dispa
r,
Cryptosporidium parvum, Giardia lam
lia, and Trichomonas vainalis are all
parasites that
are usually identi ed
y microscopic examination of specimen materials. Feces
are tested
for E. histolytica, E. histolytica/dispar, C. parvum, and G. lam
lia; urine or
vai- nal
dischare is tested for T. vainalis. However, immunoloic procedures directed t
oward identifyin
the oranism or a sol- u
le antien found in the specimen are extremely helpful.
The
identi cation rates for each test compare favora
ly with conventional identi cation
techniques,

ut immunoloic procedures offer an important advantae,
ecause a technol- o
ist who is not
experienced in parasitoloy can successfully perform them. Direct fluorescent
anti
ody (DFA)
and enzyme-linked immunosor
ent assay (ELISA) kits are avail- a
le for these or
anisms. The DFA
methods use monoclonal anti
ody la
eled with uorescein isothiocyanate aainst the
parasites
cell wall to visualize the initial anti
ody-parasite complex (see Fi. 105, color
plate 10)
Auto uorescence of Cyclospora cayetanensis is a characteristic of the oranism tha
t is used as an
additional dianostic technique for identifyin this parasite in feces,
ut it c
an lead to
confusion and incor- rect interpretation of DFA reactions performed to detect ot
her parasites,
such as C. parvum. 13 EIA methods employ enzyme conjuated to either anti- en o
r anti
ody,
dependin on the assay
ein performed. The ELISA methods capture oranisms or s
olu
le antien
y
addin patient samples to anti
ody-coated wells. The enzyme conjuate is then ad
ded. The
appropriate su
strate for the enzyme employed is then added. The wells
are washed

etween each step of the procedure to remove anti-
odies or antiens that a
re not
attached. A chane in chromoen color indicates the presence of
ound antien
. The more color

produced, the reater the concentration of the patient anti
ody or antien. (Mor
e details a
out
this procedure are iven in this chapters la
oratory exercise.) PCR techniques ha
ve
een
developed to identify species of Plasmodium, Toxoplasma, Giardia, Trypanosoma, L
eishmania,
Cryptosporidium, Entamoe
a, Microsporidia, Ba
esia, and Cyclospora. Many of t
hese procedures
are availa
le in refer- ence la
oratories
ut are not yet used in the r
outine
la
oratory. PCR testin has
een compared to microscopy in the identi cation of ma
ny parasites.
Thouh speci city is equal, most studies indicate that PCR is more sensitive. It i
s easier to
interpret
ut more dif cult and time-consumin to perform. It also provides the a

ility to
enotype. 1214 The FDA has recently approved a rapid EIA dianostic test for mala
ria. The
procedure derives results in approxi- mately 15 minutes. It allows the different
iation of
Plasmodium falciparum from less virulent malaria parasites. Thouh malaria
is rare in the
United States, cases are dianosed in individuals who travel to endemic countrie
s. Since the disease is uncommon, dianosis may
e delayed. Death can
e prevented if the diseas
e is dianosed
and treated early. Rapid testin allows faster dianosis and leads to earlier tr
eatment. 15 Other
rapid tests are availa
le for the dianosis of Cryptosporidium, Giardia,
Cyclospora, and
other parasitic Ta
le 55-2 Comparison of Tests Used TITER IIF-IG EIA-IM IN
TERPRETATION
1:16 Neative No evidence of exposure 1:16 Neative Infection pro
a
ly acquired m
ore than 1 year
ao 1:1024 1:41:256 Infection pro
a
ly acquired within the past 18 months 1:1024 1
:1024 Recent
infection, pro
a
ly acquired within the past 4 months IIF
indirect immuno uorscenc
e; EIA
enzyme immunoassay TABLE 201. Interpretation of Toxoplasma Seroloical Test Resul
ts
1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 332 CHAPTER 20 Seroloical Respons
e to Parasitic
and Funal Infections 333 infections. These tests are useful in dianosin foo
d
orne and
water
orne illnesses. Helminth Infections Phaocytosis
y macrophaes and neutro
phils can destroy
larval staes of helminths. It has
een lon reconized that eosinophils and mas
t cells play an
important role in the defense aainst tissue parasites. IE-coated mast cells re
cruit eosinophils
to the site of infection. Eosinophils deranulate and kill the oranisms thr
ouh
oxyen-dependent and oxyen-independent mechanisms. 3 Intestinal helminth infec
tions are
relatively simple to dia- nose,
ecause es, adult round worms, and sements o
f tapeworms are
easily recovered from stool specimens. However, larvae or em
ryos of some ta
peworm species
can exit the hosts intestine and mirate to other tissues. The patholoy produced

y these

wanderin parasites depends on the damae caused to the invaded oran or tissue.
Because symptoms
are often vaue or mimic other disease processes, it is dif cult to identify the p
arasite as the
causative aent. Serodianostic tests can provide helpful information when inves
tiatin these
pro
lems, especially in cysticercosis and echinococcosis. Current methods includ
e IFA tests;
slide, tu
e, and precipitin tests; complement fixation; particle alutina
tion tests; and
enzyme-linked immunoassays, amon others. Most commercial kit systems are
ased
on an ELISA
system. DNA pro
es are used in immuno
lot methods at the Centers for Disease Con
trol and
Prevention (CDC) to dianose cysticercosis, echinococcosis, paraonimiasis, and
schistosomiasis.
Ta
le 202 lists tests performed at the CDC. Samples for CDC processin must

e su
mitted
via state health la
oratories. The CDC does not accept speci- mens sent directly

y private
la
oratories or physicians. TABLE 202. Anti
ody Detection Tests for Parasites Off
ered at the
Centers for Disease Control and Prevention DISEASE ORGANISM TEST Ame
iasis E
ntamoe
a
histolytica Enzyme immunoassay (EIA) Ba
esiosis Ba
esia microti Immuno uorescence
(IFA) Ba
esia
sp. WA1 Chaas disease Trypanosoma cruzi IFA Cysticercosis Larval Taenia solium
Immuno
lot (Blot)
Echinococcosis Echinococcus ranulosus EIA, Blot Leishmaniasis Leishmania
razil
iensis IFA L.
donovani L. tropica Malaria Plasmodium falciparum IFA P. malariae P. ovale P. vi
vax
Paraonimiasis Paraonimus westermani Blot Schistosomiasis Schistosoma sp. FASTELISA S. mansoni
S. haemato
ium Blot S. japonicum Stronyloidiasis Stronyloides stercoralis EIA
Toxocariasis
Toxocara canis EIA Toxoplasmosis Toxoplasma ondii IFA-IG, EIA-IM Trichinell
osis
(Trichinosis) Trichinella spiralis EIA Courtesy of the Centers for Disease Contr
ol and
Prevention, Division of Parasitic Diseases. Send an e-mail to dpdx@cdc.ov or ca
ll the Division
of Parasitic Diseases at (770) 488-4431 for information on the followin disease
s or oranisms
for which seroloy tests are not availa
le at CDC
ut may
e availa
le elsewhere
within the
United States or a
road: African trypanosomiasis; Aniostronylus; Anisakis; Bay
lisascaris
procyonis; Echinococcus multilocularis; Fasciola hepatica; Filariasis; Gnathosto
ma.
1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 333 334 SECTION 4 Seroloical Dia
nosis of
Infectious Disease Limitations of Parasitic Seroloy As in all clinical la
orato
ry areas, it is
important in clinical immunoloy to nd the most straihtforward and economic proc
edure for each
test method. However, it is also impor- tant to consider the speci city and sensit
ivity of the
method when choosin a procedure. Because no external pro ciency testin proram i
s currently

offered in the area of parasitic seroloy except for dianosis of Toxoplasm

a and Ba
esia
microti, 11 it is very dif cult to evaluate the quality of com- mercial products.
In the
United States, commercial kit manufacturers must o
tain FDA approval
efore s
ellin their
products. The FDA requires only that a new method
e equivalent to a method that
has already
een
approved. Researchers at the CDC have expressed concern that, over time, the qua
lity of new test
kits may drift in a neative direction,
ecause a new kit may not
e quite as o
od as the one
used for comparison
ut may still receive approval. 16 Because commercial kits f
or the dianosis
of some parasitic diseases are not availa
le, individual la
oratories develop th
eir own methods
usin antien purchased from various sources. This fact and the lack of pro ciency
testin places
the
urden of quality assurance on the individual la
ora- tory. Due to the d
ifficulty in
procurin and preparin antiens that are hihly speci c for these parasites, a
lare variation
occurs in the results reported
y various la
orato- ries. Accordin to the CDC,
unexpected
results should
e con rmed
y another la
oratory. Another pro
lem affectin the ch
oice of a
procedure is timin. A procedure may detect only a certain class of anti-
ody s
uch as IM; thus,
the test must
e performed when the patient is producin IM, or the dianosis m
ay
e missed. A
particular anti
ody produced aainst a certain stae in a parasites life cycle ma
y not
e
recovered
y a iven proce- dure, so aain timin is important. It must not
e f
orotten that
related oranisms produce similar antiens, which, in turn, induce the formation
of
cross-reactin anti
odies. This pro
lem reduces the speci city of a procedure. Alt
houh all of
these pro
lems must
e considered, it should
e empha- sized that with a clear u
nderstandin of
parasitoloy and the principles of immunoloy, useful dianostic information can

e provided for
the physician. FUNGAL IMMUNOLOGY Funal infections are usually well controlled i
n humans as lon
as the immune system functions properly. Funi that are normally considered nonp
athoenic may
cause mild to very severe patholoy in immunocompromised individuals. A rapidly
rowin
population of immunocompromised indi- viduals exists as a result of several fact
ors, the most
important
ein the prevalence of immunode ciency diseases such as AIDS. Other fac
tors are the
increased use of medications such as
road-spectrum anti
iotics for
acterial in
fections and
immunosuppressive aents for treatment of cancer and trans- plantation patients.
Because of the
immunocompromised status of these individuals, a su
stantial increase in the pre
va- lence of
funal infections has occurred, and mycoses have
ecome a serious health issue a
round the world.

Accordin to the U.S. National Center for Statistics, funal infections were the
seventh most
common cause of infectious-disease- related death in 1992, and the num
er of dea
ths due to funal
disease had increased threefold since 1980. 17 This time period parallels the in
troduction of HIV
in the United States. Funal diseases are often the rst opportunistic diseases de
tected in
patients with AIDS. One of the most common opportunis- tic diseases in patients
with AIDS is
pneumonia caused
y Pneumocystis carinii, also known as Pneumocystis jiroveci. T
his oranism was
rst characterized as a parasite
ut now is des- inated a funus,
ecause it has
a reater ene
sequence homoloy with funi than with parasites. 18 Many saprophytic funi form
erly dismissed as
cultural contaminants are now reported as opportunistic pathoens. 19 Characteri
stics of Funi
Funi are eukaryotic cells with nuclei and riid cell walls. Funi have two fund
amental
structural forms, appearin either as lamentous molds or yeasts; sometimes they a
ppear in
oth
forms. Molds are composed of hyphae and conidia. Hyphae are lamentous tu
ular
ra
nchin
structures that intertwine to form a dense mat called a mycelium. Conidia are as
exual
reproductive structures produced at the tip or alon the sides of fertile hyphae
. Yeasts are
unicellular and reproduce asexually
y
uddin. Althouh most funi are monomorp
hic, some exhi
it
thermal dimorphism. These dimorphs reproduce
oth as molds, at 25C to 30C, and as
yeasts, at
35C to 37C. The mycelial mold form is the saprophytic state found in nature; the y
east form is
the parasitic or pathoenic state found in tissue in disease, with the exception
of Coccidioides
immitis, which rows as a spherule at 35C to 37C. The thermal dimorphs are the
etioloic
aents of serious systemic mycoses that can
e life-threatenin. Mycotic Infe
ctions (Mycoses)
Of the reater than 80,000 species of funi that have
een identi ed, less than 40
0 are
considered pathoens. Ninety percent of the funal infections affectin humans a
nd ani- mals are
caused
y less than 50 species. 20 Thouh most pathoenic funi are found in
water, soil,
or decayin oranic matter, some exist in humans as normal ora. Funal diseases
are enerally
not spread from person to person. Those funi that are etioloic aents of human
infection are
normally soil saprophytes that have
een traumatically intro- duced into
ody ti
ssues or
accidentally inhaled into the luns. These exoenous pathoens can row at the r
elatively
elevated temperature of the human
ody and can survive cellular defenses. A few
endoenous
oranismsfor exam- ple, the yeast Candida al
icansalso can cause infection when th
e host
defense is de cient for only a
rief period. 19 1814_Ch20_328-346.qxd 7/13/09 5:
11 PM Pae 334

CHAPTER 20 Seroloical Response to Parasitic and Funal Infections 335 Mycoses

are clinically
classi ed as super cial, cutaneous, su
cutaneous, and systemic. The most common huma
n pathoens
are Coccidioides, Blastomyces, Histoplasma, and Paracoccidioides. These ora
nisms usually
cause lun disease
ut may also
ecome systemic and damae any oran of the
ody
. 21 Due to the
increased incidence of mycoses in immunocompromised patients, a new classif
ication called
opportunistic has
een added. 20 Many funi that cause oppor- tunistic infection
s are normally
considered nonpathoenic. The most common opportunistic oranisms are Candida, A
sperillus,
Cryptococcus, and Zyomycetes. Identifyin the etioloic aent is critical for t
reatin and
manain the patient when a funal infection has
een esta
lished. Because cultu
re does not
always reveal the infectious aent, immuno- loic procedures can provide the rst
presumptive
evidence of the infection. 22 Immune Response to Funi Host immunity, includin
the innate and
adaptive responses to
acteria and viruses, has
een widely studied. Much less h
as
een done to
investiate the role of these responses in com
atin funal infections. The deve
lopment of a
funal infection depends on the virulence of the funus, the num-
er of infecti
n funi inhaled
or injected into the tissue, and the hosts immune status. 19 The a
ility of the o
ranism to
survive at 37C at a neutral pH and the production of tox- ins are factors that
affect the
virulence of the funus. 21 Natural or innate resistance to funal infection
is very hih.
Physical
arriers such as skin and mucous mem
ranes pro- vide primary defense a
ainst funal
invasion. Effector cells such as neutrophils, macrophaes, and dendritic cells p
lay an early role
in the defense aainst oranisms that
reach the physical
arriers. These cells
possess
ermline-encoded receptors that allow reconition of funi and su
sequent killin
 of the
oranisms throuh phaocytosis. 23 Althouh the
ody produces anti
odies to fun
i, humoral
immunity and complement play only minor roles in the immune response to
funi.
Opsonization is not necessary for phao- cytosis of most funi,
ut it is requir
ed for
phaocytosis of Cryptococcus neoformans. 23 Cellular immunity is the most import
ant defense
aainst funal infection. In the majority of cases, the cellular defenses of nor
mal persons are
suffi- cient, and infections resolve spontaneously. Seroloy in Mycotic Diseases
Dianosis of
funal infections depends on the patients his- tory, clinical symptoms, and the i
denti cation of
the oranism. Funi are identi ed
y culture, reconizin the oranisms rowth rat
e and the
presence of distinctive colonial and microscopic morpholoical characteristi
cs. Althouh the

humoral response to funi is not the major defense aainst funal infection, ant
i
odies are
produced aainst the invad- in oranisms. Anti
ody detection, antien detection
, and the
identi cation of funi in patient specimens or culture are the three areas in whic
h immunoloic
procedures have found major application. Because funi row so slowly, isolation
and
morpholoical identification of funi usually cannot
e accomplished in le
ss than 5 days.
Therefore, the rapid avail- a
ility of results in seroloical testin make
s them an
important tool in dianosin funal infections. Culture often fails to detect or
identify the
infectious aent. In many cases, positive seroloical test results stimulate con
tinued efforts to
isolate and identify the etioloic aent in culture. 22 Seroloical tests also c
an yield
information used to monitor the effects of treatment with therapeutic aents. Se
lectin the tests
to
e performed depends on the eti- oloic aent of the mycosis. The clinician m
ust provide the
la
oratory with all the pertinent information availa
le, includin the pat
ients symptoms,
history of other past or present infections, and medical treatments that have le
ft the patient
immunocompromised or de
ilitated. The patients occupation, place of residence, an
d record of
travel are also important,
ecause some occupations expose an individual to oppo
rtunistic or
pathoenic funi that miht not other- wise have
een considered, and many funi
are endemic to
speci c eoraphic areas. Because seroloical tests are effec- tive at different s
taes of
mycotic disease and
ecause the stae of the disease may not
e known, the
est
procedure is to
use a com
ination of seroloical tests and a variety of related antiens to iden
tify the
anti
odies of the etioloic aent. Serial dilutions of serum should
e made afte
r an ini- tial
2- to 3-week interval (of the acute phase), midway throuh an infection,
and after
recovery (the convalescent phase) to determine if and how the titer is chanin.
Titers of 1:32
or a fourfold or reater rise in titer are sini cant in makin a dianosis as lon
 as the
patient is immunocompe- tent. In immunocompromised individuals, testin for
antiens rather
than anti
odies must
e performed. Serodianostic tests are commercially ava
ila
le with
different derees of sensitivity and specificity for asperillosis,
lasto
mycosis,
candidiasis, coccidioidomyco- sis, cryptococcosis, histoplasmosis, paracoccidioi
domycosis, and
sporotrichosis. Some of these tests are availa
le in routine la
oratories
(see Ta
le
203). The followin discussion provides information on four of the funal infecti
ons for which
seroloical testin is used. Asperillosis Asperillus species are found worldwi
de in soil and

air, in decay- in veetation, and on stored rains (Fi. 202). Asperillosis is
an
opportunistic infection predominantly caused
y Asperillus fumiatus, A. avu
s, A. nier,
and A. terreus. Other pathoenic species of Asperillus are involved with less f
re- quency.
Typical clinical infections may
e colonizin, alleric, or disseminatin, depen
din on the
patholoical ndins in the host. Disseminated asperillosis usually occurs in sev
erely
immunocompromised hosts. 24 Pulmonary colonization is usually a primary conditio
n induced
y
inhalation of lare num
ers of conidia. Alleric
ronchopulmonary asperillosis
is characterized

y alleric 1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 335 336 SECTION 4 Sero
loical Dianosis
of Infectious Disease Ta
le 203. Seroloical and Molecular Testin Used for Funa
l Infections
ORGANISM SEROLOGICAL TESTING MOLECULAR DIAGNOSTIC TESTING Asperillus sp. Im
munodiffusion
Not routinely availa
le Counterimmunoelectrophoresis Enzyme immunoassay Blastomy
ces dermatitidis
Immunodiffusion Not routinely availa
le Enzyme immunoassay Complement xation Cand
ida al
icans
Immunodiffusion PNA-FISH Counterimmunoelectrophoresis Latex alutination Enzyme
immunoassay
Coccidioides immitis Complement xation Performed on isolates of the oranism (us
ed for
con rmation) Immunodiffusion (exoantiens test) Latex alutination Tu
e precipiti
n test
Precipitation Enzyme immunoassay Cryptococcus neoformans Latex alutination ant
ien test Not
routinely availa
le (research la
s only) Tu
e alutination Enzyme immunoassay I
ndirect
immuno uorescence Dematiaceous oranisms Not useful Not routinely availa
le (resea
rch la
s only)
Only useful in screenin for allery to oranisms Dermatophytes Not useful Not r
outinely
availa
le Histoplasma capsulatum Immunodiffusion (exoantiens test) Performed on
isolates of the
oranism Complement xation Skin test for delayed or immediate hypersensitivity La
tex
alutination Counterimmunoelectrophoresis Radioimmunoassay for anti
ody Direct u
orescent
anti
ody assay for antien in tissue Paracoccidiodes
rasiliensis Immunodiffusio
n Availa
le in
reference la
s Complement xation Penicillium marneffei Immunodiffusion Not routin
ely availa
le
Sporothrix schenckii Immunodiffusion (exoantiens test) Not routinely availa
le
Zyomycetes Not
useful Not routinely availa
le 1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 336
CHAPTER 20
Seroloical Response to Parasitic and Funal Infections 337 reactions to the t
oxins and the
endotoxins of Asperillus species. Allery, asthma, or transient pulmonary in ltra
tes may develop
when hypersensitive individuals are exposed repeatedly to lare num
ers of conid
ia. 19
Disseminatin or invasive asperillosis is usually found in immunocompro- mised
individuals with

chronic ranulomatous disease. 25 The followin sections provide information re

ardin seroloical testin for asperillosis. Immunodiffusion and Counterimmunoelectrophoresis
Tests Ouchterlony
immunodiffusion (ID) or counterimmunoelec- trophoresis (CIE) for anti
odies are
methods availa
le
for asperillosis testin. In the ID or CIE methods, the pres- ence of one or mo
re lines of serum
precipitins is suestive
ut not conclusive evidence of an active infection. Th
ree or more lines
are associated with asperilloma (funus
all) or with invasive asperillosis if
the patient is
not aneric. For test results to
e valid, the Asperillus reference antiserum m
ust demonstrate
three or more
ands with Asperillus ref- erence antien. Nonspecific
andin
can
e caused

y C-reactive protein. The immunodiffusion tests are positive only in immunocom
petent patients
(Fi. 203). Enzyme Immunoassay An EIA method has
een developed to detect the ser
um
alactomannan antien of Asperillus. This test is more sen- sitive than immunod
iffusion tests
and is therefore more valua
le for dianosis of asperillosis in immunocompro- m
ised patients.
Results are availa
le in a
out 3 hours rather than the usual 4 weeks requir
ed for culture.
Invasive asperillosis has a mortality rate of 50 to 100 percent. Earlier ident
i cation of the
oranism expedites the administration of antifunal drus and reduces mortality.
26 Candidiasis
Infections with Candida al
icans and several other Candida species are collectiv
ely called
candidiasis. Candida al
icans is considered the most common cause of all serious
funal diseases.
Althouh it is a normal endoenous inha
itant of the alimentary tract and the mu
cocutaneous
reions of the
ody, it can
ecome an opportunistic pathoen in de
ili- tated an
d
immunocompromised hosts. Systemic involvement is rare except when the yeasts are
seeded into the

ody in lare num
ers throuh indwellin catheters, oran trans- plantation, or
needles of dru
a
users. Proloned therapy with anti
iotics or treatment with steroids and corti
cos- teroids may
also lead to systemic involvement. 19 Intact skin and mucous mem
ranes are
arri
ers to inva- sion
of this funus. Cellular defenses protect aainst mucocutaneous infection
. Most
individuals also have some anti
odies to C. al
icans. Opsonic serum factors and
the a
il- ity of
polymorphonuclear cells to phaocytize the yeast cells are also
elieved to play
a part in
immunity. 19 Hematoenous dissemination occurs in patients with neutropenia. A n
um-
er of
seroloical tests for candidiasis have
een developed,
ut their clinical utilit
y has
een
hampered
y the occur- rence of false-positive results in patients who are colon
ized with Candida
or who have super cial infections, and the fre- quency of false-neative results i
n

immunocompromised patients who fail to produce suf cient anti
ody. 22 Some of the
seroloical
tests for candidiasis are discussed next. Immunodiffusion and Counterimmunoelect
rophoresis Tests
ID and CIE methods for anti
odies ive compara
le results, and
oth relia
ly det
ect anti
odies in
systemic candidiasis in immunocompetent hosts. A heat-sta
le cytoplasmic anti- 
en is used with
positive-control sera containin at least three of the seven known precipitins.
Formation of one
or more
ands
etween the reaent antien and patients serum is considered a posi
tive reaction.
Systemic candidiasis is pre- sumptively identified when serial dilutions of spec
imens show an
increasin num
er of precipitin
ands or convert from neative to positive. The
patients
symptoms, direct smears, and cultures should
e considered to validate posi- tiv
e ID and CIE
tests. 22 FIGURE 202. Asperillus fumiatus; LPCB stain
450. FIGURE 203. Immunodif
fusion
patterns of selected funal anti
odies. (Courtesy of Immunomycoloies, Norman, O
K.)
1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 337 338 SECTION 4 Seroloical Dia
nosis of
Infectious Disease Latex Alutination (LA) The LA test for anti
odies is quanti
tative and can
e
of dia- nostic and pronostic value. Latex particles sensitized with a homoena
te antien of C.
al
icans are reacted with patient sera and control sera. When this screenin tes
t is positive, a
serial dilution is performed and reported as the hihest dilution ivin a 2 react
ion. A titer of
1:4 suests an early infection, colonization, or a nonspeci c reaction. A titer o
f 1:8 or
reater, conversion from a neative to a positive test, or a fourfold increase i
n titer is
presumptive evidence of invasive infection. A fourfold decrease in titer may ind
icate successful
therapy. 22 Enzyme Immunoassay for Candida Antien Anti
ody detection tests have
little or no
value in immuno- compromised patients; therefore, tests for antienemia must
e
performed for
these patients. Antien tests include EIA and the reverse passive alutination
test. A
dou
le-anti
ody sandwich EIA method can
e used to detect antienemia. Reverse P
assive
Alutination Test A reverse passive LA test is also availa
le for detectin ant
i- enemia. 22 A
study found that when a titer of 1:8 was used to indicate a positive test result
, the sensitivity
was 50 per- cent and the speci city was 73 percent. 27 Coccidioidomycosis Coccidio
ides immitis is
the etioloic aent of coccidioidomy- cosis. The funus is endemic in the southw
estern United
States, especially in Californias San Joaquin Valley (where it is called valley fe
ver) and in
northern Mexico. C. immitis is a mold found primarily in alkaline desert soil in
hot, semiarid
reions. Animals, especially desert rodents, are vectors. The infectious conidia

ecome air
orne

in dust storms or when disrupted
y earthquakes or construction projects. Primar

y pulmonary
coccidioidomycosis results from inhalation of dust containin the funus. Sixty
percent of
infections are asymp- tomatic and self-limitin in immunocompetent individuals.
28 Hosts who are
symptomatic may develop an acute primary pulmonary disease resem
lin the u that
resolves
without treatment, a chronic proressive pulmonary disease, or sec- ondary infe
ctions.
Dissemination to the skin (erythema nodosum),
ones, su
cutaneous tissues, l
ymph nodes, and
menines is rare. The antien developed for seroloical identi cation of circulati
n anti
odies
of C. immitis is coccidioidin, which is an antien filtrate of
roth cultures of
mycelial rowth.
Seroloical tests are important in the dianosis and man- aement of coccidiomyc
osis. Seroloical
methods availa
le for coccidioidomycosis are discussed
elow. Complement Fixatio
n Complement
xation (CF) is the most widely used quanti- tative serodianostic method for
identifyin C.
immitis infections. 22 Complement- xin anti
odies of the IG class develop in 3
to 6 months
after the onset of symptoms. Titers of 1:2 to 1:4 are presumptive evidence of an
early infection
and should
e repeated in 3 to 4 weeks. A titer of 1:16 is indicative of an acti
ve infection,
particularly when accom- panied
y a positive ID test. Titers reater than 1:16
occur in 90 to 95
percent of patients with disseminated coccid- ioidomycosis. Titers parallel the
severity of the
mycoses. 25 Serum, cere
rospinal uid (CSF), pleura, peritoneum, and joint uids can

e assayed

y CF. A titer of 1:2 or hiher in CSF is indicative of coccidioidal meninitis
95 percent of the
time. False-neative results occur in patients with soli- tary pulmonary lesions
. Cross-reactions
in patients with acute histoplasmosis will occur as false-positive reactions. 22
Tu
e
Precipitation (TP) Test Precipitatin IM anti
odies appear in 1 to 3 weeks afte
r infection in 90
percent of symptomatic patients; therefore, a positive TP test can
e an early i
ndication of a
primary infection. If precipitins are formed in any dilution of this test, it is
considered
dianostic. The precipitins disappear within 4 to 6 months in 80 to 90 percent o
f patients with
coccidioidomycosis, even in cases with dissemination. This makes the test of lit
tle pronostic
value. The TP test is, however, hihly speci c with very few cross-reactions. 19,2
2,28,29
Immunodiffusion Aar el dou
le ID tests are as sensitive as complement x- ation
tests and
are not su
ject to anticomplementary reactions. The ID method is the m
ost commonly
used screenin test for the dianosis of coccidioidomycosis. When diffusion
and
s form lines of
identity with the reference antisera, the
ands represent the reaction of coccid
ioidin antien

and patient anti
ody. Sinle
ands typically indi- cate chronic infection. Two o
r more
ands
usually indicate active disease or dissemination. Serum dilutions can
e per- fo
rmed for
quanti cation of coccidioidin anti
odies. This test is hihly speci c when reference
antisera are
used. 19,29 Latex Alutination Latex particles sensitized with coccidioidin are
reacted with
inactivated patient serum to detect anti
odies to the oran- ism. This procedure
is not
recommended for testin CSF or diluted sera,
ecause many false reactions occur.
The LA test is
positive early in the course of the disease,
ut as many as 10 percent of the ca
ses of
coccidioidomycosis con rmed
y culture or seroloy ive false-neative results wit
h an LA test.
Because false-positive results are common, a CF test or an ID test should
e per
formed for
con rmation when LA screenin tests are positive. 19,22,29 Enzyme Immunoassay EIA
tests for IG
and IM anti
odies are availa
le for use with serum or CSF. Positive EIA tests s
hould
e con rmed
with CF or TP tests,
ecause the EIA test is not a
solutely speci c. 22 1814_Ch20_
328-346.qxd
7/13/09 5:11 PM Pae 338 CHAPTER 20 Seroloical Response to Parasitic and Fun
al Infections
339 Cryptococcosis Cryptococcus neoformans is the etioloic aent of cryptococcosis. Pieons
are the chief vector, and C. neoformans is found where pieons roost and defecat
e. After an
individ- ual inhales the yeast, cryptococcosis may
e asymptomatic and unapparen
t, or it may
develop as a symptomatic pul- monary infection. Encapsulation and the a
ility to
synthesize
melanin are the sinificant virulence factors. Untreated infections with C. neof
ormans have a
predilection for dissem- inatin to the CNS and the
rain. In disseminated disea
se, the yeast
spread is hematoenous. Any oran or tissue of the
ody may
e infected,
ut loc
alization outside
the luns or
rain is relatively uncommon. There is little humoral response elic
ited
y
infections with C. neoformans, whether the patient is immunosuppressed or not. I
n normal
patients, su
clinical infection usually is resolved rapidly
y rowth- inhi
itin
 su
stances
present in
ody uids. 19 Serious clinical disease is found in patients with de
il
itatin
illnesses and immunosuppression; AIDS patients especially are predis- posed to d
isseminated
infections. Primary and secondary cutaneous cryptococcosis are reularly encount
ered clini- cal
manifestations in immunosuppressed patients. 19,22 Meninitis occurs in approxim
ately two-thirds
of the infections that disseminate. The predilection of the funus for the CNS
is postulated
to
e due to the a
sence of inhi
itory factors in spinal uid and the minima
l phaocytic
response found there. 19 The Latex Particle Alutination (LPA) Antien Test Whe
n cryptococcosis

is rst suspected, the capsular poly- saccharide antien of C. neoformans can
e d

etected. An
india-ink stain can
e performed to demonstrate the poly- saccharide capsule of
Cryptococcus
(Fi. 204),
ut
ecause of its hiher sensitivity and specificity, latex alutin
ation for
cryptococcal antien is now recommended. 21,30 Inactivated serum or spinal ui
d and positive
and neative human reference sera are each mixed with latex particles sensitized
with ra

it
anticryptococcus lo
ulin in rins on a test slide. The test is read macroscopic
ally for
alutina- tion. To
e valid, the neative control must
e neative, and the pos
itive control
must show 2 alutination. The pres- ence of any alutination when assayin a pat
ient specimen
is considered a positive test if controls are accepta
le. Results may
e quantit
ated
y
performin a serial dilution. The titer is reported as the hihest dilution show
in 2 alutination. A titer of 1:2 suests infection,
ut a titer of 1:4 or reater is eviden
ce of an active
infection. Hiher titers cor- relate with the severity of infection. Positive ti
ters are found in
CSF in 95 percent of cases involvin the CNS. Cross-reactions may occasionally o
ccur due to
rheumatoid factor (RF) or circulatin anti
odies that
ind with nonreac- tive po
lysaccharide in
immune complexes. Treatin the serum or CSF specimens with a protease that destr
oys RF and
cleaves anti
odies in the immune complexes eliminates these reac- tions. Extract
ion of RF and
circulatin anti
odies in serum can also
e accomplished
y treatment with
ethylenediaminetetraacetic acid (EDTA) and
oilin for 5 minutes. False-positive results i
n spinal uid can

e eliminated simply
y heatin the specimen in a
oilin water
ath. 22,29 Enzy
me-Linked
Immunoassay An EIA test is availa
le to detect antiens of C. neoformans in
oth
serum and CSF.
The EIA method for antien detec- tion is more sensitive than the latex alutin
ation procedure

ut takes more time to perform. 22,29 While tests for anti
odies to C. neoforman
s are not as
widely used as antien tests, they may
e helpful in deter- minin patient pro
nosis. Two
methods availa
le for anti
ody detection are the tu
e alutination (TA) test
and the indirect
uorescent anti
ody test. Tu
e Alutination (TA) Test The TA test can
e used as

oth a
qualitative screenin test and a semiquantitative test to detect anti
odies to C
. neofor- mans.
Serum or CSF from patients with suspected C. neoformans infection is heat
inactivated to
destroy com- plement. The serum or spinal fluid, an anticryptococcus positive co
ntrol, and a
neative control of ra

it serum are each mixed in test tu
es with a Cryptococcu
s antien suspension of weakly encapsulated yeast cells. The tu
es are incu
ated and the
n refrierated

overniht. Reactions are read for alutination. The positive control must read
3 to 4 in order
for the test to
e valid. Tests with reactions reater than the neative control
are considered
positive. The test can
e made semiquantitative
y performin a serial dilution
and followin the
same procedure as that for the screenin test. The titer is reported as the hih
est dilution
showin any deree of alutination. A titer of 1:2 or reater suests a curren
t or recent
infection with C. neoformans. Approximately 90 percent of patients with pulmonar
y cryptococcosis will demonstrate anti
ody titers in the TA test. As the mycosis pror
esses, antiens

ein to appear with a decrease in anti
ody production. Followin treatment, a F
IGURE 204. India
ink preparation; Cryptococcus neoformans 1000. (From Kern, ME. Medical Mycoloy:
A
Self-Instructional Text. FA Davis, Philadelphia, 1985, p. 53, with permission.)
1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 339 340 SECTION 4 Seroloical Dia
nosis of
Infectious Disease decrease in antien titers and a reappearance of anti
odies i
ndicate a ood
pronosis. The TA test has a speci city of 89 percent with extramenineal infectio
ns. 22,29,30
Indirect Fluorescent Anti
ody Test Indirect immuno uorescence tests are also avail
a
le for the
detection of anti
odies to C. neoformans. They are most valu- a
le when antien
tests are
neative; moreover, they can
e com
ined with antien tests to determine the pat
ients pronosis. A positive test suests a recent or current infection with C. neoformans
or a
cross-reaction with another funus. IFA tests have a speci city of 77 percent and
a sensitivity
of 50 percent. Both false-neative and false-positive results may occur. Molecul
ar Testin
Screenin clinical samples for funi usin molecular test- in is not commonl
y performed.
The development of cost-effective screenin methods for funal infections is i
mpaired
y the
lare num
er of pathoens that exist. A panel of PCR primers for funal infectio
ns common in
immunocompromised patients would
e extremely useful for screenin. 25 Thouh no
t commonly
performed in rou- tine la
oratories, PCR assays are availa
le for many funi in
reference
la
oratories. Another molecular technique used for detectin funal antiens is
peptide nuclear
acid uorescent in situ hy
ridiza- tion (PNA-FISH). This technique utilizes s
mall PNA
polymer pro
es that are la
eled with a fluorescent dye to identify PNA sequences
on chromosomes.
This procedure is currently used to differentiate Candida al
icans from other Ca
ndida species. 31
See Ta
le 203 for molecular tests used for speci c mycoses. SUMMARY Seroloical tes
tin for
parasitic and funal diseases is less routine than for other infectious diseases
,
ecause test
avail- a
ility is limited and dianosis is usually made from culture and morphol

oical features
of the oranisms. However, some seroloical tests are availa
le and can
e usefu
l for detectin
these diseases. Clearly the nature of the immune response as related to parasiti
c infections is
complex. Most parasitic diseases are dianosed
y more direct methods such as fe
cal,
lood, or
urine examination. Therefore, seroloical procedures are not frequently needed.
However, clinical
manifestations of several parasitic diseases such as toxoplasmosis are not alway
s clear-cut. In
these cases, it may
e possi
le to con rm the dianosis seroloically. In the Unit
ed States,
serum for such studies is often sent to a state pu
lic health reference
la
oratory for
seroloical testin. Occasionally, a state la
oratory may send specimens to
the CDC in
Atlanta, Georia. Many private la
oratories also offer testin for the more com
mon parasites
such as Entamoe
a histolytica, Entamoe
a histolytica/dispar, Trichinella spir
alis, Giardia
lam
lia, and Toxoplasma ondii. T. ondii is also included as part of the ToRCH
(Toxoplasma,
ru
ella, cytomealovirus, and her- pes) panel, which is used to evaluate coneni
tal and neonatal
infections of new
orn infants. Tests used in parasitic seroloy cover the full r
ane of methods
availa
le in immunoloy. Ta
le 202 illustrates that everythin from latex aluti
nation to
countercurrent electrophoresis has
een used or is currently
ein used to ident
ify and quantify
anti
odies aainst parasites. New dia- nostic procedures can
e expected in the
future,
especially as molecular technoloy to detect parasitic nucleic acids devel- ops,

ut these tests
will remain expensive for some time. In underdeveloped countries in which pa
rasitic
infections are common, hih cost will limit the
enefits from these procedures.
Identi cation of
funal aents in culture, direct mounts, or histopatholoic preparations of clin
ical specimens is
not always possi
le. Seroloical tests are therefore useful for
oth the dianos
is and pronosis
of patients with funal infections. Positive seroloical tests are often the rst
su- estive
evidence of mycotic infection. Tests for funal anti
odies and funal anti
ens are
availa
le. Assays with dif- ferent derees of sensitivity and speci city for asper
illosis,
candidiasis, coccidioidomycosis, and cryptococcosis were presented in this chapt
er.
Serodianostic test ndins should
e used in conjunction with the patients symptom
s, history,
and other clinical findins. Because cross-reactions are sometimes encount
ered, it is
recommended that a
attery of tests
e utilized to more accurately interpret tes
t results. While
molecular tests have
een developed for funal infec- tions, they are mainly per
formed in
reference la
oratories at the time of this writin. Ta
le 203 provides a summary

of the mycoses
presented in this chapter and the seroloi- cal and molecular tests that may
e
useful for their
dianosis. 1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 340 CHAPTER 20 Seroloi
cal Response to
Parasitic and Funal Infections 341 An otherwise healthy new
orn develops a fe
ver and a respiratory infection a few days after
irth. He also has a slihtly enlared liver
. The family has
a cat that spends a reat deal of time outdoors. The mother cleans the cats litte
r
ox and did
so throuhout her prenancy. It is sus- pected that the child may have conenita
l toxoplasmosis.
The followin seroloical test was performed on the mother and the
a
y.
The results are
listed
elow: TEST MOTHER BABY T. ondii IG 1:256 1:512 Questions: a. Eval
uate the
a
ys
status related to T. ondii infection.
. What additional testin should
e
performed to
con rm the
a
ys status? CASE STUDY 1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 3
41 342
SECTION 4 Seroloical Dianosis of Infectious Disease GIARDIA LAMBLIA ANTIGEN TE
STING BY A
SANDWICH ELISA METHOD Principle The qualitative determination of Giardia lam
lia
tropho- zoite
and cyst antiens in feces is an enzyme immunoassay that employs ra

it and mous
e antisera.
Giardia-specific antien (GSA 65) is a 65K MW lycoprotein produced in a
undance

y Giardia
protozoa as they multiply in the intestinal tract. The test sample is added to a
microtiter
sample well. Durin the first incu
ation, GSA 65 antiens present in the stool s
upernate are
captured
y anti
ody (anti-GSA 65)
ound to the well. After washin to remove un

ound
su
stances, enzyme conjuate comprised of the second anti-Giardia anti
ody a
nd the enzyme
(horse- radish peroxidase) is added. Durin the second incu
ation, this anti
ody
sandwiches the
antien. After washins to remove un
ound enzyme conjuate, su
strate is a
dded that
develops color in the presence of the
ound enzyme complex. Presence of color in
dicates a
positive reaction. Reaents, Materials, and Equipment Test kit such as ProSpect
Giardia, which
contains the followin: Test strips: microplate containin anti-Giardia anti
ody
(eiht wells per
strip) Test strip holder Enzyme conjuatemouse monoclonal anti-Giardia anti
ody w
ith thimerosal
Positive control Neative control Su
stratetetramethyl
enzidine (TMB) in
uffer W
ash
uffer 10X
concentration Stop solution1.0 N hydrochloric acid Specimen dilution
uffer Other
materials
required
ut not provided in the kit include the followin: Stool specimen colle
ction containers
Wash
ottle or dispenser for wash
uffer Timer that measures minutes Distilled o
r deionized water
Optional materials include the followin: Microplate reader Micropipettes Plasti
c or lass
disposa
le test tu
es Sample Collection, Storae, and Preparation Specimen is co

llected in the
same manner as that used for examination of stool for ova and parasites. The spe
cimen should
e
stored at 2C to 8C and tested within 48 hours. If the sample cannot
e assayed wit
h 48 hours,
the speci- men should
e frozen at 20C to 70C. Specimens preserved with 10 p
ercent
formalin, MF, or SAF fixatives should
e tested within 2 months. Specimens that
have
een
concentrated or xed with PVA are not suita
le for testin with this assay. Diluti
on in Wells
La
el one tu
e for each specimen. Add 0.4 mL specimen dilution
uffer to each tu

e. Coat one swa

with specimen, and viorously mix into specimen dilution
uffer. Express as much
fluid as
possi
le
efore removin the swa
from the tu
e, and discard the swa
. Put a tra
nsfer pipette
into the tu
e. If specimen is watery or preserved, mix
y shak- in. No further
preparation is
necessary. Procedure* Run positive and neative controls with each run. 1. Break
off the required
num
er of wells (num
er of sam- ples plus two for controls) and place in a strip
holder. Return
the remainder of the strips to the foil pouch, and reseal tihtly. 2. Add four d
rops of the
neative control to well 1 and four drops of positive control to well 2. (Do not
dilute the
controls.) 3. Add 100 L of sample dilution
uffer to each of the remainin wells.
4. Add one
drop of the stool specimen to each test well. Samples can
e added directly or p
rediluted in
tu
es
efore addin to wells. 5. Incu
ate the microtiter plate at room temperatu
re (20C to 25C)
for 60 minutes. 6. Shake out or aspirate the contents of the wells. Wash
y llin
each well with
diluted wash
uffer. Avoid ener- atin
u

les in the wells. Decant
y shakin
out or aspiratin
contents of the wells. Repeat wash procedure for a total of three washes. After
the last wash,
decant contents. Invert strip and viorously tap it on a clean paper towel. EXER
CISE *Adapted
from the ProSpect Giardia Microplate Assay (revised January 7, 2005), Remel Inc.
, Thermo Fisher
Scienti c. 1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 342 CHAPTER 20 Seroloica
l Response to
Parasitic and Funal Infections 343 7. Add four drops (200 L) of enzyme
conjuate to
each well. 8. Incu
ate for 30 minutes at room temperature. 9. Decant and wash ea
ch well ve
times, as descri
ed in step 6. 10. Add four drops (200 L) of color su
strate to e
ach well. 11.
Incu
ate the microplate for 10 minutes. 12. Add one drop of stop solution to eac
h well. Mix wells

y tappin the strip holder on the counter. 13. Read reactions within 10 minutes
after addin
stop solution. Read visually or at 450 nm if a microtiter reader is used. Interp
retation of
Results Visual The color of each well should
e compared to the color chart incl
uded in the test
kit. Positive control: At least 1 yellow color intensity Neative control: Colorl

ess Positive
test: Any sample well that has yellow color of at least 1 intensity Neative test
: Colorless
Microtiter Reader Zero the reader on air. Read all wells at 450 nm. Su
tract the
a
sor
ance
readin of the neative control from the a
sor
ance readins of all other wells.
Positive
control: 0.300 Neative control: 0.100 Positive test: 0.050 Neative test: 0.050 (te
sts with
faint yellow color should
e repeated) Comments Giardia lam
lia is a protozoan p
arasite that
lives in the intes- tinal tract. Giardiasis is the most common parasitic
disease dianosed
in the United States and, accordin to the CDC, causes an estimated 20,000
infections each
year. Transmission of the infection occurs throuh inestion of fecally contamin
ated food or
water. This disease is common in day-care centers and in institutions where peop
le are con- fined
for extended periods. Giardiasis is also commonly dianosed in homosexual men. S
ymptoms of the
acute dis- ease include diarrhea, nausea, weiht loss, mala
sorption, a
dominal
cramps,
atulence, and anemia. Because acute, chronic, and asymptomatic infections occur a
nd
ecause
symptoms are similar to those of many other intestinal dis- eases, it is importa
nt to accurately
dianose the disease so that appropriate treatment can
e administered. Dianosi
s of iardiasis
is frequently made
y o
servation of the parasite in fecal preparations. This me
thod relies on an
experienced technoloist to correctly identify the oran- ism. When an insuf cient
num
er of
samples are examined, the dianosis is often missed
ecause the excretion of or
an- isms is
frequently intermittent. Other more invasive methods such as simoidoscopy o
r
iopsy have
also
een used. The alternate approach, an ELISA method, such as the one present
ed in this
exercise, offers a rapid noninva- sive technique that does not require the o
ser
vation and
identi cation of an intact oranism. These results suest that immunoloic proced
ures can play a
sini cant role in the dianosis of some parasitic diseases. 1814_Ch20_328-346.qxd
7/13/09 5:11
PM Pae 343 344 SECTION 4 Seroloical Dianosis of Infectious Disease 1. Compar
ed to a hosts
response to the mumps virus, over- comin a parasitic infection is more di
fficult for the
host
ecause of which of the followin characteristics of parasites? a. Lare si
ze
. Complex
antienic structures c. Ela
orate life cycle d. All of the a
ove 2. Most of the
patholoy
associated with parasitic infec- tions results from which of the followin? a. S
ym
iotic
relationships with the host
. Ela
orate parasitic life cycles c. Immune respons
e to the
offendin oranism d. Innate defense mechanisms of the host 3. Parasites are a
l
e to evade host
defenses
y which of the followin means? a. Acquisition of host antiens
. Cha

nin surface
antiens c. Sequesterin themselves within host cells d. All of the a
ove 4. The
chronic nature
of parasitic infections is due to the hosts a. ina
ility to eliminate the infecti
ve aent.
.
type I hypersensitivity response to the infection. c. a
ility to form a ranulom
a around the
parasite. d. tendency to form circulatin immune complexes. 5. Clinical informat
ion provided
y
studyin the immune response to parasitic diseases a. aids in correctly dianosi
n the disease.

. predicts the pronosis of the disease. c. determines the possi
ility of reinf
ection
y the
parasite. d. All of the a
ove 6. The presence of
oth IM and IG anti
od
y in
toxoplasmosis infections suests that the infection a. occurred more than 2 yea
rs ao.
.
occurred less than 18 months ao. c. is chronic. d. has resolved itself. 7. IE
is an important
component of the immune response to infections caused
y a. Toxoplasma ondii.

. Giardia
lam
lia. c. Cryptosporidium parvum. d. Schistosoma mansoni. 8. Which of the foll
owin are factors
that have ena
led saprophytic funi to cause infections in humans? a. Their a
il
ity to survive
the
odys cellular defenses
. Their traumatic introduction into
ody tissues c.
Use of
anti
iotics and immunosuppressive aents d. All of the a
ove 9. In conenital to
xoplasmosis, the
new
orn has elevated levels of what class of immunolo
ulin? a. IA
. IG c. I
M d. IE 10. When
a mycosis is suspected, patient information must
e acquired for all of the foll
owin except a.
symptoms and physical examination.
. occupation, residence, and travel. c. medi
cal treatment and
medications. d. an exercise proram. 11. The most sini cant defense aainst funa
l infections is
a. cellular immunity.
. humoral immunity. c. phaocytosis. d. complement activa
tion. 12.
Serodianosis is most important in makin a rapid and presumptive dianosis of f
unal infections
when a. the patient is under 12 or over 50 years of ae.
. the patient has an u
ndianosed acute
or chronic respiratory infection. c. cultures of specimens are positive. d. hist
oloical tissue
slide preparations are positive. 13. Because the stae of the mycosis is often n
ot known, what is
the
est way to proceed when initiatin serodi- anosis? a. Use a skin test in a
n endemic area,

ecause a posi- tive skin test is dianostic.
. Use a com
ination of seroloica
l tests. c.
Serial testin is excessive; a sinle test is always dianostic. d. Use a sinle
, speci c
anti
ody test. 14. After infection has
een esta
lished, serodianostic tests ar
e most likely to

e positive in which of the followin cases? a. The patient has developed a stat
e of anery.
.
The patient is immunocompromised. c. The patient is immunocompetent. d. The test
s were taken

efore anti
odies had time to develop. REVIEW QUESTIONS 1814_Ch20_328-346.qxd 7

/13/09 5:11 PM
Pae 344 CHAPTER 20 Seroloical Response to Parasitic and Funal Infections 34
5 15. Which
est
descri
es nonspeci c cross-reactions that occur in funal seroloical tests? a. Oc
cur as a result
of crude unpuri ed antiens
. Occur with only one enus of funi c. Do not interf
ere with funal
identi cation d. Tend to remain at hih titer as a mycosis develops 16. Two serolo
ical tests
currently used for the dianosis of asperillosis and candidiasis are a. complem
ent xation (CF)
and enzyme immunoassay (EIA).
. immunodiffusion (ID) and counterimmunoelec- tro
phoresis (CIE).
c. counterimmunoelectrophoresis (CIE) and complement xation (CF). d. enzyme immun
oassay (EIA)
and immunodiffusion (ID). 17. A 27-year-old man from Ohio, dianosed with AIDS,
developed chest
pains and after a short period of time also developed severe headaches with dizz
iness. His ho

y
was raisin messener pieons. His physician ordered a sputum culture and spinal
tap, and
oth
were positive for a yeastlike funus. These ndins are most consistent with infec
tion
y a.
Candida al
icans.
. Coccidioides immitis. c. Cryptococcus neoformans. d. Histop
lasma capsulatum.
18. Which of the followin seroloical tests detects the polysaccharide capsule
antien in
serum and CSF of patients with suspected infection with Cryptococcus neoforman
s? a. Complement
xation (CF)
. Hypersensitivity skin test c. Latex alutination (LA) d. Hemalu
tination test
19. What is the most widely used quantitative seroloical test for identificatio
n of anti
odies
in infection with Coccidioides immitis? a. Complement xation (CF)
. Latex alut
ination (LA) c.
Exoantien test d. Fluorescent anti
ody test ACKNOWLEDGEMENT The author would li
ke to thank
Russell F. Cheadle, MS, MT(ASCP), and Norma B. Cook, MA, MT(ASCP), for their con
tri
utions to
this chapter in previous editions. References 1. World Health Oranization. Revi
sed Glo
al Burden
of Disease (GBD) 2002 Estimates: Mortality, incidence, prevalence, YLL, YLD and
DALYs
y sex,
cause and reion, estimates for 2002 as reported in the World Health Report 2004
. 2. Garcia, LS.
Dianostic Medical Parasitoloy, ed. 5. American Society for Micro
ioloy Press,
Washinton, DC,
2007, pp. 567591. 3. Male, D, Brostoff, J, and Roitt, I. Immunoloy, ed. 7. Mos
y
Elsevier,
Philadelphia 2006, pp. 277298. 4. John, DT, and Petri, WA. Markell and Vo
es Medical
Parasitoloy, ed. 9. Saunders/Elsevier, Philadelphia, 2006, p. 112. 5. Barry, JD
. The relative
sini cance of mechanisms of antienic variation in African Trypanosomes. Parasito
l Today
13:212218, 1997. 6. Jenkins, SJ, Hewitson, JP, Jenkins, GR, et al. Modulation of
the hosts
immune response
y schistosome larvae. Parasite Immunol 27:385393, 2005. 7.
Collier, L,
Balows, A, and Sussman, M (eds). Topley and Wilsons Micro
ioloy and Micro


ial
Infections, ed. 9, vol. 5. Oxford University Press, New York, 1998, pp. 6970. 8.
Papazarharia,
M, Athanasiadis, GI, Papadoupoulos, E, et al. Involvement of NK cells aainst tu
mors and
parasites. Int J Biol Markers 22(2):144153, 2007. 9. Jones, JL, Kruszon-Moran, D,
Wilson, M, et
al. Toxoplasma ondii infection in the Unites States: Seroprevalence and risk fa
ctors. Am J
Epidemiol 154:357365, 2001. 10. Wilson, M, Jones, JL, and McAuley, JB. Tox
oplasma. In
Murray, PR, Baron, EJ, Jorensen, JH, et al. Manual of Clinical Micro
i
oloy, ed. 9,
vol. 1. American Society for Micro
ioloy Press, Washinton, DC, 2007, pp. 207020
81. 11. Wilson,
W, Schantz, PM, and Nutman, T. Molecular and immunoloic approaches to the dian
osis of parasitic
infec- tions. In Detrick, B, Hamilton, RG, and Folds, JD (eds): Manual
of Clinical
La
oratory Immunoloy, ed. 7. American Society for Micro
ioloy, Washinton,
DC, 2006, pp.
557568. 1814_Ch20_328-346.qxd 7/13/09 5:11 PM Pae 345 346 SECTION 4 Seroloic
al Dianosis of
Infectious Disease 12. Christoph, J, Kattnew, E, Seitz, HM, and Reiter-Owana I.
Strateies for
the dianosis and treatment of prenatal toxo- plasmosisa survey (a
stract). Z
Ge
urtshilfe
Und Neonatoloie 208(1):1016, 2004. 13. Moran, UM, Pallant, L, Dwyer, BW, et al.
Comparison of
PCR and microscopy for detection of Cryptosporidium parvum in human fecal specim
ens: Clinical
trial. J Clin Micro
iol 36:995998, 1998. 14. Aviles, H, Belli, A, Armijos, R, et
al. PCR
detection and iden- ti cation of Leismania parasite in clinical specimens in Ecuad
or: A
comparison with classical dianostic methods. J Parasitol 85(2):181187, 1999. 15.
FDA clears
for marketin first quick test for malaria.
http://www.fda.ov/

s/topics/NEWS/2007/New01659.html. Released June 26, 2007. A
ccessed July 27,
2007. 16. Wilson, M, and Schantz, P. Nonmorpholoic dianosis of par- asitic inf
ections. In
Balows, A, Hausler, WJ, et al. (eds): Manual of Clinical Micro
ioloy, ed. 6
. American
Society for Micro
ioloy, Washinton, DC, 1991, pp. 717726. 17. The Fifth NIAI
D Workshop in
Medical Mycoloy: Epidemioloy. http://www3.niaid.nih.ov/research/topics/ fun
al/meetins.htm.
Accessed Auust 10, 2007. 18. Striner, JR, Beard, CB, Miller, RF, and Wake eld, A
E. A new name
(Pneumocystis jerovici) for Pneumocystis from humans. Emerin infectious diseas
es [serial
online] Septem
er 8, 2002. http://www.cdc.ov/ncidod/EID/vol8no9/02-0096.htm. 19
. Rippon, JW.
Medical Mycoloy: The Pathoenic Funi and Pathoenic Actinomycetes, ed. 3. WB S
aunders,
Philadelphia, 1988. 20. Brooks, GF. Jawetz, Melnick & Adel
ers Medical Mycoloy,
ed. 24.
McGraw-Hill Medical, London, 2007. 21. For
es, BA, Sahm, DF, Weissfeld, AS.
Bailey and

Scotts Dianostic Micro
ioloy, ed. 12. Mos
y Elsevier, Philadelphia, 2007, pp. 5
42627. 22.
Reiss, E, Kaufman, L, Kovacs, A, et al. Clinical immunomy- coloy. In Rose, NR,
et al.: Manual of
Clinical Immunoloy, ed. 6. American Society for Micro
ioloy, Washinton, DC, 2
002, pp. 559583.
23. Hohl, TM, Rivera, A, and Pamer, E. Immunity to funi. Curr Opin Immunol 18(4
):465472, Au.
2006. 24. Washinton, WC, Koneman, EW, Allen, SD, et al. Konemans Color Atlas and
Text
ook of
Dianostic Micro
ioloy, ed. 6. Lippincott Williams and Wilkins, Philadelphia, 2
006, pp.
11511243. 25. For
es, BA, Sahm, DF, and Weissfeld, AS. La
oratory meth- ods in

asic
mycoloy. In Bailey and Scotts Dianostic Micro
ioloy, ed. 12. Mos
y Elsevi
er,
Philadelphia, 2007, pp. 628716. 26. FDA News. U.S. Food and Dru Association. May
16, 2003. 27.
Petri, MG, Koni, J, Moecke, HP, Gramm, HJ, et al. Epidemioloy of inva
sive mycoses in
ICU patients: A prospec- tive multicenter study in 435 non-neutropenic pat
ients.
Paul-Ehrlich Society for Chemotherapy, Divisions of Mycoloy and Pneumonia Resea
rch. Intensive
Care Med 23:317325, 1997. 28. Centers for Disease Control. Coccidiomycosis.
http://
www.cdc.ov/ncidod/d
md/diseaseinfo/coccidiomycosis. Accessed Novem
er 29, 2007.
29. McGinnis,
MR, and Tilton, RC. Immunoloic dianosis of funal infection. In Howard, DA, et
al. (eds):
Clinical and Pathoenic Micro
ioloy, ed. 2. Mos
y, St. Louis, MO, 1994, pp. 6416
48. 30. Packae
insert. Cryptococcal Latex Alutination System (CALAS), Meridian Bioscience, In
c. Rev. March
2002. 31. Wilson, DA, Joyce, MJ, Hall, LS, et al. Multicenter evalua- tion of Ca
ndida al
icans
peptide nucleic acid fluorescent in situ hy
ridization pro
e for characterizatio
n isolates from

lood cultures. J Clin Micro
iol 43(6):29092912, 2005. 1814_Ch20_328-346.qxd 7/1
3/09 5:11 PM
Pae 346 21 Spirochete Diseases Marc Golihtly, PhD, Candace Golihtly, MS, MLT(
ASCP), and
Christine Stevens 347 LEARNING OBJECTIVES After nishin this chapter, the reader
will
e a
le
to: 1. Descri
e identifyin characteristics of Treponema pallidum and Borrelia

urdorferi. 2.
Explain how syphilis and Lyme disease are transmitted. 3. Discuss the different
staes of
syphilis. 4. Discuss the advantaes of direct uorescent stainin for T. pallidum
over dark eld
examination without stainin. 5. De ne reain. 6. Distinuish treponemal from nont
reponemal tests
for syphilis. 7. Descri
e the principle of the followin tests for syphilis: Ven
ereal Disease
Research La
oratory (VDRL), rapid plasma reain (RPR), uorescent treponemal anti

ody a
sorption
(FTA-ABS), and alutination assays. 8. Give reasons for false-positive reain t
est results. 9.
Compare and contrast sensitivity and speci city of treponemal and nontreponemal te
stin for the

various staes of syphilis. 10. Discuss the advantaes and disadvantaes of poly
merase chain
reaction (PCR) and enzyme immunoassay (EIA) testin for syphilis. 11. Discuss li
mitations of
cere
rospinal uid (CSF) testin for neurosyphilis and testin for conenital syph
ilis. 12.
Descri
e early and late manifestations of Lyme disease. 13. Relate various aspec
ts of the immune
response to Lyme disease to disease staes. 14. Compare immuno uorescence assay (I
FA), EIA, and
immuno
lot testin for Lyme disease as to sensitivity and ease of performance. 1
5. Discuss causes
of false positives and false neatives in seroloical testin for Lyme disease.
KEY TERMS
Borrelia
urdorferi Chancre Conenital syphilis Erythema mirans Flocculation F
TA-ABS test
Gummas Immuno
lottin Nontreponemal tests Polymerase chain reaction (PCR) Prozon
e Reain RPR test
Tertiary syphilis Treponema pallidum Treponemal tests VDRL test 1814_Ch21_347-36
8.qxd 7/10/09
3:54 PM Pae 347 348 SECTION 4 Seroloical Dianosis of Infectious Disease Spir
ochetes are lon,
slender, helically coiled
acteria con- tainin axial filaments, or periplasmic
flaella, which
wind around the
acterial cell wall and are enclosed
y an outer sheath. 1 These
ram-neative,
microaerophilic
acteria exhi
it a characteristic corkscrew flexion or mot
ility. Diseases
caused
y these oranisms have many similarities, includin a localized skin inf
ection that
disseminates to numerous orans as the disease proresses, a latent stae, and c
ardiac and
neuroloical involvement if the disease remains untreated. This chapter discusse
s disease
manifes- tations and testin for the two major spirochete diseases, syphilis and
Lyme disease.
Seroloical testin plays a key role in dianosis of these diseases,
ecause iso
lation of the
oranism itself is dif cult to accomplish in the la
oratory, and clinical symptoms
are not always
apparent. SYPHILIS Syphilis remains the most commonly acquired spirochete diseas
e in the United
States today. 2,3 It is typically spread throuh sexual transmission. Althouh t
he incidence of
cases in the United States reached an all-time low of 2.2 cases per 100,000 indi
viduals in 2000,
4 it has
een increasin from 2000 to 2005 (latest data availa
le at time of wri
tin). 3 In this
period, there has
een a dramatic increase in primary and secondary syphilis. Ho
mosexual
transmission
etween men is responsi
le for much of this increase. 3,5,6 In fact
, in 2000, this
roup accounted for 6 percent of the syphilis cases in this country; in 2005, ov
er 60 percent of
syphilis occurred in this roup. Neurosyphilis (usually rare) is also appearin
in the homosexual
HIV-positive male popula- tion. 7 Also trou
lin has
een the rst reported increa
ses of syphilis
in the female population in over 10 years. 3 In countries such as the United Kin
dom in western

Europe, the incidence also appears to
e on the rise aain. 810 Likewise, primary
and secondary
syphilis rates in China are rapidly increasin; in 2005, they were su
stantially
hiher than
most developed countries, includin the United States. 11 Thus, despite the
current emphasis
on safe sexual practices, syphilis remains a major health pro
lem in many areas
of the world.
Early detection of the disease is of major importance. This chapter presents cha
racteristics of
the oranism and the disease and discusses the most frequently used methods of i
denti cation.
Characteristics of the Oranism The causative aent of syphilis is Treponema pal
lidum, su
species pallidum, a mem
er of the family Spirochaetaceae. Oranisms in this fami
ly have no
natural reservoir in the environment and must multiply within a livin host. Thr
ee other
pathoens in this roup are so morpholoically and antienically similar to T. p
allidum that all

ut one are clas- si ed as su
species. 12 These other oranisms are T. pallidum su

species
pertenue, the aent of yaws; T. pallidumsu
species endemicum, the cause of nonve
nereal endemic
syphilis; and T. carateum, the aent of pinta. Yaws is found in the tropics, pin
ta is found in
Central and South America, and endemic syphilis is found in desert reions. T. p
allidum (which
will hereafter
e used to refer to su
- species pallidum) varies in lenth from
6 to 20 mm and in
width from 0.1 to 0.2 mm, with 6 to 14 coils (Fi. 211). 1,13 The outer mem
rane
of T.
pallidumis a phospholipid
ilayer with very few exposed proteins. Several identi
fied mem-
rane
proteins, called treponemal rare outer mem
rane proteins (TROMPs), have
een cha
racterized. 14 It
appears that the scarcity of such proteins delays the host immune response. Mode
of Transmission
Pathoenic treponemes are rapidly destroyed
y heat, cold, and dryin out, so th
ey are almost
always spread
y direct contact. Sexual transmission is the primary mode of diss
em- ination, and
this occurs throuh a
raded skin or mucous mem
ranes comin in contact with
an open
lesion. Approximately 30 to 50 percent of the individuals who are exposed to a s
exual partner
with active lesions will acquire the disease. 13 Conenital infections can also
occur durin
CHAPTER OUTLINE SYPHILIS Characteristics of the Oranism Mode of Transmission St
aes of the
Disease Conenital Syphilis Nature of the Immune Response La
oratory Dianosis T
reatment LYME
DISEASE Characteristics of the Oranism Staes of the Disease Nature of the Immu
ne Response
La
oratory Dianosis Treatment SUMMARY CASE STUDIES EXERCISE: RPR CARD TEST FOR
SEROLOGICAL
DETECTION OF SYPHILIS REVIEW QUESTIONS REFERENCES 1814_Ch21_347-368.qxd 7/10/09
3:54 PM Pae
348 CHAPTER 21 Spirochete Diseases 349 prenancy. Transmission to the fetus is
possi
le in

mothers with clinically latent disease. Other potential means of transmission in

clude parenteral
exposure throuh contaminated needles or
lood,
ut this is extremely rare
. For the past 30
years, the lack of transfusion-transmitted syphilis in the United States has act
ually called into
question the necessity of testin poten- tial donors for presence of the disease
. 15 However,
current uidelines (2007)
y the American Red Cross for
lood donation requires
waitin 12 months
after treatment for syphilis
efore donatin. 16 Because syphilis can only
e tr
ansmitted
y
means of fresh
lood products, the use of stored
lood components has virtua
lly eliminated
the possi
ility of transfusion-associated syphilis. 13 Staes of the Disease Onc
e contact has

een made with a suscepti
le skin site, endothelial cell thickenin occurs with
areation of
lym- phocytes, plasma cells, and macrophaes. 17 The initial lesion, called a ch
ancre, develops

etween 10 and 90 days after infection, with a
out 21 days
ein the averae. 8
A chancre is a
painless, solitary lesion characterized
y raised and well- de ned
orders (Fi. 2
12). In men
these usually occur on the outside of the penis,
ut in women they may appear in
the vaina or on
the cervix and thus may o undetected. This primary stae lasts from 1 to 6 week
s, durin which
time the lesion heals spontaneously. If the initial chancre is untreated, a
out
25 percent of
such cases proress to the secondary stae, in which systemic dissemination of t
he oranism
occurs. This stae is usually o
served a
out 1 to 2 months after the primary cha
ncre disappears,
ut in up to 15 percent of reported cases, the primary lesio
n may still
e
present. 18 Symptoms of the secondary stae include eneralized lymphadenopat
hy, or
enlarement of the lymph nodes; malaise; fever; pharynitis; and a rash on the s
kin and mucous
mem
ranes. 9,13,18 The rash may appear on the palms of the hands and the soles o
f the feet. 13
Involvement of the central nervous system may occur earlier than previously
suspected,

ecause via
le oranisms have
een found in the cere
rospinal uid (CSF) of sever
al patients
with primary or secondary syphilis. 12 Approximately 40 percent of patients with
secondary
syphilis may exhi
it neuroloical sins such as visual distur
ances, hearin los
s, tinnitus, and
facial weakness. 8 Lesions persist from a few days up to 8 weeks. After this tim
e, spontaneous
healin occurs, as in the primary stae. The latent stae follows the disappeara
nce of secondary
syphilis. This stae is characterized
y a lack of clinical symptoms. It is ar
i
trarily divided
into early latent (less than 1 years duration) and late latent, in which the prim
ary infec- tion
has occurred more than 1 year previously. Patients are noninfectious at this tim
e, with the

exception of prenant women, who can pass the disease on to the fetus even if th
ey exhi
it no
symptoms. A
out one-third of the individuals who remain untreated develop tertia
ry syphilis. 8,13
This stae appears anywhere from months to years after secondary infection. Typi
cally, this
occurs most often
etween 10 and 30 years followin the secondary stae. 8,13 Te
rtiary syphilis
has three major manifestations: ummatous syphilis, cardiovascular disease, and
neurosyphilis.
Gummas are localized areas of ranulomatous in am- mation that are most often
found on

ones, skin, or su
cutaneous tissue. Such lesions can reach up to several cent
imeters in
diameter, and they contain lymphocytes, epithelioid cells, and
ro
lastic cells.
18 They may
heal spon- taneously with scarrin, or they may remain destructive areas of chro
nic in ammation.
It is likely that they represent the host response to infection. Cardiovascular
complications
usually involve the ascend- in aorta, and symptoms are due to destruction of el
astic tissue,
especially in the ascendin and transverse sements of the aortic arch. 18 This
may result in
aortic aneurysm, thickenin of the valve lea ets causin aortic reuritation, or
narrowin of
the ostia, producin anina pectoris. 13 FIGURE 211. Treponema pallidum. Electron
microraph
showin the coils and periplasmic aella. (Courtesy of CDC Archives, Atlanta, GA)
FIGURE 212.
Primary chancre in the early stae of syphilis. (Courtesy of CDC/M. Rein, VD)
1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 349 350 SECTION 4 Seroloical Dia
nosis of
Infectious Disease Neurosyphilis is the complication most often associated with
the tertiary
stae,
ut it actually can occur anytime after the primary stae and can span al
l staes of the
disease. In immunodeficient individuals such as HIV patients, there has
een a l
are rise in the
incidence of early neurosyphilis. 7 Durin the rst 2 years followin infection, h
owever, central nervous system involvement often takes the form of acute meninitis. Late m
anifestations of
neurosyphilis include ta
es dorsalis, a deeneration of the lower spinal cord, a
nd eneral
paresis, or chronic proressive dementia. It usually takes more than 10 years fo
r these to occur,

ut
oth are the result of struc- tural central nervous system damae and cannot

e reversed.
Fortunately, these symptoms are now very rare
ecause of early detection and tre
atment with
penicillin. 8,13 Conenital Syphilis Conenital syphilis occurs when a woman who
has early
syphilis or early latent syphilis transmits treponemes to the fetus. Due in lar
e measure to a
national plan launched
y the Centers for Disease Control and Prevention, the oc
cur- rence of
conenital syphilis dropped to 529 cases in the year 2000 and continued to d
rop throuh
2004, 19,20 despite increases in primary and secondary syphilis. Althouh the di

sease can
e
transmitted at any stae of prenancy, typi- cally the fetus is most affected du
rin the second
or third trimester, and fetal or perinatal death occurs in approxi- mately 10 pe
rcent of the
cases. 18 Infants who are live
orn often have no clinical sins of dis- ease dur
in the rst few
weeks of life. Some may remain asymptomatic,
ut
etween 60 and 90 percent of su
ch infants
develop later symptoms if not treated at
irth. 21 In ammation of the um
ilical co
rd, called
necrotizin funisitis, may
e the rst indication of the disease. 13,22 The infant
may exhi
it
clear or hemorrhaic rhinitis, or runny nose. Skin eruptions, in the form of a m
acropapular rash
that is especially prominent around the mouth, the palms of the hands, and soles
of the feet, are
also common. 9 Other symptoms include eneralized lymphadenopathy, hepatosplenom
ealy, jaundice,
anemia, painful lim
s, and
one a
normalities such as saddle nose or sa
er shins
. 12,13,19
Neurosyphilis may occur in up to 60 percent of infants with conenital disease.
23 Nature of the
Immune Response The primary
ody defenses aainst treponemal invasion are intact
skin and mucous
mem
ranes. Once the skin is pene- trated, T cells and macrophaes play a
key role in
the immune response. Primary lesions show the presence of
oth CD4 and CD8 T cells.
Cytokines
produced
y these cells activate macrophaes, and it is ultimately phaocytosis

y macrophaes
that heals the primary chancre. 18 The protec- tive role of anti
ody is uncertai
n, however, as
coatin of treponemes with anti
ody does not necessarily
rin a
out their destr
uction. 14 In
fact, circulatin immune complexes may actually prevent the host from synthesizi
n further specific treponemal anti
ody. 24 T. pallidum is also capa
le of coatin itself with
host proteins,
which delays the immune systems reconition of the pathoen. 13 The rare treponem
al proteins, or
TROMPS, are important in
rinin a
out complement activation that ultimately ki
lls the oranism.
14 However, the chronic nature of the disease is an indicator that the oranisms
are a
le to
evade the immune response. Treponemes may persist in the host for years if anti

iotic therapy is
not o
tained. La
oratory Dianosis Traditional la
oratory tests for syphilis can

e classi ed
into three main types: direct detection of spirochetes, nontre- ponemal seroloi
cal tests, and
treponemal seroloical tests. These vary in sensitivity and ease of performance.
Principles and
procedures of each type of testin are discussed
elow and are compared and summ
arized in Ta
le
211. Direct Detection of Spirochetes Direct detection of spirochetes can
e accom
plished
y
dark- field microscopy or fluorescent anti
ody testin. The performance of
either test
requires that the patient have active lesions. Dark-Field Microscopy Primary and

secondary
syphilis can
e dianosed
y demon- stratin the presence of T. pallidum in exud
ates from skin
lesions. 2,12 In dark- eld microscopy, a dark- eld condenser is used to keep all inc
ident liht
out of the eld except for that captured
y the oranisms themselves. It is essent
ial to have a
ood specimen in the form of serous fluid from a lesion. This is usually o
taine
d
y cleanin the
lesion with sterile saline and ru

in it with clean auze. Pathoenic treponeme
s are
identified on the
asis of characteristic corkscrew morpholoy and exin mot
ility. 13
Because o
servation of motility is the key to identi ca- tion, specimens must
e e
xamined as
quickly as possi
le,
efore they
ecome dried out. False-neative results can oc
cur due to delay
in evaluatin the slides, an insufficient specimen, or pretreatment of the patie
nt with
anti
iotics. 13 Thus, a neative test does not exclude a dianosis of syphilis.
In addition, an
experienced microscopist should perform testin. If a specimen is o
tained from
the mouth or the
rec- tal area, morpholoically identical nonpathoens can
e found, so these
must
e
differentiated from the true pathoens. Fluorescent Anti
ody Testin of a Spe
cimen The use of
a uorescent-la
eled anti
ody is a sensitive and hihly specific alternative to da
rk-field
microscopy. This can
e performed
y either a direct method, which uses a uoresce
nt-la
eled
anti
ody conjuate to T. pallidum, or an indirect method usin anti
ody speci c fo
r T. pallidum
and 1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 350 CHAPTER 21 Spirochete Dise
ases 351 a
second la
eled anti-immunolo
ulin anti
ody. 12 An advan- tae of this method is
that live
specimens are not required. A specimen can
e
rouht to the la
oratory in a cap
illary tu
e, and
xed slides can
e prepared for later viewin. Even after xin, treponemes can
e w
ashed off the
slide, so each slide must
e handled individually, and rinsin must
e care- ful
ly done. 12 The
use of monoclonal anti
odies has made this method very sensitive and speci c. 12 H
owever, monoclonal anti
odies can still cross-react with other su
species of T. pallidum, an
d this must
e
taken into account when makin a dianosis. Seroloical Tests If a patient does
not have active
lesions, as may
e the case in secondary or tertiary syphilis, then seroloical
testin for
anti
odies is the key to dianosis. Seroloical tests can
e classified as nontr
eponemal or
treponemal, dependin on the reactivity of the anti
ody that is detected. Nontre
ponemal tests,
which detect anti
ody to cardi- olipin, have traditionally
een used to screen f
or syphilis

ecause of their hih sensitivity and ease of performance. However, false-positi
ve results are
common
ecause of the nonspeci c nature of the antien. Therefore, any positive re

sults must
e
con rmed
y a more speci c treponemal test, which detects anti
odies to T. pallidum.
Nontreponemal Tests Nontreponemal tests determine the presence of reain, an ant
i
ody that forms
aainst cardiolipin, a lipid material from damaed cells. This is found in the s
era of patients
with syphilis and several other disease states. An antien that is a com
ination
of cholesterol,
lecithin, and cardiolipin is used Ta
le 211. Comparison of Tests Used for the Dia
nosis of
Syphilis TEST ANTIGEN ANTIBODY COMMENTS Direct Microscopic Dark eld T. pallidum fr
om patient None
Requires active lesion. Must have ood specimen, experienced technoloist; inexp
ensive
Fluorescent anti
ody T. pallidum from patient Antitreponemal anti
ody Requires
active lesion.
More speci c with uorescent ta than dark eld; specimen does not have to
e live Nont
reponemal
VDRL Cardiolipin Reain Flocculation; ood for screenin tests, treatment monito
rin, spinal uid
testin; false positives RPR Cardiolipin Reain Modi ed VDRL with charcoal particl
es. More
sensitive than VDRL in primary syphilis TRUST Cardiolipin Reain Uses red partic
les to visualize
the reaction; similar to RPR Treponemal FTA-ABS Nichols strain of T. pallidum An
titreponemal
Con rmatory; speci c, sensitive; may
e neative in primary stae EIA Treponemal or
recom
inant
Antitreponemal Simple to perform; can
e automated; not as sensitive as FTA-ABS
Enzyme-la
eled
treponemal Anti-IG or anti-IM, Simple to perform; sensitive in primary anti
en antitreponemal
anti
ody syphilis,
ut sensitivity decreases in from patient later staes MHA-T
P or Serodia
TP-PA Sheep RBCs or el particles Antitreponemal Not as sensitive as FTA-ABS se
nsitized with T
pallidum sonicate DNA pro
e Patient DNA matched to None Technically demandin;
very speci c;
treponemal DNA lacks sensitivity VDRL
venereal disease research la
oratory; RPR
rapid plasma
reain; TRUST toluidine red unheated serum test; FTA-ABS
uorescent treponemal ant
i
ody
a
sorption; EIA enzyme immunoassay; MHA-TP
microhemalutination assay for anti

odies to
Treponema pallidum; TP-PA
T. pallidum particle alutination; DNA
deoxyri
onucle
ic acid.
1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 351 352 SECTION 4 Seroloical Dia
nosis of
Infectious Disease in the reaction to detect the nontreponemal reainic anti-
o
dies, which are
either of the IG or IM class. The term reain should not
e confused with the
same word that
was oriinally used to descri
e IE. They are not the same. Fortunately, the ter
m reain in
reference to IE is rarely used today. The traditional nontreponemal tests are

ased on occulation reactions in which patient anti
ody complexes with the cardiolipin anti
en. Flocculation
is a specific type of precipitation that occurs over a narrow rane of antien c
oncentrations.

The antien consists of very ne particles. In eneral, nontreponemal tests are po

sitive within 1
to 4 weeks after the appearance of the primary chancre. 2,12 Titers usually peak
durin the
secondary or early latent staes. In primary disease,
etween 13 and 41 percent
of individuals
appear nonreactive,
ut
y the secondary stae, almost all patients have reactiv
e test results.
2,12 However, testin of sera from patients in the secondary stae is su
ject to
false neatives

ecause of the prozone phenomenon (anti
ody excess). Typically this creates a no
nreactive pattern that is ranular or rouh in appearance. 12 If a prozone is suspected, seri
al twofold
dilutions of the patients sera should
e made to o
tain a titer. Reain titers te
nd to decline
in later staes of the disease, even if the patient remains untreated. After sev
eral years, a
out
25 percent of untreated syphilis cases were shown to
ecome nonreactive for rea
in. 13 This
decline occurs more rapidly in individuals who have received treatment. A rst- ti
me infection,
if in the primary or secondary stae, should show a fourfold decrease in titer

y the third month
follow- in treatment and an eihtfold decrease
y 6 to 8 months. 18 Followin s
uccessful
treatment, tests typically
ecome com- pletely nonreactive within 1 to 2 years.
Examples of
nontreponemal tests include Venereal Disease Research La
oratory (VDRL), rapid p
lasma reain
(RPR), toluidine red unheated serum test (TRUST), unheated serum reain (USR), a
nd reain screen
test (RST). The RPR and the VDRL are the most widely used of these tests. Venere
al Disease
Research La
oratory Test The VDRL test, which was desined
y the Venereal Disea
se Research
La
oratories, is
oth a qualitative and quantitative slide occulation test for se
rum, and there
is a modi cation for use on spinal uid. 25 Antien for all tests must
e prepared f
resh daily
and is done so in a hihly reulated fashion. The antien is an alcoholi
c solution of
0.03 percent cardi- olipin, 0.9 percent cholesterol, and 0.21 percent lecithin.
Basically, the
antien suspension is prepared
y addin the VDRL antien via a dropper to a
uf
fered saline
solution while continuously rotatin on a at surface; attention must
e paid to
required
rotation speed and timin. A daily cali
rated Hamilton syrine is used to
deliver one drop
of antien for the slide test. If the delivery is off
y more than 2 drops out o
f 60, the syrine
must
e cleaned with alcohol and recali
rated. The serum specimens to
e tested
are heated at
56C for 30 minutes to inactivate complement, and 0.05 mL is pipetted into a ceram
ic rin of a
lass slide. Three control sera nonreactive, minimally reactive, and reactiveare p
ipetted into
rins on the lass slide in the same manner. Sera and patient samples are spread
out to ll the

entire rin. One drop (1/60 mL) of the VDRL antien is then added to each rin.
The slide is
rotated for 4 minutes on a rotator at 180 rpm. It is read microscopically to det
ermine the
presence of occu- lation, or small clumps. The results are recorded as reactive (
medium to lare
clumps), weakly reactive (small clumps), or nonreactive (no clumps or sliht rou
hness). 25 Tests
must
e performed at room temperature within the rane of 23C to 29C (73F to 85F),

ecause
results may
e affected
y tem- perature chanes. All sera with reactive or weak
ly reactive
results must
e tested usin the quantitative slide test, in which twofold dilut
ions of serum
ranin from 1:2 to 1:32 are ini- tially used. Sera yieldin positive results at
the 1:32
dilution are titered further. The VDRL test and some of the newer ELISA tests ar
e the only ones
routinely used for the testin of spinal uid. 12,13 For VDRL spinal uid testin, t
he antien
volume used is less than the serum test and is at a different concentration. In
addition,
different slides are used (Boerner alutination slides). The test is read micro
scopically, as in
the VDRL serum test. If a test is reactive, twofold dilutions are made and retes
ted followin the
same protocol. A positive VDRL test on spinal fluid is dianostic of neurosyphil
is,
ecause false
positives are extremely rare. 13 Rapid Plasma Reain (RPR) Test The RPR is a mod
i ed VDRL test
involvin macroscopic alutination. The cardiolipin-containin antien suspensi
on is
ound to
charcoal particles, which make the test easier to read. The suspension is contai
ned in small
lass vials, which are sta
le for up to 3 months after openin. The antien is s
imilar to the
VDRL antien with the addition of ethylene- diaminetetraacetic acid (EDTA), t
himerosal, and
choline chloride, which sta
ilize the antien and inactivate comple- ment so tha
t serum does not
have to
e heat-inactivated
efore use. Patient serum (approximately 0.05 mL) is
placed in an 18
mm circle on a plastic-coated disposa
le card usin a cap- illary tu
e or Dispen
stir device.
Antien is dispensed from a small plastic dispensin
ottle with a cali
rated 20
-aue needle.
One free-fallin drop is placed onto each test area, and the card is mechanicall
y rotated under
humid conditions. 2 Cards are read under a hih-intensity liht source, and if oc
- culation is
evident, the test is positive. All reactive tests should
e con rmed
y retestin
usin dou
lin
dilutions in a quan- titative procedure. The RPR test appears to
e more sensiti
ve than the VDRL
in primary syphilis. 13 Standard Treponemal Seroloical Tests Treponemal tests
detect anti
ody
directed aainst the T. pallidum oranism or aainst specific treponemal anti
- ens. The two
main types of treponemal tests include the uorescent treponemal anti
ody a
sor
ed
(FTA-ABS) test

and alutination tests. Typically, these tests are more 1814_Ch21_347-36

8.qxd 7/10/09
3:54 PM Pae 352 CHAPTER 21 Spirochete Diseases 353 dif cult to perform and are
more
time-consumin than non- treponemal tests, so they have
een traditionally used
for con rmation
rather than screenin. Fluorescent Treponemal Anti
ody A
sorption Test One of th
e most used
con rmatory tests is the FTA-ABS test, an indirect uorescent anti
ody test. A dilut
ion of heatinactivated patient serum is incu
ated with a sor
ent consistin of an
extract of
nonpathoenic treponemes (Reiter strain). This removes cross-reactivity wi
th tre- ponemes
other than T. pallidum. Slides used for this test have the Nichols strain of T.
pallidum fixed to
them. They are kept frozen until use and then are equili
rated at room tem- pera
ture for 30
minutes. Diluted patient samples and controls are measured and applied t
o individual
wells on the test slide. Slides are then incu
ated in a covered moist cham

er at 37C for 30
minutes. They are rinsed with deionized water and placed in a Coplin jar with ph
osphate-
uffered
saline for 5 minutes. After a second rinsin, the slides are air-dried, and anti

ody conjuate
(antihuman immunolo
ulin conjuated with fluorescein) is added to each well. Sl
ides are
reincu
ated as
efore, and a similar washin procedure is followed. Moun
tin medium is
applied, and coverslips are placed on the slides. They are examined under a uores
cence microscope as soon as possi
le. If speci c patient anti
ody is present, it will
ind to
the T.
pallidum antiens. The anti
ody conjuate will, in turn, only
ind where patient
immunolo
ulin
is present and
ound to the spirochetes. When slides are read under a u- orescenc
e microscope,
the intensity of the reen color is reported on a scale of 0 to 4 . No uorescence i
ndicates a
neative test, and a result of 2 or a
ove is considered reac- tive. 12 A result of
1 means that
the specimen was minimally reactive, and the test must
e repeated with a second
spec- imen drawn
in 1 to 2 weeks. 12 Experienced personnel are required to read and interpret uore
scent test
results. The FTA-ABS is hihly sensitive and specific,
ut it is time-consumin
to perform. This
test is usually positive
efore reain tests, althouh 20 percent of primary syp
hilis cases are
nonreactive. 24 In secondary and latent syphilis, tests are usually 100 percent
reactive. Once a
patient is reactive, that individual remains so for life. Althouh there are few
er false
positives compared to reain tests, reactivity is seen with other treponemal dis
eases, nota
ly
yaws and pinta. 13 Alutination Tests Several varieties of passive hemalutina
tion tests have

een used to detect speci c treponemal anti
ody. These are no loner availa
le in
the United

States, and particle aluti- nation tests such as the Serodia TP-PA test have l
arely replaced
microhemalutination tests. 22 Particle alutina- tion tests use colored 
elatin particles
coated with treponemal antiens and are more sensitive in detectin pri- mary
syphilis. 18 In
the Serodia TP-PA test, patient serum or plasma is diluted in microtiter plates
and incu
ated
with either T. pallidumsensitized el particles or unsensitized el particles as
a control.
Presence of T. pallidum anti
odies is indicated
y alutination of the sensitiz
ed el particles,
which form a latticelike structure that spreads to produce a smooth mat that cov
ers the wells
surface. In wells contain- in a sample that is neative for the anti
ody, the 
el particles
settle to the wells
ottom and form a compact
utton. Comparison and Testin Stra
tey of
Traditional Nontreponemal and Treponemal Seroloical Tests Nontreponemal tests a
re inexpensive,
simple to perform, and can yield quantitative results. Thus, they are extremely
useful as a
screenin tool, in monitorin the proress of the disease, and in determinin th
e outcome of
treatment. The main dis- advantae is that they are su
ject to false positives.
Transient false
positives occur in diseases such as hepatitis, infectious mononucleosis, varicel
la, herpes,
measles, malaria, and tu
er- culosis, and durin prenancy. 12 Chronic condition
s causin
sustained false-positive results include systemic lupus erythe- matosus, leprosy
, intravenous
dru use, autoimmune arthritis, advanced ae, and advanced malinancy. 2,18 A re
active
nontreponemal test should
e con rmed
y a more specific treponemal test. In pren
ancy, this is
espe- cially important,
ecause nontreponemal titers from a previous syphi
lis infection may
increase nonspeci cally. 2,14 Under the followin conditions, titers can
e consid
ered to
e a
nonspeci c increase: lesions are a
sent, the increase in titer is less than fourfo
ld, and
documentation of previous treatment is availa
le. 2 Althouh treponemal tests ar
e usually
reactive
efore rea- in tests in primary syphilis, they suffer from a
lack of
sensitivity in conenital syphilis and neurosyphilis. Nontreponemal tests s
hould
e used for
these purposes. 2,13 Treponemal tests, while more dif cult to perform, have
een t
raditionally
used as con rmatory tests to distinuish false- positive from true-positive reain
results. They
also help esta
lish a dianosis in late latent syphilis or late syphilis,
ecaus
e they are more
sensitive than nontreponemal tests in these staes. 2 Ta
le 212 compares sensitiv
ities of some
of the most widely used seroloical tests. Newer Technoloies in Syphilis Testin
 Enzyme
Immunoassay Several enzyme immunoassay (EIA) tests have
een devel- oped for ser
odianosis of

syphilis. One of these, Reain II, uses a cardiolipin antien similar to that us
ed in the VDRL
test. This method can easily screen lare num
er of sam- ples, and it is reporte
d to have a
sensitivity of 93 percent in primary syphilis. 26 However, it also has a slihtl
y hiher rate of
false positives than RPR tests. Other EIA tests have
een developed to capture a
spe- ci c class
of anti
ody, either IM or IG. Microtiter wells are coated with anti
ody to IM
or IG and are
reacted with patient serum. Treponemal antiens that are la
eled with an enzyme
are then added
(Fi. 213). These have a fairly 1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 353
354 SECTION 4
Seroloical Dianosis of Infectious Disease hih sensitivity in primary syph
ilis,
ut the
sensitivity decreases as the disease proresses. 27 Speci city is similar to other
treponemal
tests. EIA tests are especially useful in dianosin conenital syphilis in infa
nts, as they look
for presence of IM, which cannot cross the placenta. The EIA tests have continu
ed to improve,
with the driv- in force
ein the risin incidence of syphilis worldwide. Sensi
tivities and
specificities are
ein reported as hih as 99 percent and 98 percent, respectiv
ely, in lare
trials. The positive and neative predictive values likewise are hih (99.3 perc
ent and 97.2
percent, respectively). 28,29 With the ease of performance of EIAs, improved sen
sitivity and
speci- ficity, and
ecause false positives may exceed the true positives
in low-risk
populations, a chane in the testin stratey has
een proposed and adapted in h
ih-volume testin centers. 30 Under this scheme, the testin order is reversed from the
traditional
nontreponemal screenin fol- lowed
y con rmatory treponemal testin stratey. Pat
ients would
e
screened
y the EIA test followed
y an RPR or equivalent test. If the initial E
IA was neative,
no further testin would
e done unless early syphilis is suspected (i.e., prior
to
seroconversion). If the EIA was positive and the su
sequent RPR was positive, th
en it would
e
considered positive for syphilis. If the initial EIA was positive followed
y a
neative RPR,
then an FTA-ABS or equivalent would
e performed. If that testin was positive,
then late or
latent syphilis or previous history of syphilis would
e considered. If neative
, then it would

e considered neative for syphilis at this time, and careful evaluation of pati
ent history
should
e considered reardin possi
le reevaluation at a later date. Polymerase
Chain Reaction
Technique Polymerase chain reaction (PCR) technoloy, which involves isolati
n and amplifyin
a sequence of DNA that is unique to a particular antien, has
een applied to te
st- in of
various tissues, includin kidney, lymph nodes, eye, and skin, as well as to spi
nal uid and swa

samples for the presence of treponemes. While there are many variations Ta
le 212
. Sensitivity
of Commonly Used Seroloical Tests for Syphilis STAGE TEST PRIMARY (%) SECONDAR
Y (%) LATENT (%)
LATE (%) Nontreponemal (Reain) Tests Venereal Disease Research La
oratory Test
(VDRL) 78 100 95
71(3794) Rapid plasma reain card test (RPR) 86 100 98 73 Speci c Treponemal Tests
Fluorescent
treponemal anti
ody a
sorption (FTA-ABS) 84 100 100 96 test T. pallidum microhe
malutination
assay (MHA-TP) 76 100 97 94 Adapted from LaSalsa, et al. Spirochete infections.
In Henry, JB
(ed): Clinical Dianosis and Manaement
y La
oratory Methods, ed. 21. WB Saunde
rs, Philadelphia,
2007, Ta
le 58-1. Patient serum Capture of patient IG and IM
Anti-IG and anti
-IM on
microtiter well + Enzyme-la
eled treponemal antien Patient A
reacts with trepo
nemal antien
Su
strate Color development +
+ E E E E E E E E E E E E E E E E E E FIGURE 2
13. Anti
ody
capture enzyme-linked immunosor
ent assay (ELISA) test. Only speci c antitreponema
l anti
ody will
react with enzyme-la
eled antien. 1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae
354 CHAPTER 21
Spirochete Diseases 355 of this procedure,
asically DNA is extracted from the
sam- ple and
then ampli ed or increased usin a DNA polymerase enzyme and a primer pair that st
arts the
reaction. The newly made DNA is then su
jected to aarose el electrophoresis us
in a technique
known as Southern
lottin. Nitrocellulose paper is used to make a copy or
lot
of the el. A
la
eled pro
e, which is a nucleotide sequence speci c to the DNA sequence
ein te
sted for, is
added to the nitrocellulose paper. The pro
e will adhere only to a complementary
strand of DNA.
Presence of the radiola
el on the nitrocellulose paper con rms the presence of tre
ponemal antien
(Fi. 214). Early studies demonstrated that PCR is an extremely sen- sitive techn
ique, capa
le
of detectin as little as one treponeme in a CSF sample. 31 For other uids, espec
ially serum,
althouh the speci city appears to
e 100 percent, sensitivity is closer to 70 per
cent. 32 More
recently, in the United Kindom, it has
een offered as a dianostic test for ea
rly syphilis in
conjunc- tion with the traditional testin in a clinical trial. This study found
the sensitivity
and speci city for swa
specimens in early syphilis was also hih (94.7 percent an
d 98.6 percent,
respec- tively). 33 PCR has
een used in conjunction with ne-needle aspiration o
f material
from the inuinal lymph nodes. Preliminary results indicate that this could

e a useful new
tool for dianosis when seroloical testin is not conclusive. 34 Other specimen
s such as whole

lood, CSF, and amni- otic uid can
e tested
y PCR; however, further research is
required

efore these specimens are used in clinical prac- tice. 35 Newer advances in thi
s technoloy

include real-time PCR, which is semiautomated and extremely fast. Most important
ly, real-time
PCR can discriminate
etween azithromycin-resistant and azithromycin-suscepti

le strains of T.
pallidumin clinical samples. The implications for early proper treatment are o
v
ious. 36
Unfortunately, at this time, the lack of commercial kits and nonstandardiz
ation has
larely hampered the lare-scale use of this technoloy for syphilis. 13 Special
Dianostic Areas
Conenital Syphilis Nontreponemal tests for conenital syphilis performed on cor
d
lood or
neonatal serum detect the IG class of anti-
ody. 37 It is difficult to differe
ntiate passively
transferred IG maternal anti
odies from those produced
y the neonate, s
o there are
pro
lems in esta
lishin a de nitive dianosis. Late maternal infection may result
in a nonreactive test
ecause of low levels of fetal anti
ody. Additionally, testin the inf
ants spinal uid
for presence of treponemes often lacks sensitivity. 38 Nontreponemal titers in t
he infant that
are hiher than the mother may
e a ood indicator of conenital disease,
ut th
is does not
always occur. 12 Several approaches have focused on detectin IM in the infant.
An FTA-ABS test
for IM alone lacks sensitivity, and the test is su
ject to interference
ecause
of the presence
of rheumatoid factor. 38 However, an IM capture assay is more sensitive, and a
Western
lot
assay (see Chapter 8 for details) usin four major treponemal antiens has demon
strated a hih
sensitivity and speci city. 12 Currently, it is recommended that in hih-risk popu
la- tions,
nontreponemal tests
e performed on
oth mother and infant at
irth, reardless
of previously
neative mater- nal tests. Since symptoms are not always present at
irth, if co
nenital syphilis
is suspected
ecause of maternal history, tests should
e repeated on infan
t serum within
a few weeks. 21 If infection is present in the infant, the titer will remain th
e same or will
increase. The Western
lot test is recommended to con rm conenital syphilis. 13 C
ere
rospinal
Fluid Test CSF is typically tested to determine whether treponemes have invaded
the central
nervous system. Such testin is usually more relia
le if central nervous system
symptoms are
pres- ent. The traditional test performed on spinal fluid is the VDRL, which is
a hihly speci c
indicator of neurosyphilis. 13 If
lood contamination of the specimen is not pre
sent, a positive VDRL con rms neurosyphilis. 17 However, sensitivity is lackin,
ecause sa
mples from
fewer than 70 percent of patients with active neurosyphilis have iven posit
ive tests. 13,18
If a neative test is o
tained, other indicators such as increased lymphocyte an
d elevated total
protein ( 45 m/dL) count, are used as sins of active disease. 17 PCR has
een ad
vocated in

dianosin neurosyphilis and may play an important role in the future; however,
the clinical
availa
ility of the test is limited, as discussed a
ove. 13,35 DNA isolated from
patient sample
and cut into framents
y restriction endonuclease DNA framents separated into

ands durin
electrophoresis
A "
lot" or copy of the DNA pattern is made on nitrocellulose Ni
trocellulose
DNA pattern DNA pro
e specific for treponemal antien DNA pro
e only com
ines wi
th complementary
DNA strand Radioactive la
el
X-ray film pattern * * * * * * * * * * * FIGURE 214.
Southern

lottin technique for determinin the presence of treponemal DNA. 1814_Ch21_347
-368.qxd 7/10/09
3:54 PM Pae 355 356 SECTION 4 Seroloical Dianosis of Infectious Disease Tre
atment Penicillin
is still the dru of choice for treatin primary or secondary syphilis. A sinle
intramuscular
injection is usually effective. 39 Doxycycline can
e used as an alternative if
the patient is
alleric to penicillin. In addition, azithromycin has
een also commonly used; h
owever, resistant
strains are aris- in. 36 Neurosyphilis is treated with crystalline penicillin o
r procaine
penicillin to achieve a hih enouh concentration in the central nervous system.
Pro
enecid is
recommended for use alon with penicillin in neurosyphilis patients. 39 F
or conenital
syphilis, crystalline penicillin is administered for 10 days. LYME DISEASE Lyme
disease was rst
descri
ed in the United States in 1975 when an unusually lare num
er of cases o
f juvenile
arthri- tis appeared in a eoraphically clustered rural area around Old Lyme, C
onnecticut (hence
the disease name), in the sum- mer and fall. Two mothers reconized this and
ro
uht it to the
attention of health of cials. Due to the epidemioloical features of this newly de
scri
ed Lyme
arthritis, transmis- sion
y an arthropod vector was suested. 40 In 1982, the a
ent was
isolated and identi ed as a new spirochete. It was iven the name Borrelia
urdor
feri after
Willy Burdorfer ( rst author in the oriinal description). 41 The clinical fea- t
ures of Lyme
disease were soon reconized to o
eyond the arthritis, and it is now known to

e a multisystem
illness involvin the skin, the nervous system, the heart, and the joints. Lyme
disease is the
most common vector-
orne dis- ease in the United States with approximately 20,00
0 cases
ein
reported each year (2003 to 2005). The num
er of cases
ein reported per year h
as dou
led since
1991. Accordin to the CDC, this re ects
oth a true increase in the frequency of
disease as well
as
etter reconition and reportin. 42 Characteristics of the Oranism Several
species of
Borrelia are known to
e the causative aents of Lyme disease. In North America,
it is
exclusively B.
urdorferi sensu stricto, while in Europe several species are k
nown to cause

Lyme disease (B. afzelii, B. arinii, B. sensu stricto, and occasionally ot

her Borrelia
species). 43,44 All share similar characteristics and for simplicity will
e ref
erred to as B.

urdorferi. The oranism is a loosely coiled spirochete, 5 to 25 mm lon and 0.
2 to 0.5 mm in
diame- ter. 1,43 The outer mem
rane, which consists of lycolipid and protein, i
s extremely uid
and only loosely associated with the oranism. Several important lipoprotein ant
iens, la
eled
OSP-A throuh OSP-F, are located within this struc- ture and are actually encode
d
y plasmids. 44
There are also surface proteins that allow the spirochetes to attach to mam- mal
ian cells. Just
underneath the outer envelope are 7 to 11 endo a- ella or periplasmic flaella. T
hese run
parallel to the lon axis of the oranism and are made up of 41 kDa su
units tha
t elicit a stron
anti
ody response. This immunodomi- nant characteristic is of dianostic im
portance, since
the response is not only stron
ut is also very early. Unfortunately,
the flaellin
su
unit has homoloy to that of other nonpathoenic and pathoenic spirochetes,
nota
ly B.
recurrentis and T. pallidum, causin cross-reactivity issues in seroloical test
in. 1,45 Due to
this, a lare num
er of unin- fected people have low levels of anti
odies to the
41 kDa protein.
46 While this is usually not a pro
lem in the current dianostic scheme of testi
n, it can
ecome
an issue when these individuals
ecome ill with certain viruses that are known p
olyclonal B-cell
activators (such as Epstein-Barr virus). In this case, this normally low-level a
nti
ody
ecomes
hih enouh to cause
ioloical false positivity. The oran- ism divides
y
ina
ry ssion
approximately every 12 hours. It can
e cultured in the la
oratory in a complex
liquid medium
(Bar
our-Stoenner-Kelly) at 33C,
ut it is diffi- cult to isolate from patien
ts, and the
spirochetemia is short-lived and enerally found only early on in illness. 43 C
ultures often
must
e incu
ated for 6 weeks or loner to detect rowth and are therefore of li
ttle dianostic
utility. 1 The main reservoir host is the white-footed mouse (Peromyscus
leucopus),
althouh in California and Oreon the spirochete is also har
ored
y the dusky-f
ooted woodrat. 44
The tick infectivity rate on the West Coast is very low, thouht, in part, to
e
due to a lare
num
er of ticks feed- in on the western fence lizard whose
lood is
actericida
l for the
spirochete. 47 Vectors are several types of Ixodes ticks: I. scapularis in the N
ortheast and
Midwest United States, I. paci cus in the West, I. ricinus in Europe, and I. persu
lcatus in Asia.
The lone star tick (A. americanum) is also known to carry the oranism
ut is no
t thouht to
transmit the dis- ease. White-tailed deer are the main host for the ticks adult s
tae (Fi.

215). Nymphs and adult staes of the tick can transmit the disease. The peak
feedin is
in the late sprin/early summer and the fall, which corresponds to the peak
i
phasic occurrence
of Lyme disease. 1 Larvae forms of the ticks enerally cannot transmit the disea
se,
ecause they
have not yet had a
lood meal from which to pick up the oranisms, and ova infec
tion is extremely
rare. The tick must feed for a period of time
efore the spirochete can
e trans
- mitted. Most
aree that the risk for transmission is very low when ticks have fed for less th
an 36 hours, and
one study nds transmission is still low at 72 hours. 48 Staes of the Disease Lym
e disease
resem
les syphilis in that manifestations occur in several staes. These have
e
en characterized
as (1) localized rash, (2) early dissemination to multiple oran systems, and (3
) a late
disseminated stae often includin arthritic symp- toms. 49 Because these staes
are not always
sharply delineated, however, it may
e easier to view Lyme disease as a proressive infectious
disease that involves diverse oran systems. The clinical hallmark of early infe
ction is the rash
known as erythema mirans (EM), which appears
etween 2 days 1814_Ch21_347-368.q
xd 7/10/09 3:54
PM Pae 356 CHAPTER 21 Spirochete Diseases 357 and 2 weeks after a tick
ite.
50 EM is a small
red papule where the
ite occurred, which rapidly expands to form a lare rinli
ke erythema and
often a central area that exhi
its partial clearin (Fi. 216). The clinical dia
nosis of early
Lyme disease relies on the reconition of this characteris- tic rash, which shou
ld
e at least 5
cm in diameter. At this stae, the patient may
e asymptomatic or have nonspeci c u
like symptoms
such as malaise, headache, fever, arthral- ias, and fatiue. 13,51,52 The EM us
ually continues
to expand for over a week, and even if untreated, radually fades within 3 to 4
weeks.
Unfortunately, approximately 20 percent of patients do not develop the rash. 49,
51,52 At this
early stae, the anti
ody response is minimal and most seroloies are neative.
Early
dissemination occurs via the
loodstream and occurs in days to weeks follow
in the EM rash.
The skin, nervous system, heart, or joints may
e affected. Approximately
10 to 15 percent
of patients will display mul- tiple skin lesions. 44 There is often miratory pa
in in the joints,
tendons, muscles, and
ones. If treatment is not o
tained, neuroloical
or cardiac
involvement is seen in a
out 15 percent of patients within 4 to 6 weeks after th
e onset of
infection. 44,53 Cardiac involvement typically mani- fests itself as varyin
derees of
atrioventricular
lock. Occasionally, acute myopericarditis or mild left ventri
cular dysfunction
are also seen. 49 The most prevalent neuroloi- cal sin is facial palsy. 44,54
This is a

peripheral neuritis that usually involves one side of the face. Other peripheral
nerve
manifestations include pain and weakness in the lim
that was
itten. The centra
l nervous system
may
e involved as well, and this is termed neuro
orreliosis. Some patient
s develop sleep
distur
ances, mild chronic confusional states, or dif culty with memory and intell
ectual
functionin. 49 An aseptic meninitis, characterized
y severe headache, pho- to
pho
ia, and mild
elevation of spinal uid protein, can also
e seen. 13 Late Lyme disease may devel
op in some
untreated patients months to years after acquirin the infection. 55 The major m
anifestations of
late Lyme disease are arthritis and late neu- ro
orreliosis (peripheral neuropat
hy and
encephalomyelitis). Between 50 and 60 percent of these individuals will develop

rief attacks of
arthritis. 44,54 This usually affects the lare joints, especially the knee, and
is episodic in
nature. Many of these patients had earlier sins of dissemination of the oranis
m, includin
secondary skin lesions and neuroloical sins such as stiffness of the neck. All
of these usually
respond well to conventional anti
iotic treatment. 48 Treatment-resistant arth
ritis has
een
reported to
e associated with particular human leukocyte antien (HLADRB) allele
s. 44 Despite
res- olution of o
jective manifestations of Borrelia infection after anti
iotic
treatment, a
small percentae of patients continue to have fatiue, concentration and short-t
erm memory pro
lems, and musculoskeletal pain. Some of these symptoms are self-limitin and las
t less than 6
months, while others may last reater than 6 months. 56 There is a lare
ody of
evidence FIGURE
215. Adult tick I. scapularis, which transmits Lyme disease. (Courtesy of Steven
M. Opal, MD,
Assistant Professor of Medicine, Brown University, Providence, RI) FIGURE 216. Er
ythema
chronicum mirans rash, which appears after a tick
ite in Lyme disease. (Courte
sy of Steven M.
Opal, MD, Associate Professor of Medicine, Brown University, Providence, RI)
1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 357 358 SECTION 4 Seroloical Dia
nosis of
Infectious Disease that suests these symptoms are not due to continuin per- s
istence of
infection. The interested reader is referred to a recent article in which postLyme
disease
syndromes and chronic Lyme disease are reviewed in detail. 56 Nature of the Immune
Response
The immune response in Lyme disease is hihly varia
le and complex. A well-docum
ented humoral and
cellular response is known to exist. Spirochete lipoproteins also trier pro- d
uction of
macrophae-derived cytokines, which further enhance the immune response. 44 Howe
ver, the clinical
effec- tiveness of these responses is certainly questiona
le and not necessarily
protective,

ecause late Lyme disease occurs despite hih levels of circulatin anti
od

y and cellular
responses. La
oratory Dianosis Dianosis of Lyme disease is a clinical one with
the la
ora- tory
testin used as supportin evidence. Unfortunately, the clinical dianosis is of
ten difficult for
reasons discussed a
ove. If the characteristic rash is present, this can
e used
as a
presumptive findin,
ut as many as 20 percent of patients do not et or
do not reconize
the rash. Direct iso- lation of the oranism from skin scrapins, spinal uid, or

lood is
possi
le,
ut the yield of positive cultures is extremely low. Therefore,
culture is not
used as a routine dianostic tool. The anti
ody response is varia
le and m
ay not
e
detecta
le until 3 to 6 weeks after the tick
ite. The IM response occurs rst fo
llowed
y the
IG response. The IG response does not peak until the third and fourth weeks of
infection. 50,51
These anti
ody responses are also not mutu- ally exclusive and can
e varia
le (
e.., an IM
response can occur in late Lyme disease). In most cases of acute early Lyme dise
ase ( rst 2
weeks), seroloical testin is too insen- sitive to
e dianostically helpfu
l. 55 If
patients with symptoms are tested in less than 7 days after infection,
seropositivity
is only a
out 30 percent. 51,57 Therefore, the decision to start treatment for e
arly Lyme disease
must
e made
efore seroconversion, like many acute infectious dis- eases. Howe
ver, untreated
seroneative patients havin symptoms for 6 to 8 weeks are unlikely to have Ly
me dis- ease, and
other possi
le dianoses should
e pursued. 48 Anti
iotic therapy
eun shortly
after the
appearance of EM may delay or a
roate the anti
ody response. In chronic Lyme di
sease, neative
seroloies have
een attri
uted to previous anti
iotic therapy; however, the sci
enti c support of
this theory is not stron. 56 The CDC currently recommends a two-tiered approach
to providin
la
oratory support for the dianosis of Lyme disease. 57,58 It is recommended th
at patients with
clinical evi- dence of Lyme disease
e screened with an IFA or EIA test. If this
seroloy is
positive or
orderline, a Western
lot should
e performed on that specimen as s
upplemental testin. Under no circumstances should Lyme testin
e performed in the a
sen
ce of supportin
clinical evidence. A positive test performed under these circumstances has only
a 6 percent
positive predictive value (even when done in an endemic area), while it rises to
97 percent when
clinical symptoms and history are present and consistent with Lyme disease. 59 S
ome of the
current testin procedures are dis- cussed and compared in Ta
le 213. Immuno uoresc
ence Assay
(IFA) The IFA was the first test used to evaluate the anti
ody response in Lyme
disease, followed

y various forms of enzyme immunoassays shortly thereafter. The IFA assay is fai

rly easy to put

toether, which is why it is usually the rst assay on the market in many in
fectious
disease arenas. Basically, microscope slides are coated with antien from whole
or processed
spirochetes (supplied
y the manufac- turer already coated), and then patient se
rum (in dou
lin
dilutions) is added and allowed to react. An antihuman lo
- ulin with a uorescen
t ta attached
is added next and reacts with any speci c anti
ody
ound to the spirochetes on the
slide.
Typically, a test result is only considered positive if a titer of 1:256 is o
ta
ined, 57 althouh
this varies
etween manufacturers. As mentioned a
ove, specimens o
tained in the
rst few weeks
are usually neative, as the level of anti-
ody present is
elow the detection
limit of this
(and other) assays. 57 As miht
e expected, other closely related oran- isms s
uch as B.
recurrentis (relapsin fever), T. denticola and others (associated with periodon
tal disease), and
T. pallidum (syphilis) may cross-react and cause
ioloical false-positive resul
ts. 45 Autoimmune
connective tissue diseases such as rheumatoid arthritis and systemic lupus eryth
ematosus can also
produce false positives in the IFA assay for Lyme dis- ease and the FTA assay fo
r syphilis. 60 An
astute technoloist can reconize a false positive
y the
eaded uorescent pat- t
ern it
produces. Readin of fluorescent patterns tends to
e very su
jective and requir
es hihly trained
individuals. However, if performed correctly
y experienced personnel, the test
can provide
sensitive and accurate results. This test is ideal for low-volume testin. Enzym
e Immunoassay
(EIA) EIA testin is quick, reproduci
le (not su
jective), relatively inexpensiv
e, and lends
itself well to automation and hih- volume testin. 57 There are many forms of t
his assay
ased
on different dianostic philosophies that revolve around the antien used for th
e assay. Antien
preparations that have
een used are crude sonicates of the oranism, puri ed proteins,
synthetic proteins, and recom
inant proteins. The manufacturers selected antien
is then coated
onto 96-well microtiter plates or strips
y various proprietary methods. Patient
sera is added
and allowed to incu
ate with the anti- en. After a washin step, antihuman lo

ulin conjuated
with an enzyme ta such as alkaline phosphatase is added to each well. Addin sp
eci c su
strate
produces a color chane. Plates are read in a spectrophotometer, and the anti
od
y is
1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 358 CHAPTER 21 Spirochete Diseases
359
quantitated
ased on color intensity. EIAs are not su
jective, and the titer is

ased on a
continuum rane rather than twofold dou
lin of patients sera in the IFA. Therefo
re, a more
accu- rate measurement of the speci c anti
ody is possi
le. 57 Like the IFA, draw

acks of EIA
include a lack of sensitiv- ity durin the early staes of Lyme disease and spec
ificity pro
lems.
The sensitivity for early serum specimens has
een reported to
e anywhere from
58 to 92 percent.
61 The dif- ferences in usin various antiens is usually manifested in trade-of
fs
etween
increasin sensitivity at the expense of speci city versus increasin speci city at
the expense
of sen- sitivity. Unfortunately, the technical adaptations of the EIA have not r
esulted in hih
enouh sensitivity and speci city to replace the two-tier method of testin. 55 Th
e speci city
issues are enerally similar to the IFA assay; however, there are no clues (
ead
ed pattern) that
a particular sample may
e a false positive. Aain, false positives occur with s
yphilis and other
treponemal diseases such as yaws and periodontal disease, as well as relapsin f
ever and
leptospirosis. 1,61 If serum is a
sor
ed to decrease cross-reactivity, this also
decreases
specific Lyme anti
ody titers. Additionally, patients with infectious mononucleo
sis, Rocky
Mountain spotted fever, and other autoimmune diseases have
een also known to
e
positive with
EIA. 57 Lyme disease patients do not test posi- tive with RPR, so this may
e he
lpful if syphilis
is in the differential dianosis. 57 Western Blot Immuno
lottin, or Western
lo
ttin, is a
method that, in many systems, is a con rmatory test (e.., HIV testin). Likewise,
in Lyme
disease testin, it is the second test in the CDC-recommended two-tier testin s
cheme. 58,62
However, the Lyme disease immuno
lot is very complex (Fi. 217) and does not prov
ide the same
level of con dence as simpler systems such as with HIV. 63 In Lyme disease, the im
muno
lot is
enerally referred to as supplemental testin. The tech- nique consists of elect
rophoresis of
Borrelia antiens in an acrylamide el and then transfer of the resultin patter
n to Ta
le 213.
Comparison of Tests for the Dianosis of Lyme Disease TEST ANTIGEN ANTIBODY COMM
ENTS IFA Whole or
processed B.
urdorferi Anti-Borrelia anti
ody from Dif cult to perform; false pa
tient,
antihuman lo
ulin positives; su
jective with uorescent ta EIA Sonicated B.
ur
dorferi
Anti-Borrelia anti
ody Easy to perform; false positives; from patient, antihuma
n more sensitive
than IFA lo
ulin with enzyme ta Puri ed aellin protein Anti aellin anti
ody from
Easy to
perform; hihly speci c; patient, antihuman lo
ulin sensitive in early Lyme dise
ase with enzyme
ta C6 peptide Conserved reion of surface Easy to perform; hihly speci c; lipop
rotein (VlsE)
initial studies promisin; sensitive in early and late Lyme disease DNA pro
e Pa
tient DNA matched
to Borrelia DNA None Technically demandin, very speci c; lacks sensitivity IFA
immuno uorescence assay; EIA enzyme immunoassay; DNA
deoxyri
onucleic acid FIGURE
217.

Immuno
lot for Lyme disease. 1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 359 3

60 SECTION 4
Seroloical Dianosis of Infectious Disease nitrocellulose paper. This step is p
erformed
y the
manu- facturer, and nitrocellulose antien strips are provided in the test kit.
These strips are
reacted with patient serum and developed with an antihuman lo
ulin (either anti
-IG or anti-IM)
to which an enzyme la
el is attached. The fur- ther incu
ation with the enzy
mes su
strate
allows for visualization of any anti
ody that has
ound to a particular antien
. The reactivity
is then scored and interpreted. Ten proteins are used in the CDC-recommended int
er- pretation of
this test. They are desinated
y their molecular weihts: 18, 23, 28, 30, 39, 4
1, 45, 58, 66,
and 93 kDa. 58 For a result to
e considered positive for presence of speci c IM
anti
ody, two
of the followin
ands must
e present: 23(Osp C), 39, and 41 ( aellin) kDa. 64,6
5 An IG
immuno
lot is con- sidered positive if any 5 of the 10
ands listed a
ove are po
sitive. 64,65 Due
to the complexity of the Lyme immuno
lots, testin and interpretation of
lo
ts should
e
done only in qualified la
oratories that follow CDC-recommended evidence-
ased
uidelines on
immuno
lot interpretations. 66 As previously stated, the currently accepted test
in protocol
calls for a two-step approach with the use of EIA or IFA as initial screenin to
ols. Then, if a
test is equivocal or positive, it should
e further tested
y Western
lo
t. 58 Current CDC
recommendations do not advise testin seropositive/
orderline patients for IM
anti
odies if
they have had symp- toms for more than 4 weeks for the reasons outlined a
ove. S
eroloical
evidence of Lyme disease in these patients is indicated
y a positive result in
the IG
immuno
lot. 58 Polymerase Chain Reaction In testin for Lyme disease, the PCR ha
s found a niche
in certain scenarios. While only a few oranisms need to
e pres- ent for detect
ion under optimal
conditions, the num
er of spirochetes in infected tissues and
ody uids is low, m
akin specimen
collection, transport, and preparation of DNA crit- ical to these clinical speci
mens and
sensitivity an issue. 55 Several pro
es for taret DNA that is present only in s
trains of B.

urdorferi have
een made and used in PCR testin. 55,67 The procedure involves
extractin DNA
from the patient sam- ple followed
y ampli cation usin speci c primers, DNA polyme
rase, and
nucleotides. Once a suf cient amount of the unknown DNA is made, this is com
ined
with a known
DNA pro
e to see if hy
ridization takes place. The sinle-stranded Borrelia DNA
pro
e will
ind
only to an exact complemen- tary strand, thus positively identifyin the presenc
e of the
oranisms DNA in the patient sample. This is much more speci c than testin for ant
i
ody,


ecause there is little cross- reactivity. Speci city of recent PCR studies has ra
ned from 93
percent to 100. However, sensitivity remains an issue. In a series of studies, t
he median
sensitivity of PCR on skin
iop- sies was 69 percent; of
lood components, 14 pe
rcent; of CSF, 38
percent; and of synovial uid, 78 percent. However, the rane of sensitivities in
any one type of
specimen is quite lare, suestin the testin remains to
e standardized
. 13,55
Furthermore, it would
e hard to clinically justify a skin
iopsy for PCR as a d
ianostic method
for an EM rash in most cases. However, in dif cult dianostic neuroloical and art
hritic cases,
PCR on CSF and synovial uid is often employed. PCR for Borrelia still has limited
availa
ility.
Treatment Borrelia is sensitive to several orally administered anti
iotics, incl
udin
penicillins, tetracyclines, and macrolides. For adult early Lyme disease and ear
ly disseminated
Lyme disease, doxycycline, cefuroxime axetil, or amoxicillin are the rec- ommend
ed drus of
choice. 48,49 For youn children, amoxicillin and cefuroxime axetil are used,
and if the child
is over 8 years of ae, doxycycline. Prophylaxis, full-course treatment, or sero
loical testin
of all patients with tick
ites is not recommended. A sinle dose of doxycycline
may
e offered
to adults and children over 8 years of ae. Macrolides are not recommended as a r
st-line therapy
for early Lyme disease, as they are less effective. 48 Under rare circumstances,
an individual
with a tick
ite may
e fully treated: (1) if the tick was identi ed as a nymph or
adult I.
scapularis; (2) if the tick was attached for more than 36 hours; (3) if treatmen
t can
e started
within 72 hours of tick removal; (4) if the local infection rate of ticks is ove
r 20 percent; or
(5) if doxycy- cline is not contraindicated. 48 Neuro
orreliosis requires the us
e of intravenous
therapy. Ceftriaxone, cefotaxime, or peni- cillin G are the anti
iotics used for
this purpose. 51
Currently there are no effective vaccines for humans. A human vac- cine made wit
h the Osp-A
surface antien has had limited usefulness, has
een associated with side effect
s, and has
een
recalled from the market. 68 SUMMARY Syphilis and Lyme disease are the two major
diseases caused

y spirochetes. Spirochetes are distinuished
y the pres- ence of axial laments
that wrap
around the cell wall inside a sheath and ive the oranisms their characteristic
motility.
Syphilis is caused
y the oranism Treponema pallidum, su
species pallidum. The
disease is
acquired
y direct con- tact, usually throuh sexual transmission. The disease c
an
e separated
into four main clinical staes. An untreated patient may proress from the prima
ry stae to the
secondary stae, in which systemic dissemination of the oranism occurs. The lat
ent stae follows

the disappearance of the secondary stae. Patients are usually free of clinical
symptoms,
ut
seroloi- cal tests are still positive, and transmission to the fetus can occur
at this stae.
A
out one-third of the individuals who remain untreated develop tertiary syphili
s. Early
dianosis and treatment help to prevent later complications. Direct la
oratory d
ianosis involves
detectin the oran- ism from a lesion, usin either dark-field microscop
y, uorescence
microscopy, or PCR. If an active lesion is not present, dianosis must
e made o
n the
asis of
seroloical tests. These can
e classified as either nontreponemal or treponemal
. Nontreponemal
tests determine the presence of reain. The VDRL, RPR, and RST tests are example
s of
1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 360 CHAPTER 21 Spirochete Diseases
361 such
tests. These are ood screenin tests for low-volume testin la
oratories,
ecau
se they are
fairly sensitive and simple to perform. However, they lack speci city, and spec- i
mens with
positive results must
e con rmed with a more speci c treponemal anti
ody test. Trad
itional
treponemal anti
ody tests include the FTA- ABS and particle alutination. T
hese detect
anti
ody formed aainst the oranism itself. Treponemal tests are more specific
and sensitive in
early staes of the disease. Titers of these anti
odies remain detecta
le for li
fe, while
nontreponemal titers drop after successful treatment. New developments in testin
 include EIA
technoloy and PCR. EIAs test for anti
ody to speci c treponemal antiens, and cla
ss separation
of anti
odies is possi
le. For lare-volume testin, the EIA is now the recommen
ded screenin
test fol- lowed
y con rmation with the RPR or equivalent test. Lyme disease rep
resents the
most common vector-
orne infection in the United States today. The oranism r
esponsi
le is a
spirochete named Borrelia
urdorferi, which is transmitted
y the deer tic
k. Althouh an
expandin red rash is often the first symptom noted, the disease can
e characte
rized as a
proressive infectious syndrome involv- in diverse oran systems. Despite resol
ution of
o
jective manifestations of Borrelia infection after anti
iotic treat- ment, a s
mall percentae
of patients continue to have fatiue; concentration and short-term memory pro
le
ms; and musculoskeletal pain, which, in some are self-limitin and last less than 6 months,
and in others
last reater than 6 months. There is a lare
ody of evidence aainst these symp
toms
ein due to
continuin persistence of infection. The presence of IM and IG cannot usually

e detected until
3 to 6 weeks after symptoms initially appear. Current testin protocol involves
screenin with
IFA or EIA and follow-up of equivocal or positive tests with immuno
lot- tin. A
ll seroloical

ndins must
e interpreted carefully and in conjunction with clinical dianosis.

1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 361 362 SECTION 4 Seroloical Dia
nosis of
Infectious Disease 1. A 30-year-old woman saw her physician, complainin of repe
ated episodes of
arthritis-like pain in the knees and hip joints. She recalled havin seen a very
small tick on
her arm a
out 6 months prior to the development of symptoms. No rash was ever se
en, however. A

lood specimen was o
tained with the followin results: rheumatoid arthrit
isneative;
lupusneative; RPR neative; EIA test for Lyme diseaseindeterminate. Questions a. D
oes the
a
sence of a rash rule out the possi
ility of Lyme disease?
. What miht cause
an indeterminate
EIA test ? c. What con rmatory testin would help in determin- in the cause of th
e patients
condition? 2. A mother who had no prenatal care appeared at the emerency room i
n la
or. A
a
y

oy was safely deliv- ered, and he appeared to
e normal. A
lood sample was o
t
ained from the
mother for routine screenin. An RPR test performed on the mothers serum was positive. The
mother had no o
vious sins of syphilis and denied any past history of the disea
se. She indicated
that she had never received any treatment for a possi
le syphilis infection. Cor
d
lood from the

a
y also exhi
- ited a positive RPR result. Questions a. Is the
a
y at risk fo
r conenital
syphilis?
. What is the sini cance of a positive RPR on a cord
lood? c. How sho
uld these
results
e handled? CASE STUDIES 1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 3
62 CHAPTER 21
Spirochete Diseases 363 RPR CARD TEST FOR SEROLOGICAL DETECTION OF SYPHILIS Pr
inciple The RPR
(rapid plasma reain) 18 mm circle card test is a nontreponemal seroloical test
for syphilis
that detects rea- in, an anti
ody formed aainst cardiolipin durin the
proress of the
disease. The antien consists of cardiolipin mixed with car
on particles, choles
terol, and
lecithin. If anti-
ody is present in the patient specimen, occulation occurs wit
h
coalutination of the car
on particles, resultin in
lack clumps aainst the w
hite
ackround
of the plastic- coated card. Nonreactive specimens appear to have an even, liht
-ray color.
Sample Preparation Either plasma or serum can
e used for this procedure. Collec
t
lood
y
venipuncture usin aseptic technique. Centrifue the specimen at a force suff
icient to
sediment cellular elements. Keep the plasma or serum in the oriinal collectin
tu
e. Heat
inactivation is not necessary for this procedure. Reaents, Materials, and Equip
ment 1. Macro-Vue
18 mm Circle Card Test Kit, which contains the followin: Antien suspension: 0.
003 percent
cardiolipin, 0.020 to 0.022 percent lecithin, 0.09 percent cholesterol, EDTA (0.
0125 mol/L),
Na2HPO4 (0.01 mol/L), 0.1 percent thimerosal (preservative), 0.02 percent charco

al, 10 percent
choline chloride (w/v), and dis- tilled water. Refrierate to store. Unopened am
pules have a
shelf life of 12 months from date of manufac- ture. Once opened, antien in the
dispensin
ottle
may
e used for approximately 3 months or until the expiration date. Brewer dia
nostic cards:
specially prepared plastic-coated cards desined for use with the RPR card anti
en. Dispenstirs
Needles, 20 aue Stirrers Dispensin
ottle Seroloical pipette, 1 mL Controls:
known reactive
and weakly reactive sera pre- pared from pooled ra

it sera and nonreactive pool
ed human sera.
These need to
e stored at 4C and may
e used until the expiration date on the la

el. A rotator,
100 rpm, circumscri
in a circle 2 cm in diame- ter with an automated timer and
a cover
containin a moistened spone or
lotter. WARNING: Because no test method can of
fer complete
assurance that human immunode ciency virus, hepati- tis B virus, or other inf
ectious aents
are a
sent, specimens and these reaents should
e handled as thouh capa
le o
f transmittin an
infectious disease. The Food and Dru Administration recommends such material
e
handled at a
Biosafety Level 2. Procedure* 1. Controls, RPR card antien suspension, and test
speci- mens
should
e at room temperature for use. The antien suspension should
e c
hecked with
controls usin the reular test procedure. Only those suspensions that ive the
prescri
ed
reactions should
e used. 2. Attach the needle to the tapered ttin on the dispen
s- in
ottle.
3. To prepare the antien suspension, viorously shake the ampule for 10 to 15 s
econds to
resuspend the antien and disperse any car
on particles loded in the neck of th
e ampule.
Overaitatin will produce a coarse antien, so disreard any particles left in
the neck of the
ampule after this time. 4. Snap the ampule neck, makin sure that all the antie
n is
elow the

reak line. Withdraw all the antien into the dispensin
ottle
y collapsin th
e
ottle and
usin it as a suction device. 5. To check the delivery of the needle, place it rm
ly on a 1 mL
pipette. Fill the pipette with antien suspension. Holdin the pipette in a
vertical
position, count the num
er of drops delivered in 0.5 mL. This should
e 301 dro
ps for a yellow
20-aue needle. 6. Hold a Dispenstir device
etween the thum
and fore n- er nea
r the sealed
end. Squeeze and do not release until open end is
elow the surface of the speci
men. Hold the
specimen tu
e vertically to minimize stirrin up the cel- lular elements when us
in oriinal

lood tu
e. Release ner pressure to draw up the sample. 7. Hold Dispenstir in a
vertical
position over the card test area. Do not touch the surface of the card. Squeeze,
allowin one
drop to fall onto card. 8. Measure out controls in the same manner. 9. Gently sh

ake
antien-dispensin
ottle
efore use. Hold in a vertical position, and dispense
several drops
into EXERCISE * Adapted from the packae insert for the Macro-Vue RPR 18 mm Circ
le Card Test
(Brewer Dianostic Kit), manufactured
y Becton Dickinson and Company, Cockneysv
ille, MD.
1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 363 364 SECTION 4 Seroloical Dia
nosis of
Infectious Disease
ottle cap to make sure the needle passae is clear. Place on
e free-fallin
drop onto each test or control area. Return test droplets from the
ottle cap to
the dispens- in

ottle. Do not mix antien and specimen. Mixin is accomplished durin rotation.
10. Rotate for 8
minutes, under humidifyin cover, on a mechanical rotator at 100 rpm. 11. Immedi
ately followin
mechanical rotation,
riefly rotate
y hand with tiltin of the card (three to
four to- and-fro
motions). Read macroscopically in the wet state under a hih-intensity incandescen
t lamp or
stron dayliht. 12. Report as reactive any specimen showin characteris- tic cl
umpin, ranin
from sliht
ut de nite to marked and intense. Report as nonreactive any specimen
show- in
sliht rouhness or no clumpin. 13. Upon completion of tests, remove the needle
from the
dispensin
ottle and rinse with distilled or deionized water. Do not wipe the n
eedle,
ecause
this may remove the silicon coatin and affect accuracy of delivery. NOTE: There
are only two
possi
le reports with the card test: reactive or nonreactive, reardless of der
ee of activity.
Any specimen demonstratin sliht
ut de nite clumpin is always reported as react
ive.
Interpretation The dianosis of syphilis should not
e made on a sinle reactive
result without
the support of a positive history or clinical evidence. As with all cardiolipin
antien tests,

io- loical false positives are possi
le with a num
er of diseases and conditio
ns. Therefore,
reactive specimens should
e su
jected to further seroloical study, includin c
on rmatory
testin. RPR card tests should not
e used for spinal fluids. Additiona
lly, the Pu
lic
Health Service has indicated that little reliance may
e placed on cord
lood se
roloical tests
for syphilis. Lipemia will not interfere with the card tests. However, if the de
ree of lipemia
is so reat that antien particles are o
scured, the specimen should
e consider
ed
unsatisfactory. Hemolyzed specimens are accepta
le unless they are so hemolyzed
that printed
material cannot
e read throuh them. 1814_Ch21_347-368.qxd 7/10/09 3:54 PM P
ae 364 CHAPTER
21 Spirochete Diseases 365 1. Treponema pallidum and Borrelia
urdorferi can

e distinuished from each other on the
asis of which of the followin? a. Only T. pa
llidum has axial
laments.
. Only B.
urdorferi has an outer sheath. c. Only B.
urdorferi can

e rown in the
la
oratory on arti cial media. d. Only T. pallidum stimulates IM production to th
e mem
rane
proteins. 2. False-positive nontreponemal tests for syphilis may
e due to which
of the
followin? a. Infectious mononucleosis
. Systemic lupus c. Prenancy d. All of
the a
ove 3. In
the uorescent treponemal anti
ody a
sorption (FTA- ABS) test, what is the purpose
of a
sorption
with Reiter treponemes? a. It removes reactivity with lupus anti
ody.
. It prev
ents
cross-reactivity with anti
ody to other T. pallidum su
species. c. It prevents c
ross-reactivity
with anti
ody to nonpathoenic treponemes. d. All of the a
ove. 4. Which test is
recommended for
testin cere
rospinal uid for detection of neurosyphilis? a. RPR
. VDRL c. FTA-A
BS d. Enzyme
immunoassay 5. Advantaes of direct fluorescent anti
ody testin to T. pallidum
include all of
the followin except a. readin is less su
jective than with dark- eld testin.
.
monoclonal
anti
ody makes the reaction very speci c. c. slides can
e prepared for later read
in. d. careful
specimen collection is less important than in dark- eld testin. 6. Which of the f
ollowin is
true of reain? a. It can
e detected in all patients with primary syphilis.
.
It is anti
ody
directed aainst cardiolipin. c. Reain tests remain positive after successful t
reatment. d. It
is only found in patients with syphilis. 7. Which syphilis test detects spec
ific treponemal
anti
odies? a. RPR
. VDRL c. FTA-ABS d. Alutination 8. Which of the followin
is true of
treponemal tests for syphilis? a. They are usually neative in the primary stae
.
. Titers
decrease with successful treatment. c. In lare-volume testin, they should
e u
sed as screenin
tests. d. They are su
ject to a reater num
er of false posi- tives than reain
tests. 9. An RPR
test done on a 19-year-old woman as part of a prenatal workup was neative
ut e
xhi
ited a rouh
appearance. What should the technoloist do next? a. Report the result out as ne
ative.
. Do a
VDRL test. c. Send the sample for con rmatory testin. d. Make serial dilutions an
d do a titer.
10. Treponemal EIA tests for syphilis are characterized
y all of the followin
except a. they
are adapta
le to automation.
. they are useful in dianosin secondary or terti
ary syphilis. c.
su
jectivity in readin is eliminated. d. they can
e used to distinuish
etwee
n IG and IM
anti
odies. 11. Which of the followin tests is the most speci c dur- in the earl
y phase of Lyme
disease? a. IFA
. EIA c. Immuno
lottin d. Isolation of the spirochete 12. Fals
e-positive
seroloical tests for Lyme disease may
e due to all of the followin except a.
shared antiens

etween Borrelia roups.
. cross-reactivity of anti
odies. c. resem
lance of ae
llar antien to
that of Treponema oranisms. d. a patient in the early stae of the disease. REV

IEW QUESTIONS
1814_Ch21_347-368.qxd 7/10/09 3:54 PM Pae 365 366 SECTION 4 Seroloical Dia
nosis of
Infectious Disease 13. Advantaes of PCR testin for syphilis include all of the
followin except
a. it is extremely speci c.
. many false positives are eliminated. c. testin of
serum is
extremely sensitive. d. it can
e used on CSF. 14. A 24-year-old man who had jus
t recovered from
infec- tious mononucleosis had evidence of a enital lesion. His RPR test was po
sitive. What
should the technolo- ist do next? a. Report out as false positive.
. Do a con rm
atory
treponemal test. c. Do a VDRL. d. Have the patient return in 2 weeks for a repea
t test. 15. A
15-year-old irl returned from a campin trip. Approximately a week after
her return, she
discovered a small red area on her le that had a larer red rin around it. Her
physician had
her tested for Lyme dis- ease,
ut the seroloical test was neative. What is th
e
est
explanation for these results? a. She de nitely does not have Lyme disease.
. The
test was not
performed correctly. c. Anti
ody response is often
elow the level of detection
in early staes.
d. Too much anti
ody was present, causin a false neative. 16. Which of the fol
lowin is a true
statement a
out late manifestations of Lyme disease? a. Treatment cannot reverse
complications.

. Both central and peripheral nervous systems may
e affected. c. Cardiac or ne
uroloical damae
occurs in all cases. d. Arthritis appears only in elderly patients. 17. Pro
lems
encountered in
IFA testin for Lyme disease include all of the followin except a. cross-reacti
vity with
anti
odies to syphilis.
. false neatives in the later staes of disease. c. fa
lse positives
with rheumatoid factor. d. su
jectivity in the readin of uorescent patterns. Ref
erences 1. The
spirochetes. In For
es, BA, Sahm, DF, and Weissfeld, AS: Bailey and Scotts Diano
stic
Micro
ioloy, ed. 11. Mos
y, St. Louis, MO, 2002, pp. 595602. 2. Pope, V, Larsen,
SA, and
Schriefer, M. Immunoloical meth- ods for the dianosis of spirochetal diseases.
In Rose, NR, et
al: Manual of Clinical La
oratory Immunoloy, ed. 5. American Society for Micro

ioloy,
Washinton, DC, 1997, pp. 510525. 3. 2005 Syphilis surveillance project annual re
port (Auust 1,
2008) CDC. http://www.cdc.ov/STD/Syphilis2005/default.htm. 4. Centers for Disea
se Control and
Prevention. Primary and sec- ondary syphilisUnited States, 20002001. MMWR 51(43):
971973,
2002. 5. Centers for Disease Control and Prevention. Primary and sec- ondary syp
hilis amon men
who have sex with menNew York City, 2001. MMWR 51(38):853856, 2002. 6. Centers fo
r Disease
Control and Prevention. Out
reak of syphilis amon men who have sex wi
th menSouthern
California, 2000. MMWR 50(38):117120, 2001. 7. Symptomatic early neurosyphilis am

on
HIV-positive men who have sex with menfour cities, United States, January
2002June 2004.
MMWR 56(25):625628, 2007. 8. Schiff, E, and Lind
er, M. Neurosyphilis. Sou
th Med J
95:10831087, 2002. 9. Doherty, L, et al: Syphilis: Old pro
lem, new stratey. BMJ
325:153165,
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8.qxd 7/10/09
3:54 PM Pae 368 22 Seroloy and Molecular Detection of Viral Infections Linda
E. Miller, PhD,
I,MP(ASCP)SI 369 LEARNING OBJECTIVES After nishin this chapter, the reader will

e a
le to: 1.
Differentiate
etween the different hepatitis viruses and their modes of transmi
ssion. 2.
Correlate the various seroloical markers of hepatitis with their dianostic si
ni cance. 3.
Indicate the la
oratory methods that are most commonly used to screen for, con rm,
or monitor
hepatitis virus infections. 4. Associate the Epstein-Barr virus (EBV) with the s
peci c diseases
it causes. 5. Correlate the heterophile anti
ody and anti
odies to the EBV with
their clinical
sini cance. 6. Indicate the la
oratory methods used to test for heterophile anti

odies and EBV
anti
odies. 7. List the diseases associated with varicella-zoster virus, ru
ella
virus, ru
eola
virus, and mumps virus. 8. State the most common seroloy method used to detect
anti
odies to
these viruses. 9. Explain how seroloy is used to detect current infection with
or immunity to
each of the precedin viruses. 10. Discuss the clinical sini cance of cytomealov
irus, human T
lymphotropic virus type I, and human T lymphotropic virus type I. 11. Discuss th
e la
oratory
methods used to detect exposure to the precedin viruses. 12. Correlate viral I
M and IG
anti
odies with their clinical sini cance in terms of detectin current infection
s, conenital
infections, or immunity to infections. 13. Discuss the role of molecular tests i

n dianosin and
monitorin patients with viral infections. KEY TERMS Anti-HBe Anti-HBs Cytomeal
ovirus (CMV)
Epstein-Barr virus (EBV) HBeA HBsA Hepatitis Herpes simplex virus Human T-cell
lymphotropic
virus (HTLV) IM anti-HBc Mumps virus Ru
ella virus Ru
eola virus Varicella-zost
er virus (VZV)
1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 369 370 SECTION 4 Seroloical Dia
nosis of
Infectious Disease IMMUNE DEFENSES AGAINST VIRAL INFECTIONS AND VIRAL ESCAPE MEC
HANISMS Viruses
are su
microscopic particles, whose size is measured in nanometers. Their
asic
structure
consists of a core of DNA or RNA packaed into a protein coat, or capsid. In som
e viruses, the
capsid is surrounded
y an outer envelope of lycolipids and proteins derived fr
om the host cell
mem-
rane. It is remarka
le that these tiny particles are capa
le of causin se
vere, and
sometimes lethal, disease in humans, ranin from childhood infections, in ammator
y diseases with
predilection for a speci c oran, disseminated disease in immunocompromised patien
ts, cancer, and
conenital a
normalities. Viruses are o
liate intracellular pathoens that rely
on the host cell
for their replication and survival. They infect their host cells
y attachin to
speci c
receptors on the cell surface; penetratin the host cell mem
rane; and releasin
their nucleic
acid, which then directs the host cells machin- ery to produce more viral nucleic
acid and
proteins. These components assem
le to form intact viruses that are released
y
lysis of the cell
or
y
uddin off the cells surface. The free virions can then infect neih
orin
host cells and

ein new replication cycles that promote dissemination of the infection. Viruse
s can thus
e
present in the host as
oth freely circulatin particles and intracellular parti
cles durin the
viral replication cycle. A num
er of immunoloic mech- anisms are required to at
tack the virus in
its different states, and successful defense aainst viral infections requires a
coordinated
effort amon innate immunity, humoral immu- nity, and cell-mediated immunity. In
nate immunity
provides the first line of protection aainst viral pathoens. Two important non
speci c defenses
aainst viruses involve type I interferons and NK cells. 1,2 Virus-infected cell
s are stimulated
to produce IFN- and IFNfollowin reconition of viral RNA
y Toll-like re
ceptors
(TLR). These interferons inhi
it viral replication
y inducin the transcription
of several enes
that code for proteins with antiviral activityfor example, a ri
onucle- ase enzym
e that derades
viral RNA. IFN- and IFN- also enhance the activity of NK cells, which
ind
to virusinfected cells and release cytotoxic proteins like perforin andranzymes, which
cause the cells
to die and release their virus particles. These cell-free virions are now access

i
le to anti
ody
molecules. When innate defenses are insuf cient in preventin viral infection, spe
ci c humoral
and cell-mediated defenses are activated. 1,2 Virus-speci c anti
odies are produce
d
y B cells
and plasma cells and can attack free virus particles in sev- eral ways. Anti
odi
es play a key
role in preventin the spread of a viral infection throuh neutralization. In th
is process,
anti
odies speci c for a component of the virus that
inds to a receptor on the ho
st cell
mem
rane will
ind to the virus and prevent it from attachin to and penetratin
the cell.
Secretory IA anti
odies play an especially important role in this process,
eca
use they
neutralize viruses in the mucosal surfaces (e.., respiratory and diestive trac
ts), which often
serve as entryways for the pathoens; mean- while, IM and IG anti
odies can
i
nd to viruses in
the
loodstream and inhi
it dissemination of the infection. In addition, IG ant
i
odies promote
phaocytosis of viruses throuh their opsonizin activity and promote destructio
n of viruses
throuh anti
ody-dependent cell-mediated cyto- toxicity (ADCC). IG and IM
anti
odies also
activate complement, which can participate in opsonization via C3
or lysis of
enveloped viruses

y the mem
rane attack com- plex. IM anti
odies can also inactivate viral parti
cles
y
alutinatin them. While anti
odies can attack viruses in many different ways,
they cannot reach
viruses that have already penetrated CHAPTER OUTLINE IMMUNE DEFENSES AGAINST VIR
AL INFECTIONS AND
VIRAL ESCAPE MECHANISMS LABORATORY TESTING FOR VIRAL INFECTIONS HEPATITIS Hepati
tis A Hepatitis E
Hepatitis B Hepatitis D Hepatitis C Viruses with Uncertain Association with Hepa
titis HERPES
VIRUS INFECTIONS Epstein-Barr Virus Cytomealovirus Varicella-Zoster Virus VIRAL
INFECTIONS OF
CHILDHOOD Ru
ella Ru
eola Mumps HUMAN T-CELL LYMPHOTROPIC VIRUSES OTHER VIRAL IN
FECTIONS SUMMARY
CASE STUDIES EXERCISE: TESTING FOR THE HETEROPHILE ANTIBODY OF INFECTIOUS MONONU
CLEOSIS BY THE
CLEARVIEW MONO TEST EXERCISE: TESTING FOR THE HETEROPHILE ANTIBODY OF INFECTIOUS
MONONUCLEOSIS
BY THE PAUL-BUNNELL TEST REVIEW QUESTIONS REFERENCES 1814_Ch22_369-398.qxd 7/13
/09 2:58 PM
Pae 370 CHAPTER 22 Seroloy and Molecular Detection of Viral Infections 371 hos
t cells.
Elimination of intracellular viruses requires the action of cell-mediated immuni
ty. Cytotoxic T
cells (CTL) play a key role in this mechanism of defense. Upon activa- tion of C
D4 T helper
cells and cytokines, CD8
CTL
ecome prorammed to expand in num
er and attack th
e
virus-infected cells. 3 To reconize the virus-infected host cell, the T-cell re
ceptor on the CTL
must
ind to a viral antien complexed with MHC class I on the surface of the vi
rus-infected host
cell (Fi. 221). CD8 is a co-receptor in this interaction. Interaction of co-stim

ulatory
molecules, such as B7 and CD28, provides secondary sinals necessary for the CTL
response. These
molecular interactions stim- ulate the ranules in the CTL to release a pore-for
min protein
called perforin, which produces pores in the mem-
rane of the infected host
cell, and
proteases called ranzymes, which enter the pores. These enzymes activate apopt
osis of the host
cell, resultin in interruption of the viral-replication cycle and release of as
sem
led
infectious virions. The free virions can then
e
ound
y anti
odies. The CTL re
sponse is
powerful and involves a series of cell divisions to produce up to 50,000 times t
he oriinal num
er of cells in a period of 1 to 3 weeks. 3 Despite the variety of defenses that
are stimulated
aainst viruses, the immune response is not always successful in totally elimina
tin these
pathoens. That is
ecause viruses have developed several strateies
y which th
ey can escape the
hosts defenses. 1,2 First, viruses are rapidly dividin aents that undero frequ
ent enetic
mutations. These mutations result in the production of new viral antiens, which
are not
reconized
y the initial immune response to the virus. This stratey is used co
mmonly
y the
in uenza virus, with con- tinual antienic variation, resultin in the emerence o
f new
infectious strains that require annual development of new vaccines to protect th
e population.
Antienic variation is also employed commonly
y other viruses, such as
rhi- noviruses,
which cause the common cold, and human immunode ciency virus (HIV), which ca
uses AIDS.
Second, some viruses can evade the action of components of the immune system suc
h as interferons,
complement pro- teins, or the lysosomal enzymes in phaocytic cells. For example
, the hepatitis C
virus can
lock the deradation of viral RNA induced
y the interferons. Third,
viruses can evade
the hosts defense
y suppressin the immune system. Some viruses, like the cytome
alovirus
(CMV), ru
eola, and HIV, accomplish this
y reducin the expression of MHC molec
ules on the
surface of virus-infected cells, makin them less likely to
e reconized
y T l
ympho- cytes.
Other viruses can alter the function of certain cells of the immune system a
fter directly
infectin them. For example, the Epstein-Barr virus (EBV) causes polyclonal ac
tivation of the B
lymphocytes it infects, and HIV suppresses the function of the CD4 T helper cells.
EBV can also
sup- press the immune system
y causin a cytokine im
alance. Finally, some viru
ses, such as CMV,
varicella-zoster virus, and HIV, can remain in a latent state
y interatin the
ir nucleic acid
into the enome of the host cells they infect. In this way, the virus can remain
silent within
the host cell and is protected from the immune system, sometimes for years,
efo

re it is
stimulated to replicate aain
y exposure to other infectious aents or
y decli
ne of the hosts
immune defenses. By utilizin these mechanisms, viruses have remained successful
pathoens to
humans for many years, causin a rane of mild to life-threatenin diseases. Rap
id, relia
le
la
oratory detection of these pathoens is essential for early patient dianosis
and treatment
and for prompt implemen- tation of measures to prevent further spread of the vir
us to other
mem
ers of the population. LABORATORY TESTING FOR VIRAL INFECTIONS As our knowle
de of viruses
has increased, so has the devel- opment of la
oratory assays to detect viral inf
ections. While
seroloical assays have
een used for this purpose for many years, molecular ass
ays have
een
developed more recently and have added much to the area of viral detect
ion as they have

een interated into the clinical la
oratory. Both seroloy and molecular assays
have
ecome some
of the most important and frequently performed tests in clinical immunoloy and
micro
ioloy.
Seroloical and molecular tests for viral infections can
e easily and rapidly p
erformed
y the
clinical la
oratory; therefore, they play an essential role in helpin physician
s esta
lish a
presumptive dianosis so that treatment may
e initiated promptly. Seroloical t
ests are also
important in monitorin the course of infection, detectin past infections, and
assessin immune
status, while molecular tests have enhanced our a
ility to detect active infecti
on and are
essential in uidin antiviral therapy. In eneral, presence of virus-speci c IM
anti
odies in
patient serum indicates a current or recent viral infection, while IG anti
odie
s to the virus
sinify either a current or past infection and, in most cases, immunity. Specifi
c IM anti
ody in
the new
orns serum indicates a conenital infec- tion with a virus; on the other
hand, IG
anti
odies in the infants serum are mainly maternal anti
odies that have crossed
the placenta.
Current infections in the adult or new-
orn may also
e indicated
y immu
noassays for
viral antiens in serum or other clinical samples from the patient TcR CD8 CTL C
ostimulatory
molecules Cytotoxic proteins (perforin, TNF-) Virus-infected host cell Viral A c
omplexed with
MHC class I Lysis and release of free virions FIGURE 221. Bindin of a CTL to a v
irus-infected
host cell. 1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 371 372 SECTION 4 Serol
oical Dianosis
of Infectious Disease or
y the presence of viral nucleic acids that molecular m
eth- ods can
detect. This chapter discusses some of the most important viral infections detec
ted
y seroloy
and molecular methods. These include the hepatitis viruses, herpes viruses, meas
les, mumps,
ru
ella, and human T-cell lymphotropic viruses. La
oratory tests for the human i

mmunode ciency
virus are discussed separately in Chapter 23. HEPATITIS Hepatitis is a eneral t
erm that means
in ammation of the liver. It can
e caused
y several viruses and
y noninfectious
aents,
includin ionizin radiation, chemicals, and autoim- mune processes. The primary
hepatitis
viruses, which are listed in Ta
le 221, affect mainly the liver. Other viruses, s
uch as
cytomealovirus, Epstein-Barr virus, and herpes sim- plex virus, can also produc
e liver
inflammation,
ut it is secondary to other disease processes. This section will
focus on the
primary hepatitis viruses. The hepatitis A virus (HAV) and the hepatitis E virus
(HEV) are
transmitted primarily
y the fecal-oral route, while the hepatitis B virus (HBV)
, the hepatitis D
virus (HDV), and the hepatitis C virus (HCV) are transmitted mainly
y the paren
teral route
(i.e., throuh contact with
lood and other
ody uids). All may produce similar c
linical
manifestations. The early, or acute, staes of hepatitis are characterized
y e
neral ulike
symptoms such as fatiue, fever, myalia, loss of appetite, nausea, vomitin, di
arrhea or
constipation, and mild to moderate pain in the riht upper quadrant of the a
dom
en. 4,5
Proression of the disease leads to liver enlarement (hepatomealy) and
tenderness,
jaundice, dark urine, and liht feces. Initial la
oratory ndins typically includ
e elevations in

iliru
in and in the liver enzymes, most nota
ly alanine aminotransferase (ALT).
4,5 These
findins are nonspecific indicators of liver in ammation and must
e followed
y s
pe- cific
seroloical or molecular tests to identify the cause of hepatitis more de nitively
. The speci c
la
oratory tests used to detect each type of hepatitis are listed in Ta
le 221. H
epatitis A HAV
is a nonenveloped, sinle-stranded ri
onucleic acid (RNA) virus that
elons
to the
Hepatovirus enus of the Picornaviridae family. 46 It is responsi
le for an esti
mated 1.5
million clinical cases of hepatitis and tens of millions of HAV infections world
wide. 7 HAV is
transmitted primarily
y the fecal-oral route,
y close person-to-person contact
, or
y inestion
of contaminated food or water. 48 Conditions of poor personal hyiene, poor sanit
ation, and
overcrowd- in facilitate transmission. Rarely, transmission throuh transfusion
of contaminated

lood has
een reported and may occur durin a short period within the acute sta
e of infection
when a hih num
er of viral particles can
e found in the source
lood. 8 Follow
in an averae
incu
ation period of 28 days, the virus produces symptoms of acute hepatitis in
the majority of
infected adults; however, most infections in children are asymptomatic. 6,7 The
infection does
not proress to a chronic state and is usually self-limitin, with symptoms t

ypically
resolvin within 2 months. Massive hepatic necro- sis resultin in fulminant hep
atitis and death
are rare and occur mainly in those patients with underlyin liver disease or adv
anced ae. 6 HAV
antiens are shed in the feces of infected individu- als durin the incu
ation p
eriod and the
early acute stae of infection,
ut they usually decline to low levels shortly a
fter symptoms
appear and are not a clinically useful indica- tor of disease. 7 Seroloical tes
ts for anti
ody
are therefore critical in esta
lishin dianosis of the infection. Acute hep- at
itis A is
routinely dianosed in symptomatic patients
y demonstratin the presence of IM
anti
odies to
HAV. 4,5,7 These anti
odies are most commonly detected
y a solid- phase anti
o
dy-capture
enzyme immunoassay (EIA) in which IM in the patient serum is
ound to anti- a
nti
od- ies on
a solid phase and detected after the addition of HAV antien, followed
y an enz
yme-conjuated
anti-HAV IG. 4 IM anti-HAV is usually detecta
le at the onset of clinical symp
toms and declines
to undetecta
le levels within 6 to 12 months. 4,6,7 Tests for total HAV anti
odi
es detect predominantly IG and are availa
le in a competitive inhi
ition EIA format. 4,7 IG
anti
odies
persist for life, and a positive total anti-HAV in the context of a neative IM
anti-HAV
indicates that the patient has developed immunity to the virus, either throuh
natural
infection or vaccination. 6 Several molecular methods have also
een developed
to detect HAV
RNA, the most common format
ein reverse transcriptase polymerase chain reactio
n (RT-PCR). 4,9
These methods may
e used for early detection of HAV in clini- cal samples durin
 out
reaks of
the illness
ut are used more commonly to detect the virus in samples of food or
water suspected
of transmittin the virus. 4,9 A vaccine consistin of formalin-killed HAV was l
icensed in the
mid-1990s to prevent hepatitis A. This vaccine has resulted in a sinificant dec
rease in the
num
er of HAV infections in the United States, to the lowest num
er of cases eve
r recorded. 7,8
While the vaccine was oriinally rec- ommended for only hih-risk individual
s, it is now
recommended for routine immunization of children aed 12 to 23 months. It is als
o recommended for
persons trav- elin to eoraphical areas where hepatitis A is endemic, for homo
sexual men, for
users of illicit drus, for persons workin with HAV-infected primates or with H
AV in a research
la
oratory, for persons who need clottin factor concentrates, and for persons w
ith chronic liver
disease. 8 Routine vaccination is not likely to
e implemented
y the developin
world, where
asymptomatic HAV infection is usually acquired durin childhood and results in l
ife-lon
immunity. 7 Infection with HAV may
e prevented in nonim- munized individuals wh

o have
een
exposed to the virus
y intramuscular injection of immune lo
ulin, a sterile pr
epa- ration of
pooled human plasma that contains anti
odies to HAV, or
y prophylactic administ
ration of the
hepatitis A vaccine. 7,8 1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 372 CHAPT
ER 22 Seroloy
and Molecular Detection of Viral Infections 373 Ta
le 221. Summary of the Hepatit
is Viruses and
Their Associated Seroloical and Molecular Markers PROGRESSION SEROLOGICAL/ HEP
ATITIS TYPE/ TO
CHRONIC MOLECULAR CLINICAL VIRUS FAMILY TRANSMISSION STATE COMPLICATIONS MARKE
RS SIGNIFICANCE
Hepatitis A RNA Fecal, oral No Low risk of fulminant IM anti-HAV Acute hepat
itis A (HAV)
Picornaviridae liver disease Total anti-HAV Immunity to hepatitis A HAV RNA Dete
ction of
HAV in clinical, food, or water samples Hepatitis B DNA Parenteral, sexual, Y
es 10 to 90
percent of HBsA Active hepatitis B (HBV) Hepadnaviridae perinatal cases may de
velop
infection chronic hepatitis HBeA Active hepatitis B (dependin on ae), with
hih deree
with increased risk of infectivity for liver cirrhosis IM anti-HBc Current or
recent and
hepatocellular acute hepatitis B carcinoma Total anti-HBc Current or past hepat
itis B
anti-HBe Recovery from hepatitis B anti-HBs Immunity to hepatitis B HBV DNA Acut
e,
atypical, or occult hepatitis B; viral load may
e used to monitor effectiveness
of therapy
Hepatitis C RNA Parenteral, sexual, Yes Eihty- ve percent Anti-HCV Current or
past (HCV)
Flaviviridae perinatal develop chronic hepatitis C infection, with infection i
ncreased risk of
HCV RNA Current hepatitis C cirrhosis, infection; viral hepatocellular load ma
y
e used
carcinoma, or to monitor autoimmune effectiveness of manifestations therapy; a
lso used to
determine HCV enotype Hepatitis D RNA Mostly parenteral, Yes Increased risk o
f IM-anti-HDV
Acute or chronic (HDV) Genus
ut also sexual, developin hepatitis D Deltavir
us perinatal;
HBV fulminant hepatitis, IG-anti-HDV Recovery from infection cirrhosis, or
hepatitis D
or required hepatocellular chronic hepatitis D carcinoma HDV RNA Active HDV inf
ec- tion;
viral load may
e used to monitor effective- ness of therapy Hepatitis E RNA F
ecal, oral No
Fulminant liver IM anti-HEV Current hepatitis E (HEV) Hepeviridae failure in p
renant
infection women IG anti-HEV Current or past hepatitis E infection HEV RNA Curre
nt
hepatitis E infection 1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 373 374 SECT
ION 4 Seroloical
Dianosis of Infectious Disease Hepatitis E HEV is a nonenveloped, sinle-strand
ed ri
onucleic
acid (RNA) virus that
elons to the enus Hepevirus, in the fam- ily Hepevirid
ae. 4,10,11 Like
HAV, it is transmitted
y the fecal-oral route. Most HEV infections are rel

ated to consumption of fecally contaminated drinkin water in developin reions of A

frica, the Middle
East, Southeast Asia, and Central Asia, all of which have poor sanitation condit
ions. 10,11
Infections in the United States and other industrialized countries are rare
ut
have
een
reported. 12 Followin an incu
ation period of 3 to 8 weeks, HEV causes an acute
, self-limitin
hepatitis that lasts 1 to 4 weeks in most people who
ecome infected. 4,11 Fulmi
nant hepati- tis,
associated with rapidly proressin disease and a hih mortality rate, occurs
more commonly
in prenant women. 11,13 The reason is not totally clear,
ut is presum- a
ly d
ue to chanes in
the immune response that occur durin prenancy. Like HAV, the infection does no
t proress to a
chronic carrier state. There is no treatment for hepati- tis E,
ut a recom
inan
t vaccine to
prevent the infection is currently underoin clinical trials. 11,13 Because HEV
is not easily
cultured, dianosis of it relies on seroloy. Acute infection is indicated
y th
e presence of IM
anti-HEV, which is detecta
le at clinical onset
ut declines rapidly in the earl
y recovery
period. 4,14 These anti-
odies are typically identified
y hihly sensitive enz
yme immunoassays
that use recom
inant and synthetic HEV antiens. Speci city of the assays may
e i
ncreased
y
test- in for IA anti-HEV alon with the IM assays. 10 In patients who
are suspected of
havin hepatitis E
ut who yield a neative IM test, molecular testin for HEV
RNA can
e
performed, typically
y RT-PCR. 4 These assays should
e performed on st
ool samples
collected within 3 weeks of clinical onset. Immunoassays for IG anti-HEV, whic
h persists
loner, may
e performed to determine pre- vious exposure and seroprevalence
of the
infection. 4,14 Seroloical and molecular tests are availa
le to dianose hepati
tis E in some
parts of the world
ut are not licensed in the United States at the time of this
writin.
Hepatitis B Hepatitis B is a major cause of mor
idity and mortality throuhout t
he world. The
World Health Oranization esti- mates that HBV has infected 2
illion people wor
ldwide, causin
360 million chronic infections and
etween 500,000 and 1.2 million deaths each y
ear due to liver
disease. 15,16 The virus is hihly endemic in the Far East, parts of the Middle
East, su
-Saharan
Africa, and the Amazon areas. In the United States, which is considered a low-pr
evalence area,
HBV is responsi
le for 1.25 million chronic infections and over 5,000 deaths ann
ually. 17 HBV is
transmitted throuh the parenteral route
y inti- mate contact with HBV-contamin
ated
lood or
other
ody uids, most nota
ly semen, vainal secretions, and saliva. 5,15,16 Tran
smission has
thus
een associated with sexual contact,
lood transfusions, sharin of needles

and syrines
y
intra- venous dru users, tattooin, and occupational needle-stick injury. Inapp
arent
transmission of HBV may occur throuh close personal contact of
roken skin or m
ucous mem
ranes
with the virus. Transmission of HBV may also occur via the perinatal route, from
infected mother
to infant, most likely durin delivery. Several measures have
een introduced to
prevent HBV
infection, includin screenin of
lood donors, treatin plasma-derived products
to inactivate
HBV, implementin infection-control measures, and, most importantly, immu- nizin
 with a
hepatitis B vaccine. 15 The rst vaccine, licensed in 1982, was composed of an ant
ien called
HBsA (see
elow) that was puri ed from inactivated virus particles in plasma from
HBV-infected
donors. The current vaccines, consistin of recom
inant HBsA produced from ene
ti- cally
enineered yeast, are some of the most widely used vaccines throuhout the world
. In the United
States, hepa- titis B vaccine is administered to healthy infants and children as
part of their
routine immunization schedule and is rec- ommended for hih-risk individuals suc
h as health-care
workers, homosexual men, hemodialysis patients, persons with multiple sex partne
rs, and infants

orn to HBV-infected mothers. 15,18 This immunization stratey has
een hihly s
uccessful,
resultin in a 75 percent decline in the incidence of acute hepatitis B in the U
nited States from
1990 to 2004. 18 The vaccine can also
e administered to individuals thouht to

e exposed to the
virus, alon with HBIG (hepatitis B immune lo
ulin), a preparation derived from
donor plasma
with hih concentrations of anti
odies to HBV, adminis- tered as a means of
passive
immunization to provide temporary protection. 18 Despite the preventative meas
ures that have

een imple- mented, a su
stantial num
er of HBV infections continue to occur, as
discussed a
ove.
Infection with HBV results in an averae incu
ation period of 45 to 90 days, fol
lowed
y a
clinical course that varies in different ae roups. 4,15,19 Over 90 percent o
f new
orns with
perinatal HBV infection remain asymptomatic, while typical symptoms of acute
hep- atitis are
o
served in 5 to 15 percent of children aed 1 to 5 years and in 33 to 50 percen
t of older
children, adoles- cents, and adults. Symptoms may last several weeks to
several months.
Most HBV-infected adults recover within 6 months and develop immunity to the vir
us,
ut a
out 1
percent develop fulminant liver disease with hepatic necro- sis, which has a hi
h rate of
fatality. Development of chronic HBV infection, in which the virus persists in t
he
ody for 6
months or more, is independently related to ae: chronic hepatitis B develops in
a
out 10 percent of infected adults, 25 to 50 percent of youn children, and 80 to 90 percen

t of infected
infants. Chronic infection is also more likely to develop in persons who are imm
unosup- pressed
and those who have HIV. 15 Chronic infection with the virus results in in ammation
and damae to
the liver, and places the patient at increased risk of developin cirrhosis 1814
_Ch22_369-398.qxd
7/13/09 2:58 PM Pae 374 CHAPTER 22 Seroloy and Molecular Detection of Viral
Infections 375
(seen in 20 percent of patients with chronic infection) or hepa- tocellular car
cinoma (100
times the risk of non-HBV carriers). 20 The virus responsi
le for hepatitis
B, HBV, is a DNA
virus
elonin to the Hepadnaviridae family. 4,5,20,21 Eiht enotypes, desina
ted A throuh H,
have
een identi ed; enotype A is the predominant one in the United States and th
rouhout the
world. The intact virion is a 42 nm sphere consistin of a nucleocapsid core sur
rounded
y an
outer envelope of lipoprotein. The core of the virus contains cir- cular partial
ly
dou
le-stranded DNA; a DNA-dependent DNA polymerase enzyme; and two proteins, th
e hepatitis B
core antien and the hepatitis Be antien (HBeA). A pro- tein called the hepati
tis B surface
antien (HBsA) is found in the outer envelope of the virus. HBsA is produced i
n excess and is
found in noninfectious spherical and tu
ular parti- cles that lack viral DNA and
circulate freely
in the
lood. These antiens, or anti
odies to them, serve as seroloi- cal mark
ers for hepatitis
B and have
een used in differential dianosis of HBV infection, in monitorin t
he course of
infection in patients, in assessin immunity to the virus, and in screenin
loo
d products for
infectivity. The levels of these markers vary with the amount of viral replicati
on and the hosts
immune response, and they are useful in esta
lishin the initial dianosis of he
patitis B and in
monitorin the course of infection. Typical patterns of these markers dur- in a
cute and chronic
hepatitis B are shown in Fiures 222 and 22-3 and are descri
ed
elow. 4,5,1921 Th
e hepatitis B
surface antien, or HBsA, is the first marker to appear,
ecomin detecta
le 2
to 10 weeks after
exposure to HBV. Its levels peak durin the acute staes of infection, then rad
ually decline as
the patient devel- ops anti
odies to the antien and recovers. Serum HBsA usual
ly
ecomes
undetecta
le
y 4 to 6 months after the onset of symptoms in patients with acute
hepatitis B. In
patients with chronic HBV infection, HBsA remains elevated for 6 months o
r more. HBsA is
thus an indica- tor of active infection and is an important marker in d
etectin initial
infection, monitorin the course of infec- tion and proression to chronic disea
se, and screenin
of donor
lood. The hepatitis Be antien, or HBeA, appears shortly after HBsA
and disappears
shortly
efore HBsA in recov- erin patients. It may
e elevated durin chronic

infection.
Incu
ation (averae 813 weeks) Acute Infection (2 weeks3 months) Symptoms Duration
HBsA HBeA
anti-HBc Total anti-HBc IM Time anti-HBs anti-HBe Early Recovery (36 months) (612
months)
(Years) Recovery R e l a t i v e
C o n c e n t r a t i o n
FIGURE 222. Typical seroloical markers in acute hepatitis B. 2008 A

ot
La
oratories,
A

ot Park, IL. Used with permission. Acute Infection (6 Months) Duration Chron
ic Infection
(Years) Weeks after exposure Years T i t e r
HBeA Total anti-HBc IM anti-HBc 0 4 8 12 16 20 24 28 32 36 52
anti-HBe HBsA
FIGURE 223. Typical seroloical markers in chronic hepatitis B. 2008 A

ot La
ora
tories, A

ot
Park, IL. Used with permission. 1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 37
5 376 SECTION 4
Seroloical Dianosis of Infectious Disease This marker is present durin period
s of active
replication of the virus and indicates a hih deree of infectivity. The HBcA i
s not detecta
le
in serum,
ecause the viral enve- lope masks it. As the host develops an immune
response to the
virus, anti
odies appear. First to appear is IM anti
ody to the core antien, o
r IM anti-HBc.
This anti
ody indicates current or recent acute infection. It typically appears
1 to 2 weeks
after HBsA durin acute infection and persists in hih titers for 4 to 6 months
and then
radually declines. This marker is useful in detectin infection in cases in whi
ch HBsA is
undetecta
lefor example, just prior to the appearance of anti
odies to the a
ntien, in
neonatal infec- tions, and in cases of fulminant hepatitis. It is therefore used
in addition to
HBsA for the screenin of donor
lood. IG anti
odies to the core antien are p
roduced
efore
IM anti- HBc disappears and then persist for the individuals lifetime. They are
the predominant
anti
odies detected in the test for total anti-HBc, and they can
e used to indi
cate a past HBV
infection. The appearance of anti
odies to the HBe antien, or anti- HBe, occurs
shortly after
the disappearance of HBeA and indicates that the patient is recoverin from HBV
infection.
Anti
odies to HBsA, or anti-HBs, also appear dur- in the recovery period of ac
ute hepatitis B,
a few weeks after HBsA disappears. These anti
odies persist for years and provi
de protective
immunity. Anti-HBs are also pro- duced after immunization with the hepatitis B v
accine.
Protective titers of the anti
ody are considered to
e 10 mIU/mL of seru
m or hiher.
15,18 Anti-HBs are not pro- duced durin chronic HBV infection, in which immunit
y fails to
develop. Seroloical markers for hepatitis B are most commonly detected
y comme

rcial
immunoassays. These are availa
le in a variety of formats, such as enzyme immuno
assay and
chemiluminescent immunoassay. They are typically auto- mated to ease
atch t
estin in the
clinical la
oratory, and have excellent sensitivity and speci city. 4,22 An examp
le of an
immunoassay for detectin HBsA is shown in Fiure 224. Because false-posit
ive results can
occur, any initial positive results should
e veri ed
y repeated testin of the s
ame specimen in
duplicate, followed
y con rmation with an additional assay, such as an HBsA neut
ralization test
or
y a molecular test that detects HBV DNA. Several molecular methods have
een
developed to
detect HBV DNA in serum or plasma and are
ased on taret amplification,
ranch
ed DNA sinal
amplification, hy
ridization, or real-time PCR. 2125 The most sensitive of these
is real-time
PCR, which can detect as few as 10 copies of HBV DNA per ml. 21 HBV DNA can
e d
etected in the
serum a
out 21 days
efore HBsA and may
e a useful adjunct in detectin acute
HBV infection in
certain situa- tions, such as when HBsA test results are equivocal or in cases
of occupational
exposures. 21 There has
een much de
ate a
out the value of testin for HBV DNA
in
lood donors;
however,
ecause of the marinal ain that this testin would provide over the h
ihly sensitive
HBsA assays in place, nucleic acid testin is currently not used to screen
loo
d donors. 26
Other uses of HBV DNA testin are to evaluate the effectiveness of antiviral the
rapy in patients
with chronic hepatitis B, to dianose atypical cases of hep- atitis B oriinatin
 from mutations
in the HBV enome that cause HBsA tests to
e neative, to dianose cases of oc
cult hepatitis B
infection in which patients are positive for HBV DNA
ut neative for seroloica
l markers for
hepatitis B, and to determine the state of virus replication in patients with ch
ronic infection.
20,21,27 HBV enotypin assays have also
een developed. They are currently used
as a research
tool
ut may
e employed in clinical settins in the future to help determine pa
tient
responsiveness to speci c therapies. 4 Hepatitis D Hepatitis D, also known as delt
a hepatitis, is
a parenterally transmitted infection that can occur only in the presence of hepa
titis B. This is

ecause HDV is a defective virus that requires the help of HBV for its r
eplication and
expression. The only mem
er within the Deltavirus enus, HDV consists of a c
ircular RNA
enome and a sinle structural protein called hepatitis delta antien within
its core,
surrounded
y a viral envelope that is of HBV ori- in and contains the HBsA. 4
,5,28,29 In
low-prevalence areas, such as the United States and northern Europe, HDV is lar
ely confined to
intravenous dru users, whereas in endemic areas of the world, such as the Medit

erranean
asin
and parts of South America and Asia, most cases appear to result from inappa
rent parenteral
routes. 4,30 * * * * * *
*
= HBsA = Enzyme-la
eled anti-HBs Solid phase coated with anti-HBs HBsA

inds to anti-HBs
Add patient serum Wash Incu
ate Un
ound proteins removed Add enzyme- la
eled an
ti-HBs conjuate
Wash To remove un
ound conjuate Add su
strate Incu
ate Stop reaction Read a
sor

ance Incu
ate
FIGURE 224. Detection of H
s antien
y chemiluminescent microparticle immunoassa
y.
1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 376 CHAPTER 22 Seroloy and Molecu
lar Detection of
Viral Infections 377 Althouh a
out 20 million people worldwide are
elieved to

e infected with
HDV, the num
er of new infections appears to
e declinin, most likely due to th
e implementation of the hepatitis B vaccine. 4,5 Hepatitis D can either occur as a
co-infection
with hepatitis B, in which infection of HDV and HBV occurs simultaneously, or as
a
superinfection, in which HDV infects individuals who are already chronic HBV car
riers.
Clinically, co-infections usually resem
le infection with HBV alone, with most p
atients
experiencin an acute self-limited hepatitis in which
oth viruses are cleared w
ithin a few
months. 4,30 Only 1 to 3 percent of cases proress to a chronic state. In contra
st,
superinfections result in a reater risk of developin fulmi- nant hepatitis or
chronic liver
disease with an accelerated proression toward cirrhosis, liver decompensation,
and hepatocellular carcinoma. 4,30 Detection of hepatitis D has
een aided tremendously

y the
development of molecular methods to detect HDV RNA, a marker of active vir
al replication
that is present in all types of active hepatitis D infections. 4 HDV RNA is dete
cted
y
reverse-transcriptase PCR assays, which are hihly sensitive, speci c, and quantit
ative. 4,31
These assays can
e used not only to detect HDV infection,
ut also to monitor p
atients durin
antiviral therapy. Hepatitis D infection is also indicated
y the presence of an
ti-HDV in the
patients serum, which can
e detected
y immunoassays employin hepatitis D anti
en. 4,30,31
While IM anti-HDV may
e used to detect acute hepatitis D infections, its appea
rance may
e
delayed, it may persist for only a short period of time, and it may
e missed. 4
,5 In con- trast,
hih titers of IM and IG anti
odies are associated with chronic infection. 4,3
0 Hepatitis C
Hepatitis C is a major pu
lic health pro
lem, havin infected over 4 mil
lion people in
the United States and over 170 million people worldwide. 5,32 It is the cause of

the major- ity

of infections previously classi ed as nonA-nonB
efore the discovery of HCV in 1989.
5
Hepatitis C is transmitted mainly
y exposure to contam- inated
lood, with intr
avenous dru use

ein the main source of infection. 5,33 Blood transfusion was also a major sour
ce of infection

efore 1992, when routine screenin of
lood donors for HCV anti
ody was impleme
nted,
ut testin
has reduced this risk to a
out 0.001 to 0.01 percent per unit of
lood trans- fu
sed. 33 Other
risk factors for acquirin hepatitis C include oran transplantation
efore 1992
, occupational
exposures to contaminated
lood; chronic hemodialysis; and possi
ly intrana
sal cocaine use,

ody piercin, and tattooin. Sexual transmission of HCV is thouht to
e less c
ommon
ut is
hiher in those who have had multiple sex partners or a history of sexually tran
smitted diseases.
5,3335 Perinatal transmission has
een estimated to occur at a rate of a
out 6 pe
rcent. 33,35
HCV has an averae incu
ation period of 7 weeks (rane is 2 to 30 weeks) and pro
duces symptoms of
acute hepatitis in only a
out 20 percent of cases. 5,33,35 Althouh the m
ajority of
infections are asymptomatic, the infection is pro
lematic,
ecause a
out 85 perc
ent of persons
develop chronic infection, which leads to cirrhosis in a
out 20 per- cent of the
se individuals.
33,35 Cirrhosis develops slowly, over 20 to 25 years, causin damae to the live
r and posin an
increased risk of developin hepatocellular carcinoma. End- stae liver disease
related to HCV is
now the leadin cause for liver transplantation. 3437 Patients with chronic HCV i
nfection may
also develop extra-hepatic manifestations, includin mixed essential cryolo
uli
nemia with immune
complexes containin HCV particles; lomerulonephritis, vasculitis, or other aut
oimmune
manifestations; neuropathy; ophthalmoloical symptoms; and dermatoloic manifest
a- tions. 33
Early detection of HCV would help in preventin these complications
ut occur
s infrequently,
due to the asymptomatic nature of the infection in most individuals. Clearance
of the infection
may occur spontaneously or may require treatment with antiviral drus, the stand
ard treat- ment
involvin a com
ination of peylated interferon- and ri
avirin. 4,38,39 HCV is an
enveloped,
sinle-stranded, positive-sense RNA virus
elonin to the family Flaviviridae a
nd the enus
Hepacivirus. 4,5,33 Its enome contains an open readin frame whose 3 end codes f
or the
nucleocapsid core protein (C) and the envelope proteins (E1 and E2/NS1) and whos
e 5 end encodes
nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). 4,5 Scientists ha
ve discovered six
different enotypes of the virus, desinated 1 throuh 6, and numerous su
types
for each,
indicated
y lowercase let- ters. 4,5,33 Genotype 1 is predominant in the United

States. HCV has

a hih mutation rate, which allows it to escape the immune response and persist
in the host. This
has also cre- ated dif culty in developin an effective vaccine. Because HCV is a
dif cult virus
to culture, demonstra- tion of the infection has relied on other la
oratory meth
ods. Both
seroloical tests and molecular tests have
een devel- oped to identify persons
with HCV
infection. Seroloical tests are used in the screenin of
lood and oran donors
for HCV
infection and in the initial dianosis of sympto- matic patients. 4,39,40 This t
estin involves
detection of HCV IG anti
ody
y third-eneration enzyme immunoassays or chemilu
minescent
immunoassay methods, which use recom
inant and synthetic antiens developed from
the con- served
domains of the C, NS3, NS4, and NS5 proteins. 4,41 Improvements in the seroloic
al assays for
anti-HCV over the years have ena
led anti
odies to
e detected earlier than prev
ious
methodsa
out 4 to 6 weeks after infection. 4,5 While the speci city of these method
s is
excellent, false- positive results may occur due to cross-reactivity present in
persons with
other viral infections or autoimmune disor- ders; therefore, any positive re
sults from an
anti-HCV screenin test should
e con rmed. 4,39 The traditional con- rmatory metho
d was the
recom
inant immuno
lot assay (RIBA), which detects anti
odies to different HCV a
ntiens that have

een immo
ilized onto a nitrocellulose strip
y a 1814_Ch22_369-398.qxd 7/13/09
2:58 PM Pae
377 378 SECTION 4 Seroloical Dianosis of Infectious Disease colorimetric react
ion. 35 However,
RIBA has
een replaced in many la
oratories
y molecular methods, which are more
sensitive and
less la
or-intensive. 5,33,36 In recent years, detectin and monitorin HCV infe
ction have
een
sini cantly improved
y the implementation of molecular tests. Molecular assays t
hat detect HCV
RNA have
een developed as qualitative and quantitative tests. Qualitative tests
distinuish

etween the presence or a
sence of HCV RNA in a clinical sample and are used to
con rm infection
in HCV-anti
ody-positive patients, to detect infection in anti
ody-neative pati
ents who are
suspected of havin HCV, to screen
lood and oran donors for HCV, and to detect
peri- natal
infections in
a
ies
orn to HCV-positive mothers. 36 The most commonly used qua
litative method
is reverse tran- scriptase polymerase chain reaction (RT-PCR),
ut a hihly sens
itive
transcription mediated ampli cation (TMA) test has recently
een developed as well
. 36 These
tests can detect HCV infection within 1 to 3 weeks after exposuremuch earlier tha
n seroloical
methods. 36,37 Quantitative tests are performed
y RT-PCR, real-time PCR, or
ra
nched DNA
amplification (
DNA). 36,42 They are used to monitor the amount of HCV RNA, or vi

ral load,
carried
y patients
efore, durin, and after antiviral therapy in chronically i
nfected
individuals. The ultimate oal of such therapy is to achieve a sustained virolo
ical response
(SVR), in which the patient continuously tests ne- ative for HCV RNA 6 months a
fter therapy is
completed. 36,38 The initial viral load level has also
een used as a pronos- t
ic tool, since
those with a low initial viral load are most likely to achieve an SVR. 36,43 Ano
ther type of
molecular assay for HCV is the eno- typin test, which determines the exac
t enotype and
su
type of the virus responsi
le for the patients infection. Genotypin tests are
ideally
performed
y sequence analy- sis, althouh other methods, includin a su
type-sp
ecific RT-PCR and
a line-pro
e assay have also
een developed for this purpose. 4,36 It is importa
nt to determine
the patients HCV enotype in order to determine optimal treatment in terms of the
dose of
antiviral drus administered and the duration of therapy. Studies have shown tha
t patients with
enotypes 1 and 4 require more aressive treatment, with hiher doses of antivi
ral drus
administered for a loner period of time, in contrast to patients with enotypes
2 and 3, who are
more likely to reach an SVR with a shorter course of treatment usin lower d
ru doses. 3638
In the future, molecular tests will most likely play an important role in eval
uatin the
effectiveness of new antiviral drus in erad- icatin this devastatin virus. Vi
ruses with
Uncertain Association with Hepatitis Durin the 1990s, researchers discovered tw
o additional
viruses in the sera of patients with hepatitis who were not infected with any of
the
well-characterized hepatitis viruses descri
ed a
ove. 4,4446 These viruses, terme
d hepatitis G
virus (HGV, also known as GB virus type C or GBV-C) and tor- quetenovirus (TTV,
also known as
transfusion-transmitted virus), are prevalent throuhout the world in persons wi
th or without
clinical hepatitis,
ut their role as etioloic aents of hepatitis remains unde
r de
ate. 4,4749
HGV is an enveloped, sinle-stranded RNA virus in the Flaviviridae family. 4,48
It is transmitted

y the
lood
orne route, perinatal route, and possi
ly throuh sexual contact. D
etection of the
virus has
een
ased primarily on RT-PCR methods, which amplify HGV RNA. 4,50 Se
roloy tests

ased on EIA have also
een developed and, like RT-PCR, are used larely as a re
search tool. 4,50
Studies indicate that anti-
odies to the HGV envelope protein E2 may
e associa
ted with recovery
from hepatitis G infection. TTV is a sinle-stranded non-enveloped DNA virus
el
onin to the
enus Annellovirus. 4,49 Parenteral transmis- sion of the virus throuh con
taminated
lood
has
een clearly evidenced, and presence of the virus in stool,
ile, and saliv

a, suest
transmission
y the fecal-oral and respi- ratory routes as well. Detection of TT
V is performed
y
PCR, primarily for research purposes. 4,51 The selection of primers for the PCR
is especially
important in in uencin the test results, since the virus has a tremendous amount
of enetic
diversity, with over 30 different enotypes discov- ered. To date, there are no
validated
seroloical tests to detect anti
ody to TTV. 4,49 HERPES VIRUS INFECTIONS The he
rpes viruses are
lare, complex DNA viruses that are surrounded
y a protein capsid, an amorphous
teument, and an
outer envelope. 52,53 These viruses are all capa
le of esta
lishin a latent inf
ection with
lifelon persistence in the host. The Herpesviridae family includes eiht viruse
s that can cause
disease in humans: the herpes simplex viruses (HSV-1 and HSV-2); varicella-zoste
r (also known as
human herpes virus-3 or HHV-3); the Epstein-Barr virus (HHV-4); cytomealovirus
(HHV-5); and the
human herpes viruses 6, 7, and 8 (HHV-6, HHV-7, and HHV-8), the latter of which
has
een
associated with Kaposis sarcoma. This chap- ter covers clinical manifestations an
d la
oratory
dianosis of some of these viruses. Epstein-Barr Virus The Epstein-Barr virus (E
BV) causes a wide
spectrum of dis- eases, includin infectious mononucleosis, lymphoproliferative
disease, and
several malinancies. 5456 EBV infections most commonly result from intimate cont
act with
salivary secre- tions from an infected individual. While transmission of the vir
us can occur
throuh other mechanisms, such as
lood transfusions,
one marrow and solid ora
n transplants,
sexual contact, and perinatal transmission, these routes appear to
e much less
frequent. 5457
In developin nations of the world and lower socioeco- nomic roups livin under
poor hyiene
conditions, EBV 1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 378 CHAPTER 22 Ser
oloy and
Molecular Detection of Viral Infections 379 infections usually occur durin earl
y childhood,
whereas in industrialized nations with hiher standards of hyiene, infections a
re typically
delayed until adolescence or adult- hood. However,
y adulthood, more than 90 pe
rcent of
individuals have
een infected, as evidenced
y the presence of EBV anti
odies i
n their serum.
EBV selectively infects host cells that are positive for the CD21 molecule, whic
h serves as a
receptor for
oth the virus and the C3d protein of complement. 53,55 The virus i
nitially infects
epithelial cells in the oropharynx, where it enters a lytic cycle, characterized

y viral replication, lysis of host cells, and release of infectious virions until the acute i
nfection is
resolved. 54 The virions also infect B lymphocytes, which spread the virus throu
hout the
lymphoreticular system. The virus-infected B cells
ecome polyclonally activated

, proliferatin
and secret- in a num
er of anti
odies, includin EBV-specific anti
odies;
heterophile
anti
odies; and autoanti
odies such as cold alutinins, rheumatoid factor, and
antinu- clear
anti
odies. 5456 In healthy individuals, this process is kept in check
y the imm
une response of
natural killer cells and specific cytotoxic T cells. However, EBV can persist in
the
ody
indefinitely in a small percentae of B cells, in which it esta
lishes a latent
infection.
Periodic reactivation of the virus results in viral sheddin into the saliva and
enital
secretions, even in healthy, asympto- matic individuals. 5456 Several antiens ha
ve
een
identified in EBV-infected cells that are associated with different phases of th
e viral
infection, and anti
odies to these antiens have
ecome an important dianostic
tool. 5456
Antiens produced durin the initial staes of viral replication in the lytic cy
cle are known as
the early antiens (EA). These antiens can
e fur- ther classi ed into two roups

ased on their
location within the cells: EA-D, which has a diffuse distri
ution in th
e nucleus and
cytoplasm, and EA-R, which is restricted to the cytoplasm only. The late antien
s of EBV are
those that appear durin the period of the lytic cycle followin viral DNA synt
hesis. They
include the viral capsid antiens (VCAs) in the protein capsid and the mem
r
ane antiens in
the viral envelope. Antiens appearin durin the latent phase include the EBV n
uclear antien
(EBNA) proteins, EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, and EBNA-LP, and t
he latent
mem
rane proteins (LMPs), LMP-1, LMP-2A, and LMP-2B (Ta
le 222). The clinical m
anifestations
of EBV vary with the hosts ae and immune status. Infections in infants and youn
chil- dren are
enerally asymptomatic or mild, while primary infections in healthy adolescents
or adults
commonly result in infectious mononucleosis (IM). 55-59 More than half of patien
ts with IM
present with three classic symptoms: fever, lymphadenopathy, and sore throat. Ot
her symptoms,
includ- in splenomealy, hepatomealy, and perior
ital edema, may also
e seen.
Symptoms usually
last for 2 to 4 weeks,
ut fatiue, myalias, and need for sleep can persist for
months. Althouh
these symptoms are essential in dianosin IM, they can also
e caused
y many o
ther infectious
aents, so la
oratory testin plays an important role in differentiatin IM from
other
infections. Characteristic la
oratory
ndins in patients with IM include an
a
solute
lymphocytosis of reater than 50 percent of the total leukocytes and at least 10
percent atypical
lym- phocytes (which are predominately activated cytotoxic T cells). 55,58,
59,60 Seroloical
ndins include presence of a het- erophile anti
ody and anti
odies to certain EBV

antiens. By
de nition, heterophile anti
odies are anti
odies that are capa
le of reactin with
similar
antiens from two or more unrelated species. The heterophile anti
odies associated with IM are
IM anti
odies produced as a result of polyclonal B-cell activation and are capa

le of reactin
with horse red
lood cells, sheep red
lood cells, and
ovine red
lood cells. T
hese anti
odies
are produced
y 40 percent of patients with IM durin the rst week of clinical il
lness and
y up
to 90 percent of patients
y the fourth week. 55 They disappear in most patients

y 3 months
after the onset of symptoms
ut can
e detected in some patients for up to 1 yea
r. 55 Because the
heterophile anti
ody is present in most patients durin the acute phase of illne
ss, testin for
this anti
ody is typically performed as a screenin test for IM in patients who
present with
symptoms of the disease. For many years, the heterophile anti
ody of IM
was detected
y a
rapid slide alutination method called the Monospot, which tested the a
ility of
serum
a
sor
ed with uinea pi kidney or
eef erythrocyte antiens to alu- tinate ho
rse red
lood
cells. The anti
ody could then
e titered
y incu
atin serial dilutions of the
patients serum
with sheep red
lood cells in the Paul-Bunnell test. These methods have
een rep
laced today
y
rapid latex alutina- tion tests or solid phase immunoassays usin puri ed
ovine
red
lood cell
extract as the antien. Studies have shown that the sensitivity of these kits va
ries from 71 to
95 percent in adults
ut is sini cantly lower in children under 12 years (25 to 5
0 percent). 59
The speci city of the kits ranes from 82 to 99 percent, dependin on the assay. 5
9
False-positive results, althouh uncommon, can occur in patients with lym- phoma
, viral
hepatitis, malaria, and autoimmune disease. 55 Neative heterophile anti
ody
results occur
in a
out 10 percent of adult patients with IM and up to 50 percent of Ta
le 552 Comparison of
Tests Used EARLY ACUTE PHASE LATE PHASE LATENT PHASE EA-R (early antien VCA (v
iral capsid EBNA
(EBV restricted) antien) nuclear antiens) EA-D (early antien MA (mem
rane E
BNA-1, -2, -3A,
diffuse) antien) -3B, -3C, -LP Latent mem
rane proteins (LMP-1, -2A, -2B) Ta
le
222.
Epstein-Barr Virus Antiens 1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 379 38
0 SECTION 4
Seroloical Dianosis of Infectious Disease children less than 12 years old
. 59 In these
patients, who demonstrate symptoms of IM
ut are neative for the het- erophile
anti
ody,
further testin for EBV-speci c anti
odies is indicated. 54,55,59 These anti
odies
are most often
detected
y indirect immunofluorescence assays (IFA) usin EBV- infected cells o
r ELISA
techniques usin recom
inant or synthetic EBV proteins. 54,59 While
oth methods

have a hih
level of sensitivity (95 to 99 percent), IFA tests have a hiher level of speci ci
ty and are
considered the old standard of EBV seroloy methods. However, many la
oratories p
refer ELISA
tests,
ecause they are less time-consumin and easier to interpret. 54 Mo
re recently,
methods employin chemiluminescence-
ased detection of the anti
odies have also

ecome availa
le.
58,61 IM anti
ody to the VCA is the most useful marker for acute IM,
ecause it
usually appears
at the onset of clinical symptoms and disappears
y 3 months. 54,55 IG
anti-VCA is also
present at the onset of IM
ut persists for life and can thus indicate a past in
fection.
Anti
odies to EA-D are also seen durin acute IM, and anti- EBNA appears durin
 convalescence.
54,55 A summary of seroloical responses durin acute, convalescent, and postIM is shown in
Ta
le 223. Some individuals develop chronic active EBV infection, with severe, of
ten
life-threatenin IM-associated symptoms that persist or recur for months after t
he acute illness.
60,62 In addition, EBV has
een associated with several malinancies,
oth hemat
oloic (e..,
Burkitts lymphoma and Hodkins disease) and nonhematoloic (e.., nasopharyneal c
arcinoma and
astric carcinoma). 55,58,60 EBV can also cause lymphopro- liferative disorders
in
immunocompromised patients, includin central nervous system lymphomas in patie
nts with AIDS,
X-linked lymphoproliferative disease in males with a rare enetic mutation, and
post-transplant
lymphoprolifera- tive disorders (PTLD) in patients who have received hema
topoietic stem
cell or solid oran transplants. 55,60,63 These disorders result from the ina
il
ity of
immunosuppressed patients to control primary EBV infection, leadin to mas- sive
polyclonal
expansion of the EBV-infected B cells and life-threatenin illness with a hih r
ate of mortality.
EBV- associated malinancies can
e dianosed with the help of seroloy tests fo
r EBV anti
odies,
immunohistochemical tests to detect EBV antiens in tissue
iopsies, and molecul
ar methods to
detect EBV DNA in
lood and tissue sam- ples. 54,55,60,63,64 Typical patt
erns of EBV
anti
odies seen in some of these disorders are shown in Ta
le 223. Molecular test
s may
e more
relia
le than seroloy in immunocompromised patients who may not demonstrate a 
ood humoral
response, and they are also useful in monitorin viral load in patients with EBV
-related
malinancies who are underoin therapy. Cytomealovirus Cytomealovirus (CMV)
is a u
iquitous
virus with worldwide distri
ution. Nearly all persons have
een exposed

y their elderly
years,
ut communal livin and poor personal hyiene facilitate spread earlier i
n life. 65,66 CMV
is spread throuh close, proloned contact with infectious
ody secretion

s; intimate
sexual contact;
lood transfusions; solid oran transplants; and perinatally, fr
om infected
mother to infant. The virus has
een isolated in saliva, urine, stool, vainal a
nd cervical
secretions, semen,
reast milk, and
lood. 6567 Primary, or initial, infections i
n healthy
individuals are usually asymptomatic
ut result in a self-limitin, heterophile
anti
ody-neative IM-like illness with fever, myalias, and fatiue in a sma
ll percentae of
cases. 66,67 An immune response aainst the virus is stimulated,
ut the virus p
ersists in a
latent state in monocytes, dendritic cells, myeloid proenitor cells, and periph
eral
lood
leukocytes and may
e reactivated at a later time in the individuals life. 68 Ta

le 223.
Seroloical Responses of Patients with Epstein-Barr VirusAssociated Diseases ANTI
-VCA ANTI-EA
HETEROPHILE CONDITION IM IG IA EA-D EA-R ANTI-EBNA ANTIBODY (IM) Uninfected

IM
Convalescent IM
Past
infection IM
Chronic active infection IM
Post-transplant lymphoproliferative disease
Burkitts lymphoma
Nasopharyneal carcinoma
Adapted fr
Straus, SE, et al. Epstein-Barr virus infections: Bioloy, pathoenesis, and man
aement. Ann
Intern Med 118:45, 1993, with permission. VCA
viral capsid antien; EA
early ant
ien; EBNA
EBV nuclear antien; IM
infectious mononucleosis 1814_Ch22_369-398.qxd 7/13/09
2:58 PM Pae
380 CHAPTER 22 Seroloy and Molecular Detection of Viral Infections 381 The clin
ical consequences
of CMV infection are much more serious in the immunocompromised host, most
nota
ly
oran-transplant recipients and patients with AIDS. CMV is the most important in
fectious aent
associated with oran transplantation, with infections resultin from reac- tiva
tion of CMV in
the recipient or transmission of CMV from the donor. 66,67,69 CMV infection of a
previously unexposed recipient from the donor oran poses a hih risk for symptomatic disease,
which can induce
a variety of syn- dromes (e.., fever and leukopenia, hepatitis, pneumonitis, a
strointestinal
complications, and retinitis) and is associ- ated with an increased risk fo
r alloraft
failure. While com
ination antiretroviral therapy has reduced the inci- dence o
f CMV-related
illness in patients with HIV infection, CMV remains a major opportunistic patho
en in patients
with low CD4 T cell counts, causin retinitis or dissem- inated disease involvin
the liver,
luns, astrointestinal tract, or central nervous system. 66,68 CMV is also t
he most common
cause of conenital infections, occurrin in approximately 1 percent of
all neonates.
67,70,71 Transmission of the virus may occur throuh the placenta,
y passae of
the infant
throuh an infected
irth canal, or
y postnatal contact with
reast milk or oth

er maternal
secretions. 66 A
out 10 percent of infants with con- enital CMV infection are s
ymptomatic at

irth. 71 Mothers who acquire primary CMV infection durin their prenancy have
a sini cantly
hiher risk of ivin
irth to a sympto- matic or severely affected infant than
do women in whom
CMV was reactivated durin prenancy. Symptomatic infants present with a multitu
de of symptoms
that re ect platelet dysfunction and central nervous system involvement, includ- i
n petechiae,
jaundice, hepatosplenomealy, and neuroloical a
normalities such as microcephal
y and lethary,
with an asso- ciated mortality rate of a
out 5 percent. 65,67,71 Over one-half o
f the survivin
infants will develop clinical sequelae, such as hearin loss, visual impairment,
and mental
retardation, in their early childhood years. While 90 percent of infants with co
nenital CMV
infection are asymptomatic at
irth, a
out 10 percent of these will develop sens
orineural hearin
loss that is slowly proressive durin the rst 5 to 10 years of life, and a small
er percentae
may develop other a
normalities such as neuromuscular defects and chorioretiniti
s. 67,71 Several
la
oratory methods have
een developed to detect CMV infection, includin viral
culture, viral
antien assays, molecular assays, and seroloy. Isolation of the virus in cul- t
ure, or
demonstration of CMV antiens or DNA from appropriate clinical specimens,
are the preferred
dianostic methods. 66,68 For example, the standard reference method for detecti
n conenital CMV
infection is to isolate the virus from the urine or saliva of the neonate within
3 weeks of

irth. 67,68 The traditional method of viral culture involves o
servin characte
ristic cytopathic
effects (CPE) in human
ro
last cell lines inoculated with CMV-infected specimens
. While this
method provides de nitive results when positive, it is limited,
ecause CPE do not
appear until a
few days to several weeks after inoculation, dependin on the viral titer. Imple
mentation of the
rapid centrifuation-enhanced (shell vial) method has reduced the time of detect
ion to 16 to 72
hours after inoculation. 72 This assay is an immuno uo- rescence method that uses
monoclonal
anti
odies to detect immediate early CMV antiens in infected cells rown on cov
erslips in shell
vials. Detection of the CMV lower matrix protein pp65 in CMV-infected leukocytes
in the
peripheral
lood or cere
ral spinal fluid
y immunocytochemical or immunofluores
cent stainin has
allowed for more rapid dianosis and treatment of CMV infection in immunocom- pr
omised patients.
66,68,73 Hihly sensitive molecular methods are increasinly
ein used to
detect CMV
infection in conjunction with, or as an alternative to, culture methods. 68 PCR
ampli cation of
CMV DNA has
een extremely useful for detectin CNS infections in immunode cient h

osts, for
detectin CMV in amniotic uid, and for esta
lishin the dianosis of CMV infectio
n in transplant
recipients. 67,72,74 Quantitative PCR, which detects CMV copy num
er in the peri
pheral
lood, is
used to monitor the effectiveness of antiviral treatment in immunocompromised ho
sts and to
identify patients at risk for developin disseminated CMV disease. 67,68,74 The
usefulness of
this assay in predictin pronosis in coneni- tal CMV infections is
ein inves
tiated. 67,72
While seroloy tests for CMV have
een commercially availa
le for many years, th
eir clinical
utility is limited. The seroloy methods performed most commonly are semi- or fu
lly automated
enzyme immunoassays (EIAs) that employ microtiter plates or microparticle system
s. 68 Assays for
CMV IG are most useful in documentin a past CMV infection in healthy individua
ls, such as
lood
and oran donors, in order to reduce post-transfusion/post-transplant primary CM
V infection in
sero-neative recipients. 68 A fourfold rise in anti
ody titers of serial serum
specimens in
persons older than 6 months of ae suests a recent infection, 50,51
ut rapid
detection of
infection is not possi
le
y this process. Assays for IM CMV anti
odies have
e
en developed
ut
are limited in value
ecause of the potential for false-neative results in new

orns and
immunocompromised patients and for false-positive results due to other infection
s or the presence of rheumatoid factor. 5052 In addition, IM anti
odies may not necessarily
indicate
primary CMV infection,
ecause they can also
e produced as a result of CMV re
ac- tivation and
may persist for up to 18 months. 47,50 Seroloical methods that use recom
inant
antiens to
detect CMV anti-
odies of different avidities are
ein developed in an attempt
to increase the
sensitivity and speci city of tests for CMV disease. 47,51 Because of the limitati
ons of seroloy
testin, direct methods of detectin CMV infection are essential. Varicella-Zost
er Virus The
varicella-zoster virus (VZV) is the cause of two dis- tinct diseases: varicella,
more commonly
known as chickenpox, and herpes zoster, or shinles. The virus is transmitted pr
i- marily
y
inhalation of infected respiratory secretions or aerosols from skin lesions asso
ciated with the
infection; transplacental transmission to the fetus may also occur. 7577 1814_Ch2
2_369-398.qxd
7/13/09 2:58 PM Pae 381 382 SECTION 4 Seroloical Dianosis of Infectious Dis
ease Primary
infection with VZV results in chickenpox (i.e., varicella), a hihly conta
ious illness
characterized
y a
listerlike rash with intense itchin and fever. 7578 H
istorically,
the majority of varicella cases have occurred durin childhood. In a typical inf
ection, vesicular
lesions rst appear on the face and trunk, then spread to other areas of the
ody.

Over a period
of hours to a few days, the skin lesions evolve from maculopapules to uid-contain
in vesi- cles
and finally
reak down to form a sca
. The illness is usually mild and self-limi
tin in healthy
children
ut may in some cases produce complications, the most common of which a
re secondary

acterial skin infections due to scratch- in of the lesions. Primary infections
in adults,
neonates, or prenant women tend to
e more severe, with a larer num-
er of le
sions and a
reater chance of developin other complications such as pneumonia. Varicella in
fection in
prenant women may also cause premature la
or or con- enital malformations if t
he infection is
acquired durin the first trimester of prenancy or may cause severe neonatal in
fection if
transmission of the virus occurs around the time of delivery. 7578 Infections in
immunocompromised patients are more likely to result in disseminated disease, wi
th exten- sive
skin rash, neuroloical conditions (e.., encephalitis), pneumonia, or hepatitis
. 7578 Durin
the course of primary infection, VZV is thouht to travel from the skin to the s
ensory nerve
endins to the dorsal anlion cells, where it esta
lishes a latent state. 78 Re
activation of the
virus occurs in 15 to 30 percent of per- sons with a history of varicella infect
ion, pro
a
ly as
a result of a decrease in cell-mediated immunity; the num
er of cases increases
with ae or
development of an immunocompro- mised condition. 79 Reactivation results in the
virus movin down
the sensory nerve to the dermatome supplied
y that nerve, resultin in eruption
of a painful
vesicular rash known as herpes zoster, or shinles, in the affected area. 7779 Th
e rash may
persist for weeks to months and is more severe in immunocompromised and
elderly
individuals. A sini cant num
er of patients with herpes zoster develop complicati
ons, the most
common
ein postherpetic neuralia, character- ized
y de
ilitatin pain that p
ersists weeks,
months, or even years after resolution of the infection. 77,79 Life-threatenin
complications
such as herpes ophthalmicus leadin to
lind- ness, pneumonia, and visceral invo
lvement are more
common in immunosuppressed persons. In 1995, a vaccine consistin of a strain of
live, attenuated varicella virus was licensed in the United States for use in children aed
12 months or
older, adolescents, and adults. Implementation of the vaccine has resulted in a
sini cant
decline in the incidence of chickenpox and its associated complications. 77,80 H
owever,

reakthrouh cases of varicella in vaccinated persons have
een reported. 77,81
Althouh most
patients experience mild illness, they are still contaious, and some individual
s may experience
more severe disease. This has prompted the CDC to recommend addin a
oo
ster dose of the

vaccine to routine childhood immuniza- tion schedules. 77 In 2005, a vaccine was

licensed for use
in healthy children that com
ines the varicella vaccine with that for measles, m
umps, and
ru
ella. 77 In addition, a sinle- aent VZV vaccine was licensed in 2006 for pr
evention of
herpes zoster in persons aed 60 or older, presuma
ly
y
oostin the immune res
ponse. 79,82
Because these vaccines all contain a live aent, they are not recommended for us
e in
immunocompromised persons. These patients should receive an antiviral dru or in
jections of
varicella immune lo
ulin within 96 hours after exposure to the virus. 77 When v
aricella was
commonplace in the United States, dianosis of the disease was
ased primarily o
n clinical ndins, and la
oratory testin was usually unnecessary. Because the incidence of v
aricella has
decreased as a result of immu- nization, la
oratory dianosis has
ecome an incr
easinly
important tool in reconizin the infection when it does occur. 83 De nitive dian
osis is
ased
on identifyin VSV or one of its products in skin lesions, tissue, or vesicular u
ids. Rapid
identi cation of the virus can
e performed
y micro- scopic examination of smears
made from the

ase of the vesicles and stained with hematoxylin-eosin, Wriht- Giemsa, to
luidine
lue, or
Papanicolaous stain to reveal multinucleated iant cells called Tzanck cells; how
ever, this
procedure cannot distinuish
etween VZV and herpes simplex virus (HSV). 83,84 C
ulture of the
virus and o
ser- vation of characteristic CPE may
e performed in a num
er of ce
ll lines
ut is
time-consumin and may not yield pro- ductive results if clinical specimens do n
ot contain
suf cient infectious virus. 83 Direct immunofluorescence stainin of scrapins fro
m vesicular
lesions with monoclonal anti
od- ies directed aainst VZV antiens provides a mo
re rapid and
sensitive means of detectin the virus. 83 The most accurate and sensitive metho
d of detectin
the infection is throuh the use of PCR to detect VZV DNA from clinical speci- m
ens. 83 PCR usin
cere
ral spinal fluid is also helpful in detectin VZV in atypical manifestation
s of the
infection involvin the central nervous system. 85 Seroloy testin is of limite
d use in
detectin current infections,
ecause accurate detection requires demonstra- tio
n of a fourfold
rise in anti
ody titer
etween acute and convalescent samples, a process that ta
kes 2 to 4 weeks
to perform. 75,83 In addition, testin for VZV IM is not per- formed routinely
for several
reasons: IM anti
odies to VZV may not
e detecta
le until the convalescent sta
e of illness,
they cannot distinuish
etween primary and reactivated infection, and they may
not
e free of
IG anti
odies when serum is processed for testin. 83,84 Seroloy is most usefu
l in determinin

immunity to VZV in individuals such as health-care workers and in identify- in

VSV-suscepti
le
persons in out
reak settins who may
enefit from prophylactic treatment. 83 The
refore, most
seroloy tests detect total VZV anti
ody, which consists pri- marily of IG. Sev
eral methods have

een developed for this purpose. Rapid tests for point-of-care testin include l
atex
alutination and mem
rane-
ased EIAs. While these have ood sensitivity, the la
tex
ead method
may
e prone to false-positive results. 83 The most sensitive and relia
le metho
d of detectin
VZV anti
ody is a fluorescent test called FAMA ( uorescent anti
ody to mem
rane an
tien)
1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 382 CHAPTER 22 Seroloy and Molecu
lar Detection of
Viral Infections 383 that detects anti
ody to the envelope lycoproteins of the
virus. 83 While
FAMA is considered to
e the reference method for VZV anti
ody, it require
s live,
virus-infected cells and is not suita
le for lare-scale routine testin. An ELI
SA method that
detects an anti
ody to a hihly puri ed solution of a VZV envelope lycoprotein ha
s
een developed and has a sensitivity and specificity compara
le to FAMA. 83,86 This method
is more
sensitive than older ELISA methods that employ a whole antien extract and is li
kely to
e useful
in clinical settins. VIRAL INFECTIONS OF CHILDHOOD Ru
ella The ru
ella virus is
a
sinle-stranded, enveloped RNA virus of the enus Ru
ivirus,
elonin to the fa
mily Toaviridae.
8790 It is transmitted throuh respiratory droplets or throuh transplacental inf
ection of the
fetus durin prenancy. This virus is the cause of the typically
enin, self-li
mited disease
that is also known as German measles. Prior to wide- spread use of the ru
ella v
accine, this was
mainly a disease of youn children. However, today it occurs most often in youn
, unvaccinated
adults. 87,91 Followin an incu
ation period of 12 to 23 days, the virus replica
tes in the upper
res- piratory tract and cervical lymph nodes, then travels to the
loodstream. I
t produces a
characteristic erythematous, mac- ulopapular rash, which appears rst on the face,
then spreads
to the trunk and extremities and usually resolves in 3 to 5 days. 8789 In adolesc
ents and
adults, this is usually preceded
y a prodrome of low-rade fever, malaise, swol
len lands, and
upper respiratory infection lastin 1 to 5 days. However, up to 50 percent of ru

ella infections
are asymptomatic. 72,88 The infection usually resolves without complications. A
si- nificant
num
er of infected adult women experience arthralias and arthritis,
ut ch
ronic arthritis
is rare. 87 Other clinical manifestations, includin encephalitis, throm
ocy- to
penia with
hemorrhae, and neuritis, are infrequent. 87 Ru
ella infection durin prenancy
may have severe

con- sequences, includin miscarriae, still
irth, or conenital ru
ella syndrom

e (CRS). 8789,92
The likelihood of severe con- sequences increases when infection occurs earlier
in the prenancy,
especially durin the rst trimester. Infants
orn with CRS may present with a num

er of
a
normalities, the most common of which are deafness; eye defects, includin cat
aracts and
laucoma; cardiac a
normalities; mental retar- dation; and motor disa
ilities. I
n mild cases,
symptoms may not
e reconized until months to years after
irth. A vaccine cons
istin of live,
attenuated ru
ella virus was developed with the primary oal of preventin infec
tion of prenant
women
y preventin dissemination of the virus in the population as a whole. 88,
93 The vaccine is
part of the routine immunization schedule in infants and children and is usually
iven in
com
ination with vaccines for measles and mumps (measles/mumps/ru
ella [MMR] vac
cine) and
possi
ly with varicella (MMRV). Followin licensure of the vaccine in 1969, the
num
er of ru
ella
infections and cases of CRS in the United States has dropped dramatically, with
only limited
out
reaks occurrin, mostly amon unvacci- nated youn immirants to this countr
y. La
oratory
testin is helpful in confirmin suspected cases of German measles, whose sympto
ms may mimic
those of other viral infections. It is essential in the dianosis of CRS and in
the determination
of immune status in other individuals. Ru
ella virus can
e rown in cultures in
ocu- lated with
respiratory secretions or other clinical specimens; however, rowth is slow and
may not produce
characteris- tic cytopathic effects upon primary isolation, requirin at least t
wo successive
su
passaes. 72,89,90 Demonstration of the virus may also
e accomplished
y its
a
ility to
interfere with the rowth of another virus added to the culture and
y neutraliz
ation of this
effect with speci c ru
ella anti
ody; however, this method is also time-consumin
and la
orintensive. For these reasons, viral culture is not routinely used to dianose ru

ella infections,
and seroloical testin is the method of choice. 72,89,90 Several methods have

een developed to
detect ru
ella anti-
odies, includin hemalutination inhi
ition (HI), passive
hemalutination, complement xation, latex alutination, and immunoassays. 72,90
Althouh HI
was once the standard technique for measurin ru
ella anti
odies, the most commonly used
method today is the ELISA
ecause of its sensitivity, speci city, ease of p
erformance, and
adapta
il- ity to automation. 72,88,90 More speci c solid-phase capture ELISAs can

e used to
detect IM ru
ella anti
odies. Newer immunoassays to detect ru
ella anti
odies e
mploy methods
such as chemiluminescence and immuno uorescence and can
e run on autoanalyzers. 9
0,94 EIAs that
use dried venous
lood spots or oral uid samples have also
een developed and may


e useful in
the dianosis of CRS, especially in resource-poor countries. 95,96 Seroloy test
s can
e used in

oth the dianosis of ru
ella infections and in screenin for ru
ella immunity.
IM and IG
anti
odies to ru
ella appear as the rash of German measles
eins to fade. 90 I
M anti
odies
enerally decline
y 4 or 5 weeks
ut may persist in low levels for a year or mo
re in some cases.
72 IG anti
odies provide immunity and persist for life. Primary ru
ella infecti
on is indicated

y the presence of ru
ella-speci c IM anti
odies or
y a fourfold rise in ru
ella
-speci c IG
anti
ody titers
etween acute- and convalescent-phase samples. 87,88,90 Presence
of IG anti
odies indicates immunity to ru
ella as a result of natural infection or immun
ization. An
anti
ody level of 10 to 15 IU/mL is considered to
e protective. 72 While I
M assays have a
hih level of sensitivity and speci- city, 90,94 false-positive results can occur
in individuals
with other viral infections, heterophile anti
ody, or rheumatoid factor, and in
some cases, IM
titers may persist lon after primary infection or immunization. 72,88 It has th
erefore
een
recommended that positive IM results, particularly in pre- nant women,
e con rm
ed
y a more
speci c test, such as an enzyme immunoassay that measures the avidity of ru
ella I
G anti
odies,
in order to distinuish
etween recent and 1814_Ch22_369-398.qxd 7/13/09 2:58
PM Pae 383 384
SECTION 4 Seroloical Dianosis of Infectious Disease past ru
ella infections. 7
2,97,98 In these
assays, low anti
ody avidity indicates a recent infection, while hih avidity is
seen in past
infections, re ectin the normal chane in anti
ody avidity durin the course of a
n immune
response. La
oratory dianosis of conenital ru
ella infection
eins with
seroloical
evaluation of the mothers anti
od- ies and measurement of ru
ella-speci c IM anti

odies in
fetal
lood, cord
lood, or neonatal serum, dependin on the ae of the fetus/in
fant. To enhance
the relia
ility of a CRS dianosis, any positive IM results should
e con rmed
y
viral culture;

y demonstration of persistently hih titers of ru
ella IG anti
odies after 3 t
o 6 months of
ae; or, more commonly now,
y reverse transcription and PCR- ampli cation of ru
e
lla nucleic
acid (RT-PCR). 72 RT-PCR is a hihly sensitive and speci c aid in prenatal or post
natal dianosis
and can
e used to detect ru
ella RNA in a vari- ety of clinical samples, includ
in chorionic
villi, placenta, amniotic uid, fetal
lood, lens tissue, products of concep- tion
, pharyneal
swa
s, spinal uid, or
rain tissue. 72,90,99 Ru
eola The ru
eola virus is a sinl
e-stranded RNA
virus
elonin to the enus Mor
illivirus in the Paramyxoviridae family. 89,100
,101 It is spread

y direct contact with aerosolized droplets from the respiratory secretions of i

nfected
individuals. Ru
eola virus is the cause of the disease commonly known as
measles.
Followin an incu
ation period of a
out 10 to 12 days, the virus produces prodro
mal symptoms of
fever, couh, coryza (runny nose), and conjunctivitis, which last 2 to 4 days. 8
9,100,101 Durin
the prodromal period, char- acteristic areas known as Koplik spots appear on the
mucous mem
ranes
of the inner cheeks or lips; these appear as ray- to-white lesions aainst a
r
iht red

ackround and persist for several days. The typical rash of measles appears a
o
ut 14 days after
exposure to the virus and is characterized
y an erythematous, maculopapular eru
ption that
eins
on the face and head and spreads to the trunk and extremities, and lasts 5 to 6
days. Measles is
a systemic infection that can result in compli- cations. These are most common i
n adults,
children less than 5 years of ae, and immunocompromised persons, and include di
arrhea, otitis
media, croup,
ronchitis, pneumo- nia, and encephalitis. 89,100,101 Rarely, a fa
tal deenerative
disease of the central nervous system, called su
acute scleros- in panencephal
itis (SSPE), can
result from persistent replication of measles virus in the
rain, with onset
of symp- toms
typically appearin 7 to 10 years after primary measles infection. 101,102 Measl
es infection
durin prenancy results in a hiher risk of premature la
or, spontaneous a
orti
on, or low
irth
weiht,
ut unlike ru
ella, it is not associated with a de ned pattern of conenit
al
malformations in the new
orn. 101 The incidence of measles has
een reatly redu
ced in developed
nations of the world since the introduction of a live, attenuated measles virus
vaccine. A
vaccine consistin of killed ru
eola virus was oriinally licensed in 1963
ut w
as ultimately
ineffective. A more effective vaccine consist- in of live, attenuated ru
eola v
irus was licensed
in 1968 and is used in the routine immunization schedule of infants and children
, either in
com
ination with ru
ella and mumps (MMR) or in com
ination with ru
ella, mumps,
and varicella
(MMRV). 101 Recommended administration of the vaccine is in two doses, the rst
e
tween the aes
of 12 and 15 months and the second
etween aes 4 to 6. Administration of the rst
dose prior to
the ae of 12 months may result in vaccine failure,
ecause the presence of mate
rnal anti
odies
can inter- fere with the infants immune response. Althouh measles continues to

e a lo
al
concern, immunization aainst ru
e- ola has resulted in a sinificant decr
ease in the
num
er of cases in the United States, with most cases resultin from failure to
immunize
infants and youn children due to reliious or other reasons, or from
cases
rouht
into the United States
y unvaccinated individuals from other countries. 101 The

dianosis of
measles has typically
een
ased on clin- ical presentation of the patient.
However, this

asis for dianosis has
een complicated
y physicians decreased a
il- ity to re
conize the
clinical features of measles due to the reduction in the num
er of measles cases
as a result of
our immunization proram. 89,101,103 In addition, atypical presenta- tions of me
asles can occur
in individuals who received the earlier form of measles vaccine, who have low an
ti
ody titers, or
who are immunocompromised. 89,100,101 La
oratory tests are therefore of value in
ensurin rapid,
accurate dianosis of spo- radic cases; in addition, they are important for epid
emioloical
surveillance and control of community out
reaks. 89,101,103 Isolation of ru
eola
virus in
conventional cell cultures is technically dif cult and slow and is not enerally p
erformed in the
routine dianosis of measles,
ut it may
e useful in epidemioloical surveillan
ce of measles
virus strains. 101,103 The optimal time to recover measles virus from nasopha- r
yneal
aspirates, throat swa
s, or
lood is from the prodrome period up to 3 day
s after rash
onset. From urine, it is 1 week after appearance of the rash. 89,101 Seroloical
testin provides
the most practical and relia
le means of con rmin a measles dianosis. 89,101,103
In conjunction with clinical symptoms, a dianosis of measles is indicated
y the
presence of
ru
eola-speci c IM anti
od- ies or
y a fourfold rise in the ru
eola-speci c IG an
ti
ody titer

etween serum samples collected soon after the onset of rash and 10 to 30 days l
ater. 101 SSPE is
associated with extremely hih titers of ru
eola anti
odies. 100,103 IM anti-

odies are
preferentially detected
y an IM capture ELISA method, which is hihly sensitiv
e and has a low
incidence of false-positive results. 89,101 IM anti
odies
ecome detecta
le
3 to 4 days
after appearance of symptoms and persist for 8 to 12 weeks. 103 Samples colle
cted
efore 72
hours may yield false-neative results, and repeat test- in on a later sample i
s recommended in
that situation. 101 A variety of methods have
een developed to detect IG ru
eo
la anti
odies,
includin hemalutination inhi
ition, microneutralization, plaque reduction neu
tralization, complement fixation, indirect fluorescent anti
ody tests, and 1814_Ch22_369-398.qxd
7/13/09 2:58
PM Pae 384 CHAPTER 22 Seroloy and Molecular Detection of Viral Infections 385
ELISA. The most
commonly used is ELISA. 89,101,103 IG anti-
odies
ecome detecta
le 7 to 10 da
ys after the
onset of symptoms and persist for life. 103 Presence of ru
eola-speci c IG anti
o
dies indicates
immunity to measles due to past infection or immunization. 89,103 Testin for I
G anti
odies is
therefore routinely performed
y clinical la
oratories in order to detect immune

status of
individuals such as health- care workers to the virus. Molecular methods to dete
ct ru
eola RNA
can
e used in cases in which seroloical tests are inconclusive or inconsis- te
nt and can
e
used to enotype the virus in epidemioloical studies. 103,104 The preferred mol
ecular technique
is reverse transcriptase PCR (RT-PCR), performed
y traditional or real-time PCR
methodoloies.
These assays are sensitive, can
e performed on a variety of clinical samples or
on infected cell
cultures, and can detect viral RNA within 3 days of rash appearance. 103 Mumps T
he mumps virus,
like ru
eola, is a sinle-stranded RNA virus that
elons to the Paramyxovi
ridae family
(enus Ru
ulavirus). It is transmitted from person to person
y infected respira
tory droplets and
possi
ly fomites, and repli- cates initially in the nasopharynx and reion
al lymph nodes.
103108 Followin an averae incu
ation period of 14 to 18 days, the virus sp
reads from the

lood to various tissues, includin the menines of the
rain, salivary lands,
pancreas, testes,
and ovaries, producin inflammation at those sites. 106 In ammation of the parotid
lands, or
paroti- tis, is the most common clinical manifestation of mumps, occurrin in 30
to 40 percent of
cases. 106 The swellin of parotitis results in earache and tenderness of the ja
w, which can
e

ilateral or unilateral and resolves in 7 to 10 days. 105,106 Althouh parotitis
is the classic
symptom of mumps, a
out 20 percent of infections are asymptomatic, and another 4
0 to 50 percent
have nonspeci c or respiratory symptoms with no parotitis. 106 Complications of mu
mps infections
include asympto- matic meninitis (50 to 60 percent of cases); symptomatic menin
itis (10 to 30
percent of cases); testicular in amma- tion, or orchitis (20 to 50 percent of post
pu
ertal
males); ovarian inflammation (in a
out 5 percent of postpu
ertal females); and d
eafness (in a
out
1 case per 20,000). 105107 Prior to routine immunization, mumps was one of the mo
st common
causes of aseptic meninitis and sensorineural deaf- ness in children. 106 Infre
quent
ut
important complications of mumps include pancreatitis, encephalitis, myocarditis
, polyarthritis,
and throm
ocytopenia. 65,105107 Mumps infec- tion in prenant women results in in
creased risk
for fetal death when it occurs in the rst trimester of prenancy,
ut it is not a
ssociated with
conenital a
normalities. 109 The num
er of mumps cases in the United States has
declined
sinificantly since the introduction of a live attenuated mumps virus vac
cine in 1967 and
its routine use in child- hood immunization schedules in 1977. 107,109,110 The v
accine is most
commonly com
ined with the vaccines for ru
ella and mumps (MMR) or is used in co
m
ination with
the ru
ella, mumps, and varicella vaccines (MMRV). The dianosis of mumps is usu

ally made on the

asis of clinical symptoms, especially parotitis, and does not require la
orator
y con rmation.
89,105107 However, la
oratory test- in is very useful in cases in which parotiti
s is a
sent or
when differentiation from other causes of parotitis is required. Within the rst f
ew days of
illness, mumps virus can
e iso- lated from saliva, urine, cere
rospinal uid, or
swa
s from the
area around the excretory duct of the parotid land. It can then
e rown in she
ll vial cultures
of rhesus monkey kidney cells or human em
ryonic lun
ro
lasts and iden- tified

y stainin
with fluorescein-la
eled monoclonal anti
odies. 103,105 However, culture metho
ds require
experi- enced personnel and specialized reaents and may not
e performed in the
routine clinical
la
oratory. Reverse transcriptase polymerase chain reaction (RT- PCR) methods ha
ve
een developed
to detect viral RNA in specimens collected from the
uccal cavity, throat, cere

ral spinal fluid,
or urine of patients with a suspected mumps infection. 103,108,111 These methods
have not
een
standard- ized
ut may
e useful in con rmin infection in cases where viral isola
tion is not
successful. 103 Seroloical testin provides the most simple and prac- tical me
ans of
confirmin a mumps dianosis, when indicated. 89,103,106 Althouh a variety
of methods have

een developed to detect mumps anti
odies, includin comple- ment fixation, hema
lutination
inhi
ition, hemolysis in el, neutralization assays, immunofluorescence assay, a
nd ELISA, the
latter two methods are used most commonly,
ecause they are sensitive, speci c, co
st-effective,
and read- ily performed
y the routine clinical la
oratory. 103,106 Use of solid
-phase IM
capture assays reduces the incidence of false-positive results due to rheumatoid
factor. Current
or recent infection is indicated
y the presence of mumps- speci c IM anti
ody in
a sinle serum
sample or
y at least a fourfold rise in speci c IG anti
ody
etween two speci- m
ens collected
durin the acute and convalescent phases of illness. 103,105,106,108 Cross-react
ivity
etween
anti
odies to mumps and related paramyxoviruses has
een reported in tests for I
G
ut is usually
not a pro
lem
ecause of differ- entiation in clinical symptoms. 89,103 IM anti

odies can
e
detected within 3 to 4 days of illness and can persist for at least 8 to 12 week
s. 103 IG
anti
odies
ecome detecta
le within 7 to 10 days and persist for years. The pres
ence of speci c
IG anti
odies indicates immunity to mumps, either as a result of natural i
nfection or
immunization. In the United States, most seroloy tests for mumps are performe
d to evaluate
immune status in health-care workers or in recip- ients of the mumps vaccine. 10
3 HUMAN T-CELL
LYMPHOTROPIC VIRUSES Human T-cell lymphotropic virus type I (HTLV-I) and human T

-cell
lymphotropic virus type II (HTLV-II) are closely related retroviruses. These vir
uses have RNA as
their nucleic acid and an enzyme called reverse transcriptase, whose 1814_Ch22_3
69-398.qxd
7/13/09 2:58 PM Pae 385 386 SECTION 4 Seroloical Dianosis of Infectious Dis
ease function is
to transcri
e the viral RNA into DNA. The DNA then
ecomes interated into the h
ost cells enome
as a provirus. The provirus can remain in a latent state within infected cells f
or a proloned
period of time. Upon activation of the host cell, the provirus can proceed to co
m- plete its
replication cycle to produce more virions. Both HTLV-I and HTLV-II infect T lymp
hocytes,
especially those that are CD4 , that cause T-cell proliferation, and that have the
potential to
esta
lish persistent infection. HTLV-II can infect macrophaes as well. 112 Both
viruses have
three structural enes, called a, pol, and env, and two major reulatory enes
, called tax and
rev. 113115 The a ene codes for the viral core proteins, the pol ene codes fo
r the reverse
transcriptase enzyme, and the env ene codes for the envelope lycoproteins. HTL
V-I and HTLV-II
can
e transmitted
y three major routes:
lood
orne (mainly throuh transfusion
s contain- in
cellular components or throuh intravenous dru a
use), sexual contact, and moth
er-to-child
(mainly throuh
reast- feedin). 112117 HTLV-I infection is endemic in southern
Japan, the
Cari

ean islands, South and Central Africa, the Middle East, parts of South Ame
rica, and
Melanesia. 112117 In the United States and Europe, infections result mainly from
immirants from
endemic areas. HTLV-II infections are hihest in various Native American populat
ions and in
intravenous dru a
users in North America and Europe. 112 HTLV-I is associated w
ith
oth
malinant and nonmali- nant disorders that have in ammatory or proliferativ
e
characteristics. 112117 Most nota
ly, infection with HTLV-I may lead to the devel
opment of adult
T-cell leukemia/lymphoma (ATL), a spectrum of T-cell malinancies that can
e cl
assi- ed into
four different su
types: acute, chronic, smolderin, and lymphoma type. The risk
of developin
ATL is hihest in those who acquired the infection prior to adulthood, with the
disease typically
appearin at least 20 years after initial infection. 112,116 HTLV-I infection ha
s also
een
associated with a 2 percent lifetime risk of developin a proressive neuroloical disorder
called HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a 2.5
percent risk of
developin an intraocular in ammation of the eyes called HTLV uveitis, a reater r
isk of
developin HTLV-I-associated sicca syndrome characterized
y dry eyes and dry mo
uth, and an
infective der- matitis in children associated with severe eczema. 112117 HTLV-II

infection is
thouht to
e associated with less severe disease, includin a chronic encephalo
myelopathy
similar to HAM/TSP, various forms of eneralized neuro- loical dysfunction, rec
urrent
ladder
and kidney infections, funal infections, and pneumonia. 112,118 Seroloical tes
tin plays an
important role in detectin HTLV-I and HTLV-II infections,
ecause culture of th
e viruses
requires sophisticated techniques that cannot
e per- formed in routine clinical
la
oratories.
ELISA tests for HTLV-I and HTLV-II anti
odies are used initially to detect HTLV
infections in
individuals and to screen
lood donors. 112,117 These tests use a com
inatio
n of HTLV-I and
HTLV-II a and env proteins as the source of antien
ound to a solid phase. Be
cause
false-positive results may also occur, any sample producin a reactive result in
the ini- tial
ELISA screen is retested
y ELISA and su
sequently tested
y a more speci c, con rma
tory method.
The con rmatory method of choice is the Western
lot, which identifies anti
odies
to separate
HTLV antiens. Specimens are considered positive for HTLV-I or HTLV-II
y this t
est if
ands
representin anti
odies to the a pro- tein p24 and the env lycoproteins
of either
virus are present. 112,114,117 Samples that are reactive
y ELISA
ut ne- ativ
e
y Western
lot
should
e tested
y the more sensitive radioimmunoprecipitation assay (RIPA). 11
2 PCR can
e used
to detect HTLV-I or HTLV-II RNA in various samples in order to clarify repeatedl
y indeterminate
results, demon- strate the presence of virus in different tissues, and d
etermine viral
load in monitorin disease proression. 112,114 OTHER VIRAL INFECTIONS See Ta
le
224 for
la
oratory methods used to detect infec- tions with other viruses and their clin
ical sini cance.
SUMMARY Viruses are o
liate intracellular parasites that can produce a wide ran
e of diseases in
humans. Viruses can exist as either free infectious virions or intracellular par
ticles after they
have infected host cells. These different states require the com
ined effort of
innate, humoral,
and cell-mediated immune responses to successfully defend the host aainst viral
infections.
Important innate defenses are carried out
y interferons, which inhi
it the repl
ication of
viruses, and NK cells, which release cytotoxic proteins that destroy virus- infe
cted host cells.
Anti
odies directed aainst speci c viral antiens can prevent the spread of viral
infection in
several ways: Anti
odies can neutralize a virus and prevent it from
indin to h
ost cells, can
opsonize a virus to make it more likely to
e phaocytized, can activate complem
ent-mediated
mechanisms of destruction, and can alutinate viruses. Cell-mediated immunity i
s required to
eliminate intracel- lular viruses. This is accomplished
y virus-speci c CTL, whic

h
inds to
virus-infected host cells and releases cytotoxic proteins that cause the cel
ls to undero
apoptosis. Despite these immunoloic mechanisms, the immune system is not alw
ays successful
in eliminatin a viral pathoen from the host,
ecause viruses have evolved s
ev- eral ways
of evadin the hosts defenses. Many viruses accomplish this
y underoin f
requent enetic
mutations, which result in the production of new viral antiens that are not rec
onized
y
previous host responses. Some viruses can evade the action of interferons, compl
ement, or other
components of the immune system, or can cause suppres- sion of the immune system

y different
means. Finally, some viruses are capa
le of interatin their nucleic acid into
the hosts enome
and remainin inside the host cells 1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pa
e 386 CHAPTER 22
Seroloy and Molecular Detection of Viral Infections 387 Ta
le 22-4. La
oratory
Tests for
Dianosis of Other Viral Infections VIRUS MODES OF TRANSMISSION DISEASE ASSOCIAT
IONS LABORATORY
TESTS Adenoviruses Respiratory or fecal-oral Fe
rile acute respiratory disease,
Culture and
con rmation with astroenteritis, myocarditis, roup-speci c uorescent- pericarditis
,
keratoconjunctivitis, la
eled anti
odies hemorrhaic cystitis Rapid methods to
detect viral
antien or nucleic acid Seroloical tests to detect anti
odies to human adenovir
uses (EIA,
IIF); a fourfold rise in anti
ody titer is dianostic Ar
ovirus Arthropod-
orne
Encephalitis
IM capture ELISA is preferred method for dianosis; IIF also availa
le Serum ne
utralization
test used to con rm other seroloical test results Antien capture ELISA used to d
etect current
infection Immunohistochemical stainin, in situ hy
ridization, and nucleic acid
ampli cation
tests also availa
le Herpes HSV-1: close personal HSV-1: inivostomatitis, D
ianosis made

y viral culture simplex (HSV) contact, perinatal conjunctivitis, keratitis, of
swa
s taken from
HSV-2: sexual, perinatal herpetic whitlow, encephalitis mucocutaneous lesions an
d HSV-2: enital
tract infection direct immuno uorescence for herpes antiens Seroloical tests req
uire a
sini - cant rise in anti
ody titer to indicate current infection (EIA, indirect
hemalutination, immuno
lottin, IFA, NT availa
le) PCR for HSV nucleic acid is
the method of
choice for detectin active infection In uenza Inhalation of air
orne Respiratory
illness,
pneumonia Identi cation of in uenza virus droplets; contact with can
e made
y uore
scent
respiratory secretions anti
ody stainin of viral antiens in epithelial cells f
rom respiratory
tract specimens
y direct IFA, rapid immunoassays to detect in uenza antiens in c
linical
samples, or culture of nose and throat swa
s Seroloical tests (e.., HI, EIA,
microneutralization) can indicate infection
y a fourfold rise in anti
ody titer

when direct
virus detection is not successful RT-PCR can
e used to monitor enetic and anti
enic
character- istics of circulatin strains Continued 1814_Ch22_369-398.qxd 7/13/0
9 2:58 PM Pae
387 388 SECTION 4 Seroloical Dianosis of Infectious Disease Ta
le 22-4. La
ora
tory Tests for
Dianosis of Other Viral InfectionsContd VIRUS MODES OF TRANSMISSION DISEASE ASSOC
IATIONS
LABORATORY TESTS Parain uenza Inhalation of air
orne droplets; Respiratory illnes
s, croup
Virus isolation can
e contact with respiratory accomplished
y culture of secr
etions
respiratory secretions in cell lines; nasal washes are the specimen of choice Ra
pid detection
of virus antiens can
e made
y direct IFA or EIA usin respiratory secretions
or middle-ear
effusions RT-PCR can
e used to detect viral RNA in respiratory secretions Serol
oical assays
(e.., ELISA, NT, direct IFA, IIF, CF, HI) can
e used to assess immune status;
recent infection
is indicated
y a four- fold rise in anti
ody titer Parvovirus B19 Respiratory r
oute,
lood
Fifth disease, red cell aplasia in IFA, RIA, and ELISA used to detect transfusi
ons, perinatal
immunocompromised hosts, B19 antien and anti-B19 conenital anemia, hydrops a
nti
ody fetalis,
spontaneous a
ortion B19 nucleic acid can
e detected
y PCR or in situ hy
ridiz
ation usin
tissues or cells Respiratory Contact with respiratory Acute respiratory infect
ion, Virus
isolation can
e syncytial secretions
ronchiolitis, pneumonia, accomplished

y culture of
virus (RSV) croup respiratory secretions in cell cultures; nasal washes are the
specimen of
choice Rapid detection of virus antiens can
e made
y direct IFA or EIA usin
respiratory
secretions or middle-ear effusions RT-PCR can
e used to detect viral RNA in res
piratory
secretions Seroloical assays (e.., ELISA, NT, direct IFA, IIF, CF, HI) can
e
used to assess
immune status; recent infection is indicated
y a four- fold rise in anti
ody ti
ter Rotavirus
Fecal-oral Gastroenteritis, diarrhea EIA is used to detect rotavirus anti
ody an
d for
serotypin EIA and latex alutination can
e used for detection of roup A rota
virus antien
in fecal samples RT-PCR can detect rotavirus RNA CF
complement xation; CNS
centra
l nervous
system; DNA deoxyri
onucleic acid; EIA
enzyme immunoassay; IFA
immuno uorescence a
ssay; HI
hemalutination inhi
ition; IIF
indirect immuno uorescence; NT
neutralization tes
t; PCR
polymerase chain reaction; RNA
ri
onucleic acid; RT-PCR reverse transcriptase po
lymerase
chain reaction; RIA
radioimmunoassay. in a silent state for lon periods of time

efore
ein
reac- tivated at a later date. Seroloical tests for viral anti
odies are amon
the most
important tests performed
y the clinical immunoloy la
- oratory. These tests

can
e used
to indicate current infections, conenital infections, and previous exposure t
o viruses or the
vaccines used to prevent viral infections with su
sequent immunity. Molecular me
thods have played
an increasinly important role in the dianosis and manae- ment of viral infect
ions in recent
years. These tests provide a sensitive means of rapidly detectin viral infectio
ns and of
quantitatin viral load to uide patient therapy. This chapter 1814_Ch22_369-398
.qxd 7/13/09
2:58 PM Pae 388 CHAPTER 22 Seroloy and Molecular Detection of Viral Infection
s 389 discussed
the seroloical and molecular methods used
y the clinical la
oratory in the dia
nosis and
manaement of some of the most important viral infections. The hepatitis viruses
are those whose
primary effect is in ammation of the liver. Hepatitis A and E are transmitted
y t
he fecal-oral
route, while hepatitis B, C, D, and G are transmitted primarily
y the parentera
l route.
Hepatitis B, C, and D may lead to chronic infections. Seroloic markers of hepat
itis infections
consist of virus-speci c anti
odies and antiens which are commonly detected

y automated
immunoassays. IM anti-HAV anti
odies indicate current or recent hepatitis A inf
ection, while IG
anti
odies indicate immunity to hepatitis A. Hepatitis B infection is indicated

y the presence
of the antien HBsA; HBeA indicates hih infectivity. IM anti
odies to hepati
tis B core
antien are pres- ent in acute hepatitis B, while IG anti-HBc is present durin
past or chronic
hepatitis B infection. Anti
odies to HBsA can
e present as a result of past he
patitis B
infection or immunization with the hepatitis B vaccine and indicate immu- nity.
Exposure to
hepatitis C can
e detected
y ELISA measurin anti-HCV, with molecular te
sts
ein used to
con- rm infection in anti
ody-positive patients, to detect infection in anti
odyneative
patients, to detect perinatal infections, and to screen
lood and oran donors f
or HCV. Hepatitis
D occurs as a super- or co-infection with hepatitis B and is indi- cated
y anti

odies to
hepatitis D or molecular tests to detect HDV RNA. Seroloical and molecular test
s for hepatitis E
have
een developed
ut are not widely availa
le. There are eiht human herpes v
iruses, includin
the Epstein-Barr virus, cytomealovirus, and varicella-zoster virus. The Epste
in-Barr virus
is the cause of infectious mononucleosis, several hematoloic and nonhematol
oic
malinancies, and lymphoproliferative disorders in immuno- suppressed individua
ls. Most
patients with infectious mononucleosis produce heterophile anti
odies, which c
an react with
antiens from
eef, horse, or sheep red
lood cells. Althouh these anti
odie
s were once
routinely screened for
y the Monospot test, they are now commonly detected
y r

apid
immunochromatoraphic or alutina- tion methods used to detect anti
odies to
o
vine antiens.
ELISA or immuno uorescence assay (IFA) tests for EBV- specific antiens are used t
o detect
heterophile-neative cases of infectious mononucleosis and to dianose other EBV
-associated
diseases. Molecular tests are useful in detect- in EBV DNA in immunocompromised
patients who may
not develop a ood anti
ody response and in monitorin viral load in patients wi
th EBV-related
malinancies durin therapy. CMV infection is asymptomatic in most healthy indiv
id- uals
ut may
produce a mononucleosis-like syndrome in some. The virus can remain latent for y
ears and
ecome
reactivated later in life. CMV infection can have more seri- ous consequences in
immunocompromised individuals or conenitally infected infants. Active CMV infec
tion is
est
detected
y shell vial assays to identify CMV antiens
y immuno uorescence,
y CM
V antienemia
assays for pp65 antien, or
y molecular assays to detect CMV viral load. Quanti
tative PCR is
useful in determinin the CMV DNA copy num
er in immunocompromised hosts undero
in antiviral
treatment. Seroloical assays for CMV anti
ody are most helpful in documentin a
past infection
in poten- tial
lood and oran donors. Primary infection with varicella virus ca
uses chickenpox
(varicella), while reactivation of the virus in nerve cells sup- plyin the skin
causes shinles
(zoster). Dianosis of current varicella virus infection is usually
ased on cli
nical ndins,

ut detection of varicella virus antiens
y shell vial assay and immuno uorescenc
e or of
varicella DNA
y PCR may
e helpful in some clinical settins. Seroloical metho
ds, most
commonly ELISA, are used mainly to document immunity to varicella virus. I
mmunization
prorams have reatly reduced the inci- dence of three childhood infections: ru

ella, ru
eola,
and mumps. Ru
ella infection is the cause of German measles
ut can result in se
vere conenital
a
normalities if it occurs durin prenancy. Ru
eola viruses cause measles, a sy
stemic infection
that can cause complications in some individuals. Mumps virus is the cause of mu
mps, whose
classic feature is swellin of the parotid lands; complications may occur. Alth
ouh the
dianosis of these three infections is usually
ased on clinical ndins, la
orato
ry testin may

e help- ful in con rmation. Because culture of these viruses is slow and dif cult,
seroloy is
most often used for this purpose. Current infections are indicated
y the presen
ce of IM
anti
odies speci c for the appropriate virus or
y a fourfold rise in virus-speci c
IG
anti
odies in two separate speci- mens collected durin the acute and convalesce
nt phases of
disease. However, testin for IG anti
odies is most com- monly performed to scr
een for immunity

to these viruses. RT-PCR is a useful adjunct to seroloy in detectin viral RNA

in patients with
inconclusive seroloy or viral culture results, in epidemioloical studies, and
in the detection
of conenital ru
ella infections. The human T-cell lymphotropic viruses, HT
LV-I and HTLV-II,
are retroviruses that infect T lymphocytes. HTLV-I is the cause of adult T-cell
leukemia/lymphoma, HTLV-I-associated myelopathy/tropical spastic paraparesis (HA
M/TSP), and other
in ammatory disorders. HTLV-II is thouht to
e associated with a HAM/TSP-like dis
ease and
several other disorders of neuroloical dysfunction. ELISA tests are used routin
ely to screen

lood donors for anti
odies to HTLV-I and HTLV-II and to detect exposure to HTLV
in other
individuals. Positive results are con rmed
y Western
lot. RIPA or PCR can
e use
d to clarify
inde- terminate results. Seroloical and molecular tests have also
een develope
d to detect
exposure to other viruses, includin adenoviruses, ar
ovirus, herpes simplex-1 a
nd -2,
parain uenza, parvovirus B19, respiratory syncytial virus, and rotavirus. 1814_Ch2
2_369-398.qxd
7/13/09 2:58 PM Pae 389 390 SECTION 4 Seroloical Dianosis of Infectious Dis
ease 1. A
25-year-old male had
een experiencin ulike symp- toms, loss of appetite, na
usea, and
constipation for 2 weeks. His a
domen was tender, and his urine was dark in col
or. Initial
testin revealed elevations in his serum alanine aminotransferase (ALT) and aspa
rtate
aminotransferase (AST) levels. Questions a. What la
oratory tests should
e used
to screen this
patient for viral hepatitis?
. If the patient tested positive for hepatitis B,
which tests
should
e used to monitor his condition? c. If the patient developed chronic hep
atitis B, which
markers would
e present in his serum? 2. A 5-pound infant was
orn with microce
phaly, purpuric
rash, low platelet count, cardiovascular defects, and a cataract in the left eye
. The infants
mother recalled experiencin ulike symptoms and a mild skin rash early in her pre
nancy. She had
not souht medical attention at the time. The infants physician ordered tests to
inves- tiate
the cause of the new
orns symptoms. Questions a. What virus is the most likely ca
use of the
infants symptoms?
. What la
oratory tests would you suest the doctor order on
the mother
to support your suested dianosis? c. What tests should
e performed o
n the infants
serum to support this dianosis? CASE STUDIES 1814_Ch22_369-398.qxd 7/13/09 2:
58 PM Pae 390
CHAPTER 22 Seroloy and Molecular Detection of Viral Infections 391 TESTING FOR
THE HETEROPHILE
ANTIBODY OF INFECTIOUS MONONUCLEOSIS BY THE CLEARVIEW MONO TEST* A rapid test fo
r the
qualitative detection of infectious mononu- cleosis (IM) heterophile anti
odies
in whole
lood,
serum, and plasma for professional in vitro dianostic use only. Intended Use Th

e Clearview
Mono test is a rapid chromatoraphic immunoassay for the qualitative de
tection of
infectious mononucleosis heterophile anti
odies in whole
lood, serum, or
plasma to aid
in the dianosis of infectious mononucleosis. Summary Infectious mononucleos
is is caused
y
the Epstein-Barr virus, which is a mem
er of the herpesvirus family. Symptoms of
IM are fever,
sore throat, and swollen lymph lands. In very rare cases, heart or central nerv
ous system
pro
lems may occur. Dianosis of IM is made
ased on the presence of heterophile
anti
odies.
Infectious mononucleosis heterophile anti
odies
elon to the IM class. They ar
e present in 80
to 90 percent of acute IM cases and can
e detected in 60 to 70 percent of patie
nts durin the
rst week of clinical illness. The Clearview Mono test is simple and utilizes an e
xtract of

ovine erythrocytes to qualitatively and selectively detect IM heterophile anti

odies in whole

lood, serum, or plasma in just minutes. Principle The Clearview Mono test is a
qualitative,
mem
rane-strip-
ased immunoassay that detects IM heterophile anti
odies in whol
e
lood, serum,
or plasma. In this test procedure,
ovine erythrocyte extracted antien is coate
d on the
test-line reion of the device. The sample reacts with
ovine erythrocyte extrac
ted
antien-coated particles that have
een applied to the la
el pad. This mixture m
irates
chromatoraphically alon the lenth of the test strip and interacts with the co
ated
ovine
erythrocyte extracted antien. If the sample contains IM anti
odies, a colored l
ine will appear
in the test-line reion, indicatin a positive result. If the sample does not co
ntain IM
heterophile anti
odies, a colored line will not appear in this reion, indicatin
 a neative
result. To serve as a procedural control, a colored line will always appear at t
he control-line
reion, indicatin that proper volume of specimen has
een added and mem
rane wi
ckin has
occurred. Reaents The test device contains
ovine erythrocyte extracted a
ntien-coated
particles and
ovine erythrocyte extracted antien-coated mem
rane. Sample Colle
ction and
Preparation The Clearview Mono test can
e performed usin whole
lood (from ven
ipuncture or
nerstick), serum, or plasma. Materials Provided Test devices Disposa
le sample d
roppers
Disposa
le heparinized capillary tu
es and dispensin
ul
s Positive control (di
luted human
plasma containin IM heterophil anti
odies, 0.09 percent sodium azide) Neative
control (diluted
human plasma, 0.09 percent sodium azide) Sample
uffer Packae insert Materials
Required But not
Provided Sample-collection container (for venipuncture whole
lood) Lancet (for n
erstick whole

lood only) Centrifue (for serum or plasma only) Timer Procedure Allow the test

device, sample,

uffer, and controls to reach room temperature (15C to 30C)
efore testin. 1. Rem
ove the test
device from the foil pouch and use it as soon as possi
le. For
est results, per
form the test
imme- diately after openin the foil pouch. 2. Place the test device on a clean
and level
surface. (See Fi. 225 for a diaram illustratin the procedure.) For whole
lood
(venipuncture)
samples: Hold the dropper upriht and add two drops of whole
lood (a
out 50 L) t
o the sample
well (S) of the test device. Then add one drop of sample
uffer to the sample we
ll. Start the
timer. For whole
lood ( nerstick) samples: Add one capil- lary tu
e of
lood (a

out 50 L) to
the sample well (S) of the test device. Then add one drop of sample
uffer to th
e sample well.
Start the timer. For serum or plasma samples: Hold the dropper upriht and add o
ne drop of serum
or plasma (a
out 25 L) to the sample well (S) of the test device. Then add one dr
op of sample

uffer to the sample well. Start EXERCISE 1814_Ch22_369-398.qxd 7/13/09 2:58 P
M Pae 391 392
SECTION 4 Seroloical Dianosis of Infectious Disease Invalid No line appears in
the control-line
reion (C): If this occurs, read the directions aain and repeat the test with a
new test device.
If the result is still invalid, stop usin the test kit and contact Inverness Me
dical Technical
Support at (800) 637-3717. Internal Quality Control Internal procedural controls
are included in
the test. A red line appearin in the control reion (C) is an internal pos- iti
ve procedural
control. It con rms suf cient sample volume and correct procedural technique. A clea
r
ackround
is an internal neative
ackround control. If the test is workin properly, the

ackround in
the result area should
e white to liht pink and should not interfere with the
a
ility to read
the test result. *Reprinted with permission from Clearview Mono whole
lood seru
m, plasma kit
packae insert, Inverness Medical Professional Dianostics, San Dieo, CA, 2006.
C X 3 T C X 3 T
C X 3 T OR OR 50 L 2 drops venipuncture whole
lood + 1 drop
uffer 50 L finersti
ck whole

lood + 1 drop
uffer 1 drop serum or plasma + 1 drop
uffer Buffer Buffer Buffe
r FIGURE 225.
Clearview Mono test procedure. 2008 Inverness Medical. Used with permission. Rea
d in 5 minutes
Positive Neative Interpret results Invalid C T C T C T C T C T FIGURE 226. Clear
view Mono test
results. 2008 Inverness Medical. Used with permission. the timer. Avoid trappin
air
u

les in
the sample well. (See Fi. 225.) 3. Wait for the red line(s) to appear. The resul
t should
e
read at 5 minutes. The
ackround should
e clear
efore the result is read. NOT
E: Low titers of
IM heterophile anti
odies miht result in a weak line appearin in the test-line
reion (T) after
a lon period of time. Do not read the result after 10 minutes. Interpretation o

f Results Refer
to Fiure 226. Positive* Two distinct red lines appear: One line should
e in the
control-line
reion (C), and another line should
e in the test-line reion (T). A positive r
esult means that
IM heterophile anti
odies were detected in the sample. *NOTE: The shade of the r
ed color in the
test line reion (T) will vary
ased on the amount of IM heterophile anti
odies
in the sample.
Any shade of red in the test line reion (T) should
e considered positive. Nea
tive One red line
appears in the control-line reion (C): No apparent red or pink line appears in
the test-line
reion (T). A neative result means that IM het- erophile anti
odies were not fo
und in the sample
or are
elow the detection limit of the test. 1814_Ch22_369-398.qxd 7/13/09 2:
58 PM Pae 392
CHAPTER 22 Seroloy and Molecular Detection of Viral Infections 393 TESTING FOR
THE HETEROPHILE
ANTIBODY OF INFECTIOUS MONONUCLEOSIS BY THE PAUL-BUNNELL TEST Principle This tes
t is used to
determine the titer of the heterophile anti
ody in patients with infectious mono
nucleosis. Serial
dilutions of patient serum are prepared and incu
ated with sheep red
lood cells
. The titer of
the anti
ody is the recip- rocal of the last dilution to show alutination of t
he red
lood
cells. Reaents, Materials, and Equipment 0.9 percent saline solution Sheep eryt
hrocytes stored
in Alsevers solution. Refrierate until use. Positive control serum containin he
terophile
anti
ody of infectious mononucleosis Patient serum to
e tested 13 100 mm lass
test tu
es 0.1
mL and 1.0 mL pipettes Procedure* 1. Inactivate complement in sera to
e tested

y heatin in a
56C water
ath for 30 minutes. 2. Place sheep red
lood cells into a tu
e and was
h the cells
three times in saline. 3. Prepare 5 mL of a 2 percent sheep red
lood cell suspension
y
pipettin 0.1 mL of washed, packed red
lood cells into 4.9 mL of saline. 4. Set
up and la
el two
rows of 10 test tu
es in a rack. One row is for a positive-control serum and the
other for the
patient serum to
e tested. La
el the control tu
es C1 throuh C10, and la
el th
e patient tu
es
P1 throuh P10. 5. Pipette 0.4 mL of saline in the rst tu
e of each row and 0.25
mL of saline in
each remainin tu
e. 6. Pipette 0.1 mL of positive control serum into tu
e 1 of
the control row.
7. Mix and serially transfer 0.25 mL throuh tu
e 9, dis- cardin 0.25 mL fro
m tu
e 9. Tu
e
10 of this row contains no serum and serves as a neative control. 8. O
tain
a sample of
patient serum to
e tested. Pipette 0.1 mL of patient serum into tu
e 1 of the p
atient row. 9.
Mix and serially transfer 0.25 mL throuh tu
e 10, dis- cardin 0.25 mL from tu

e 10. 10. Add 0.1
mL of the 2 percent sheep red
lood cell sus- pension (prepared in step 3 a
ove)
to each tu
e in
the control and patient rows. 11. Shake the tu
es to o
tain an even mixture. Cov

er the tu
es with

para lm and incu
ate at room temperature for at least 15 minutes. A more accurate
readin may
e
o
tained
y allowin the tu
es to incu
ate for 2 hours. 12. Followin the
incu
ation
period, read each tu
e indi- vidually for macroscopic alutination, as fol
lows: ently
shake the tu
e to resuspend the red
lood cells, tilt the tu
e, and hold up to l
iht. Compare
each tu
e to the neative control tu
e and record results. Interpretation of Res
ults The anti
ody
titer is reported as the reciprocal of the dilution in the last tu
e, which show
s visi
le
alutination (Fi. 227). Titers less than or equal to 56 are considered normal.
EXERCISE *From
Paul, JR, and Bunnell, WW. The presence of heterophile anti
odies in infectious
mononucleosis. Am
J Med Sci 183:90, 1932. Positive Neative FIGURE 227. Interpretation of Paul-Bu
nnell test
results. 1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 393 394 SECTION 4 Serolo
ical Dianosis of
Infectious Disease 1. An individual with hepatomealy, jaundice, and elevated li
ver enzymes has
the followin la
oratory results: IM anti-HAV (neative), HBsA (positive), IM
anti-HBc
(positive), and anti-HCV (neative). These ndins sup- port a dianosis of a. hep
atitis A.
.
acute hepatitis B. c. chronic hepatitis B. d. hepatitis C. 2. Which of the follo
win hepatitis
viruses is transmitted
y the fecal-oral route? a. Hepatitis B
. Hepatitis C c.
Hepatitis D d.
Hepatitis E 3. Quantitative tests for HCV RNA are used to a. screen for hepatiti
s C.
. determine
the HCV enotype. c. differentiate acute HCV infection from chronic HCV infectio
n. d. monitor
hepatitis C patients on antiviral therapy. 4. The serum of an individual who rec
eived all doses
of the hepatitis B vaccine should contain a. anti-HBs.
. anti-HBe. c. anti-HBc.
d. all of the
a
ove. 5. A 12-year-old irl presented to her physician with a sore throat, lymp
hadenopathy, and
fatiue. Her la
oratory results were 5000 lymphocytes/ L with 10 percent aty
pical
lymphocytes, CMV anti
ody neative, and het- erophile anti
ody screen neativ
e. These
la
oratory results a. con rm a dianosis of infectious mononucleosis.
. indicate
that the
dianosis is not infectious mononucleosis,
ecause the heterophile anti
ody scre
en is neative.
c. suest a dianosis of infectious mononucleosis
ut should
e followed
y a h
eterophile
anti
ody titer to strenthen the dianosis. d. suest a dianosis of infectious
mononucleosis

ut should
e followed
y a test for IM anti-VCA to strenthen the dianosis. 6
. In the
la
oratory, heterophile anti
odies are routinely detected
y their reaction with
a. B
lymphocytes.
.
ovine erythrocyte antiens. c. sheep erythrocyte antiens. d. E
pstein-Barr virus
antiens. 7. Presence of IM anti-ru
ella anti
odies in the serum from an infant


orn with a rash

suests a. a dianosis of measles.
. a dianosis of German measles. c. coneni
tal infection
with the ru
ella virus. d. passive transfer of maternal anti
odies to the infants
serum. 8. A
prenant woman is exposed to a child with a ru
ella infection. She had no cl
inical symptoms

ut had a ru
ella titer performed. Her anti
ody titer was 1:8. Three weeks lat
er, the test was
repeated, and her titer was 1:128. She still had no clinical symptoms. Was the l
a
oratory ndin
indicative of ru
ella infection? a. No, the titer must
e reater than 256 to
e
sini cant.
.
No, the chane in titer is not sini cant if no clinical sins are present. c. Yes
, a reater
than fourfold rise in titer indicates early infection. d. Yes,
ut clinical symp
toms must also
correlate with la
oratory ndins. 9. The most common cause of conenital infectio
ns is a. CMV.

. ru
ella. c. VZV. d. HTLV-I. 10. A positive result on a screenin test
for HTLV-I
anti
ody a. is hihly speci c for HTLV-I infection.
. should
e followed
y PCR.
c. must
e
con rmed
y Western
lot. d. must
e con rmed
y viral culture. REVIEW QUESTIONS
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, Van Dooren,
SV, Vandamme, A-M, Vanham, G, and Gatuzzo, E. Human T-lymphotropic virus I: Rece
nt knowlede
a
out an ancient infection. Lance Infect Dis 7:266281, 2007. 115. Lal, RB. Deline
ation of
immunodominant epitopes of human T-lymphotropic viruses types I and II and their
usefulness in
developin seroloic assays for detection of anti
odies to HTLV-I and HTLV-II.
J AIDS Hum
Retro 13(suppl 1):S170S178, 1996. 116. Proietti, FA, Carneiro-Proietti, ABF, Cat
alan-Soares,
BC, and Murphy, EL. Oncoene 24:60586068, 2005. 117. Thorstensson, R, Al
ert, J,
and Andersson,
S. Strateies for dianosis of HTLV-I and -II. Transfusion 42:780791, 2002. 118.
Araujo, A, and
Hall, WW. Human T-lymphotropic virus type II and neuroloical disease. Ann Neuro
l 56:1019, 2004.
1814_Ch22_369-398.qxd 7/13/09 2:58 PM Pae 398 23 La
oratory Dianosis of HIV
Infection Linda
E. Miller, PhD, I, MP(ASCP)SI 399 LEARNING OBJECTIVES After nishin this chapter,
the reader
will
e a
le to: 1. Explain conditions under which transmission of human immunod
e - ciency virus
(HIV) can occur. 2. Descri
e the makeup of the HIV particle. 3. Differentiate th
e three main
structural enes of HIV and their products. 4. Descri
e replication of the HIV v
irus. 5. Descri
e
the effects of HIV on the immune system. 6. Discuss antiretroviral treatments an
d the impact they
have had on HIV infection. 7. Discuss ow cytometric methods for CD4 T-cell enumer
ation. 8.
Discuss the roles of various la
oratory tests in dianosin and monitorin HIV i
nfection. 9.
Compare rst-eneration, second-eneration, third-eneration, and fourth-eneratio
n enzyme-linked
immunosor
ent assay (ELISA) tests for HIV anti
ody. 10. Give reasons for false p
ositives and
false neatives in HIV anti
ody testin. 11. De ne positive predictive value and r
elate this to
HIV anti
ody testin. 12. Descri
e the Western
lot test. 13. Interpret a Wester
n
lot test,
iven the types of reactive
ands. 14. Discuss advantaes and disadvantaes of p
24 antien
testin. 15. Differentiate
etween reverse transcriptase polymerase chain reacti
on (RT-PCR),
real-time RT-PCR,
ranched DNA (
DNA) ampli cation, and nucleic acid sequence-
ase
d ampli cation

(NASBA) testin for HIV nucleic acid. 16. Discuss the clinical utility of HIV vi
ral load testin
and dru-resistance testin. 17. Discuss the protocol for HIV testin of infants
and children
youner than 18 months of ae. KEY TERMS AIDS Amplicon Branched chain DNA CD4 T
cell ELISA Env
Flow cytometry Ga HAART HIV Hy
ridization NASBA p24 antien Pol Positive predic
tive value
Reverse transcriptase Seroconversion Viral load tests Western
lot test 1814_Ch2
3_399-426.qxd
7/10/09 5:20 PM Pae 399 400 SECTION 4 Seroloical Dianosis of Infectious Dis
ease Human
immunode ciency virus (HIV) is the etioloic aent of the acquired immunodeficienc
y syndrome, or
AIDS, a disease that has posed one of the reatest medical challenes worldwide.
Accordin to the
World Health Oranization, in 2007, an estimated 33.2 million people were livin
with HIV
infection, 2.5 million people
ecame newly infected, and 2.1 million people died
of AIDS. 1
Althouh the major- ity of infected persons reside in developin countries, HIV
infection has
also created a sini cant pro
lem in developed nations. For example, in the United
States, over 1
million cases of AIDS and more than 565,000 AIDS-related deaths were reported fr
om 1981 (when the
rst cases of AIDS were identi ed) throuh the year 2006. 2 Accurate dianosis is es
sen- tial for
early intervention and haltin the spread of the disease. This chapter emphasize
s techniques for
la
oratory diano- sis of HIV infection, presents some characteristics of the vi
rus, and outlines
immunoloic manifestations of the disease. The virus that is responsi
le for cau
sin AIDS, HIV-1,
was identified independently
y the la
oratories of Luc Montanier of France and
Ro
ert Gallo and
Jay Levy of the United States in 1983 and 1984 respectively. 35 It was formerly
called human
T-cell lymphotrophic virus-type III (HTLV-III), lymphadenopathy-associated
virus (LAV), and
AIDS-associated retrovirus (ARV). Isolates of HIV-1 have
een classified into th
ree roups: roup
M (the main or major roup), roup N (the non-M/non-O, or new roup), and roup
O (the outlier
roup). 6,7 Group M viruses are respon- si
le for the majority of HIV-1 infectio
ns worldwide.
This roup contains nine su
types or clades, desinated A throuh D, F th
rouh H, J, and
K. 8 Su
type C is the most predominant su
type worldwide, and su
type B is the m
ost prevalent
su
type in the United States and Europe. 810 Groups N and O are larely con n
ed to Central
Africa. A related
ut enetically distinct virus, HIV-2, was dis- covered in 198
6. 9 The majority
of HIV-2 infections have occurred in West Africa, althouh the virus has also
e
en identi ed in
patients in other parts of the world. 8 HIV-2 is transmitted in the same manner
as HIV-1 and may
also cause AIDS,
ut it is less pathoenic and has a lower rate of transmission.
6,8 Althouh

this chapter discusses the differ- ences
etween the two viruses, our focu
s is on HIV-1,

ecause it is much more prevalent throuhout the world. In this chapter, the ter
m HIV is used to
refer to HIV-1, and HIV-2 is so named. HIV TRANSMISSION Transmission of HIV occu
rs
y one of
three major routes: (1) intimate sexual contact; (2) contact with
lood or other

ody fluids;
or (3) perinatally, from infected mother to infant. 1012 The majority of
cases of HIV
infection have occurred
y sexual transmission, throuh either vainal or anal
intercourse.
Worldwide, a
out 85 percent of cases of HIV infection can
e attri
uted to heter
osexual contact,
while in the United States, the larest num
er of cases has resulted from anal i
ntercourse in
homosexual males. 2,1013 The pres- ence of other sexually transmitted diseases su
ch as syphilis,
onorrhea, or enital herpes increases the likelihood of trans- mission
y disru
ptin protective
mucous mem
ranes and increasin immune activation in the enital areas. 10,12,13
The second route
of transmission is
y parenteral expo- sure to infected
lood or
ody fluids. Th
is has occurred
throuh the sharin of contaminated needles
y intravenous dru users,
lood tra
nsfusions or the
use of clottin factors
y hemophiliacs, occupational injuries with needle stick
s or other sharp
o
jects, or
y mucous mem
rane contacts in health-care workers exposed to infect
ious uids.
12,14,15 The virus has also
een acquired
y transplantation of infected tissue.
Screenin of

lood and oran donors for HIV has dramatically decreased the incidence of infec
tion in recipients of
lood transfusions, clottin factors, and oran transplants to an
estimated one
transmission per 2 million
lood donations. 13,16 Studies
y the Centers for Dis
ease Control and
Prevention (CDC) have estimated the averae risk of transmission to health-care
workers to
e
approxi- mately 0.3 percent after a percutaneous exposure to CHAPTER OUTL
INE HIV
TRANSMISSION CHARACTERISTICS OF HIV Composition of the Virus Structural Genes Vi
ral Replication
IMMUNOLOGIC MANIFESTATIONS Immune Responses to HIV Effects of HIV Infection on t
he Immune System
CLINICAL SYMPTOMS TREATMENT LABORATORY TESTING FOR HIV INFECTION CD4 T-Cell Enum
eration Detection
of HIV Anti
ody Detection of HIV Antien Nucleic Acid Testin Virus Isolation Te
stin of Infants
and Youn Children SUMMARY CASE STUDY EXERCISE: SIMULATION OF HIV-1 DETECTION RE
VIEW QUESTIONS
REFERENCES 1814_Ch23_399-426.qxd 7/10/09 5:20 PM Pae 400 CHAPTER 23 La
orato
ry Dianosis of
HIV Infection 401 HIV-infected
lood and a
out 0.09 percent after a mucous mem
r
ane exposure.
Body fluids considered to
e poten- tially infectious include
lood, semen, vai
nal secretions,
cere
ral spinal uid, synovial uid, pleural uid, peritoneal uid, pericardial uid, amni
otic

uid, and other uids con- tainin visi
le
lood. Saliva, sputum, nasal secretions,
tears, sweat,
urine, vomitus, and feces are not considered to
e infectious unless they contai
n visi
le
lood.
14,15,17 The third route of transmission is perinatal, from infected mother to h
er fetus or
infant. Transmission
y this route can occur durin prenancy,
y transfer of
l
ood durin
delivery, or throuh
reastfeedin. 10,18 Perinatal transmis- sion has
een mark
edly reduced
throuh HIV screenin durin prenancy, administration of antiretroviral drus t
o HIV-positive
prenant women and their new
orn
a
ies, and use of infant formula
y mothers wh
o are infected
with the virus. These measures have reduced the rate of perina- tal transmission
to less than 2
percent, as compared to rates of 25 to 30 percent in untreated mothers. 18 CHARA
CTERISTICS OF HIV
Composition of the Virus HIV
elons to the enus Lentivirinae of the virus fami
ly Retroviridae.
11,12,19 It is classified as a retrovirus,
ecause it contains ri
onucleic acid
(RNA) as its
nucleic acid and a unique enzyme, called reverse transcriptase, that tran- scri

es the viral RNA
into DNA, a necessary step in the viruss life cycle. HIV is a spherical particle,
100 to 120 nm
in diameter, which contains an inner core with two copies of sinle-stranded RNA
, surrounded
y a
protein coat or capsid and an outer envelope of lycoproteins em
edded in a lipi
d
ilayer.
11,12,19,20 The lycoproteins are kno
like struc- tures that are involved in
in
din the virus to
host cells durin infection. Fiure 231 shows the structure of the HIV virion. St
ructural Genes
The enome of HIV includes three main structural enes a, env, and poland a
num
er of
reulatory enes. Fiure 232 shows the relative locations of the major HIV enes
and indicates
their ene products. The a ene codes for p55, a precursor protein with
a molecular
weiht of 55 kd, from which four core structural proteins are formed: p6, p9, p
17, and p24.
12,19 All four are located in the nucleocap- sid of the virus. The capsid that s
urrounds the
internal nucleic acids contains p24, p6, and p9, while p17 lies in a layer
etwe
en the protein
core and the envelope, called the matrix, and is actually em
edded in the intern
al portion of the
envelope. 19 The env ene codes for the lycoproteins p160, p120, and p41, wh
ich are found in
the viral envelope. Gp160 is a precursor protein that is cleaved to form p120 a
nd p41. Gp120
forms the numerous kno
s or spikes that protrude from the outer envelope, while
p41 is a
transmem
rane lycoprotein that spans the inner and outer mem
rane and attaches
to p120. Both
p120 and p41 are involved with fusin and attachin HIV to receptors on host c
ells. 11,12,19
The third structural ene, pol, codes for enzymes neces- sary for HIV replicatio
n, 11,12,19

namely p66 and p51. These are su
units of reverse transcriptase p31, or interas
e, which mediates interation of the viral DNA into the enome of infected host cells, and of
p10, a protease
that cleaves protein precur- sors into smaller active units. The p66 protein is
also involved in
deradation of the oriinal HIV RNA. These proteins are located in the core, in
association with
the HIV RNA. Several other enes in the HIV enome code for products that have r
eulatory or
accessory functions. 11,12,19 Althouh these products are not an interal part o
f the viral
structure, they serve important functions in controllin viral replica- tion and
infectivity. The
tat (transactivator) ene codes for p14, a reulatory protein that activates tra
nscription of HIV
proviral enes. Rev (which reulates expression of virion proteins) codes for p1
9, a protein that
transports viral RNA out of the nucleus and into the cytoplasm for translation.
Nef codes for
p27, which has multiple functions, includin modi cation of the host cell to enhan
ce viral
replication and make it less likely to
e destroyed
y the immune system. Vpu (v
iral protein U)
codes for p16, a protein with mul- tiple roles, includin ef ciently assem
lin an
d
uddin the
virions off infected host cells and promotin host cell death. Vpr (viral protei
n R) codes for
p15, which helps interate HIV DNA into the host cell nucleus. Vif codes for p23
, which acts as a
viral infectivity factor
y sta
ilizin newly synthesized HIV DNA and facilitati
n its transport
to the nucleus. Ta
le 231 summarizes the major HIV-1 enes, their products, and t
heir functions.
HIV-2 has a, env, pol, and reulatory/accessory enes that have similar functi
ons to those seen
in HIV-1. The homoloy
etween the enomes of the two viruses is approxi
mately 50
percent. 19,21 The a and pol reions are most similar, while the env reion di
ffers reatly.
Thus, the viruses can most easily
e distinuished on the
asis of anti- enic d
ifferences in
their env proteins. Western Blot Pattern p160 p120 envelope protein p66 revers
e transcriptase
p55 p51 p41 transmem
rane protein p31 interase p24 core protein p17 FIGURE 231
. Structure of
HIV virion, showin some of the major components. (Modi ed from Sloand, E, et al.
HIV testin:
State of the art. JAMA 266:2862, 1991, with permission.) 1814_Ch23_399-426.qxd
7/10/09 5:20 PM
Pae 401 402 SECTION 4 Seroloical Dianosis of Infectious Disease Viral Replica
tion The rst
step in the reproductive cycle of HIV is the virus attachin to a suscepti
le ho
st cell. This
interaction is medi- ated throuh the host-cell CD4 antien, which serves as a r
eceptor for the
virus
y
indin the p120 lycoprotein on the outer envelope of HIV. T he
lper cells are
the main taret for HIV infection,
ecause they express hih num-
ers of CD4 m
olecules on their

cell surface and
ind the virus with hih af nity. 12 Other cells such as macropha
es, monocytes,
dendritic cells, Lanerhans cells, and microlial
rain cells can also
e infect
ed with HIV,

ecause they have some surface CD4. HIV viruses that preferentially infect T cel
ls are known as
T-tropic or X4 strains, while those strains that can infect
oth macrophaes and
T cells are
called M-tropic or R5 strains. Entry of HIV into the host cells to which
it has
attached requires an additional
indin step involvin coreceptors that promote
fusion of the HIV
envelope with the plasma cell mem
rane. These coreceptors
elon to a family of
proteins known as
chemokine receptors, whose main function is to direct white
lood cells to sites
of inflammation.
The chemokine receptor CXCR4 is required for HIV to enter T lymphocyt
es, while the
chemokine receptor CCR5 is required for entry into macrophaes. 11,12,19
Bindin of the
coreceptors allows for HIV entry
y inducin a conformational chane in the p41
lycoprotein,
which mediates fusion of the virus to the cell mem
rane. After fusion occurs, th
e viral particle
is taken into the cell, and uncoatin of the particle exposes the viral enome.
11,12,19 Action
of the enzyme reverse transcriptase produces complementary DNA from the
viral RNA.
This DNA
ecomes interated into the host cells enome and is called a provirus
(Fi. 233).
The provirus can remain in a latent Ta
le 55-2 Comparison of Tests Used GENE PRO
TEIN PRODUCT
FUNCTION a p17 Inner surface of envelope p24 Core coat for nucleic acids p9 Co
re-
indin
protein p7 Binds to enomic RNA env p120 Binds to CD4 on T cells p41 Transmem

rane protein
associated with p120 pol p66 Su
unit of reverse transcriptase; derades oriina
l HIV RNA p51
Su
unit of reverse transcriptase p31 Interase; mediates interation of HIV DNA
into host enome
p10 Protease that cleaves a precursor tat p14 Activates transcription of HIV p
rovirus rev p19
Transports viral mRNA to the cytoplasm of the host cell nef p27 Enhance HIV repl
ication vpu p16
Viral assem
ly and
uddin vpr p15 Interation of HIV DNA into host enome vif p
23 Infectivity
factor RNA ri
onucleic acid Ta
le 23-1. Major HIV Genes and Their Products Codes
for
nucleocapsid and core proteins *Reulatory enes Codes for viral enzymes p55 pre
cursor p6 p9 p17
p24 Codes for viral envelope proteins p160 precursor p120 p160 p66 p51 p31 p1
0 5LTR 3LTR pol
vpr* vif* vpu* rev* tat* env nef* a FIGURE 232. The HIV-1 enome. The relat
ive locations
of the major enes in the HIV-1 enome are indicated, as well as their ene prod
ucts.
1814_Ch23_399-426.qxd 7/10/09 5:20 PM Pae 402 CHAPTER 23 La
oratory Dianosi
s of HIV
Infection 403 state for a lon period of time, durin which viral replica- tion
does not occur.

Eventually, expression of the viral enes is induced when the infected host cell
is activated
y

ind- in to antien or
y exposure to cytokines. Viral DNA within the cell nucl
eus is then
transcri
ed into enomic RNA and messener RNA (mRNA), which are transported to
the cyto- plasm.
Translation of mRNA occurs, with production of viral proteins and assem
ly of vi
ral particles.
The intact virions
ud out from the host cell mem
rane and acquire their enve- l
ope durin the
process. These viruses can then proceed to infect additional host cells. Viral r
eplication occurs
to the reatest extent in antien-activated T helper cells. Because viral replic
ation occurs very
rapidly, and the reverse tran- scriptase enzyme lacks proofreadin activity,
enetic
mutations commonly occur, producin distinct isolates that exhi
it antienic var
iation. These
isolates can vary in their suscepti
ility to the hosts immune responses. 19 IMMUN
OLOGIC
MANIFESTATIONS Immune Responses to HIV When HIV infects a healthy individual, th
ere is typically
an initial
urst of viral replication followed
y a slowin down of virus produc
tion as the
hosts immune response develops and keeps the virus in check. 12,1921 This initial
viral
replication can
e detected in the la
oratory
y the pres- ence of increased lev
els of p24
antien and viral RNA in the hosts
loodstream (see discussion later). As the vir
us replicates,
some of the viral proteins produced within host cells form complexes with major
histocompati
ility com- plex (MHC) class I antiens and are transported to the c
ell surface,
where they stimulate lymphocyte responses. While HIV can stimulate
oth humoral
and cell-mediated
immune responses, the interactions
etween HIV and the immune system are complex
, and the
relevance of the responses enerated to clearin the virus are not completely un
der- stood, 20,21
as will
e discussed
elow. B lymphocytes are stimulated to produce anti
odies t
o HIV, which can
usually
e detected in the hosts serum
y 6 weeks after primary infection. 1921 Th
e rst
anti
odies to
e detected are directed aainst the a proteins such as p24, fol
lowed
y
production of anti
odies to the envelope, pol, and reulatory proteins. The most
immunoenic proteins are in the viral envelope and elicit the production of neutralizin anti
o
dies. These
anti
odies appear within the rst 6 months of infection and prevent the virus from
infect- in
neih
orin cells. Anti
odies to the envelope proteins have also
een shown to

ind to FC
receptors on NK cells and participate in ADCC-mediated killin of HIV-infected c
ells. However,
the in vivo role of these anti
odies is unclear, as they are una
le to completel
y eliminate the
virus from the host, for reasons that are discussed
elow. T cellmediated immunit
y is thouht to
play an important role in the immune response to HIV, as it does in other viral

infections. 1923
CD8 cytotoxic T lymphocytes, also known as cytolytic T cells (CTLs), appear withi
n weeks of HIV
infec- tion and are associated with a decline in the amount of HIV in the
lood
durin acute
infection. While anti
odies can attach only to virions circulatin freely outsid
e of host cells,
CTLs can attack host cells har
orin viruses internally. This process involves t
he
indin of CTL
containin HIV-speci c antien receptors to HIV proteins associated with MHC class
I mol- ecules
on the surface of infected host cells. HIV-speci c CTL Translation into viral prot
eins assem
ly
Spliced transcripts Full-lenth transcripts Transcription mRNA Interation into
host DNA
Transport to nucleus Circular d.s. DNA Dou
le-stranded DNA cDNA Viral RNA Uncoat
in Buddin New
virion p120 CD4 Entry via CD4 receptor Cytoplasm Nucleus Proviral DNA 5 3 5
3 FIGURE
233. Replication cycle of HIV. (From Crowe, S, and Mills, J. Virus infections of
the immune
system. In Parslow, TG, et al: Medical Immunoloy. Lane Medical Books, McGraw-H
ill, New York,
NY, 2001, p. 646, with permission.) 1814_Ch23_399-426.qxd 7/10/09 5:20 PM Pa
e 403 404 SECTION
4 Seroloical Dianosis of Infectious Disease are stimulated to develop into mat
ure, activated
clones throuh the effects of cytokines released
y activated CD4 T helper cells
, a process that
is common to immune responses aainst other viruses (see Chapter 5 for details).
After the CTLs

ind to HIV-infected host cells, cytolytic enzymes are released from their ranu
les and destroy
the taret cells. Free virions are released from the damaed cells and can
e
o
und
y anti
odies. CTL can also suppress replication and spreadin of HIV
y producin cyto
kines like
interferon- , which have antiviral activity. Effects of HIV Infection on the Immun
e System
Althouh the humoral and cell-mediated immune responses of the host usually redu
ce the level of
HIV replication, they are enerally not suf cient to completely eliminate the viru
s. This is

ecause HIV has developed several mechanisms
y which it can escape immune respo
nses. 1922,24
CTL and anti-
ody responses to HIV are hindered
y the viruss a
ility to undero
rapid enetic
mutations, eneratin escape mutants with altered antiens toward which the hosts
initial immune
responses are ineffective. In addition, HIV can down- reulate the produc
tion of MHC class
I molecules on the surface of the host cells it infects, protectin them from CT
L reconition.
HIV can also
e har
ored as a silent provirus for lon periods
y numerous cells
in the
ody,
includin restin CD4 T cells, dendritic cells, cells of the monocyte/macrophae
lineae, and
microlial cells in the
rain. In this proviral state, HIV is protected from att
ack
y the immune
system until cell activation stimulates the virus to multiply and display its vi

ral antiens. The

a
ility of HIV to evade the immune response results in a persistent infection th
at can destroy
the immune system. Because the viruss prime tarets are the CD4 T helper cells, t
hese cells are
most severely affected, and a decrease in this cell population is the hallmark f
eature of HIV
infection. 25 CD4 T helper cells are killed or rendered nonfunctional as a resul
t of HIV
infection. These effects occur from a variety of mech- anisms, includin loss of
plasma mem
rane
interity due to viral
uddin, destruction
y HIV-speci c CTL, and viral inductio
n of apoptosis.
19,22 Infected T cells turn over much more rapidly than they can
e replaced, ha
vin a half-life
of 12 to 36 hours. 22 In addition to reducin T-cell num
ers, HIV also causes a

normalities in T
helper cell function and impair- ment of memory T helper cell responses. 12,19,2
1,25 Because T
helper cells play a central role in the immune system
y reulatin the activiti
es of B and T
lymphocytes (see Chapter 5), destruction of these cells results in decreased eff
ectiveness of

oth anti
ody- and cell-mediated immune responses. HIV proteins actually stimula
te polyclonal
acti- vation of B cells, resultin in maturational and functional defects, with
increased
circulatin immunolo
ulin levels, immune complexes, and auto-anti
odies. 12,19,
21,26 However, B
cells in HIV-infected individuals have a reduced a
ility to mount anti
ody respo
nses after
exposure to specific anti- ens, due to the decrease in T-cell help. 12,19,21 Ce
ll-mediated
immunity is also affected
y the reduction in T helper activity, resultin in a
decline in CTL
activity and delayed-type hypersensitivity responses to specific antiens.
12,19,21,22,24
Altered production of cytokines and chemokines have also
een seen, includin
 increases in
the levels of some cytokines durin the early staes of disease, followed
y dec
linin levels of
IL-2 and interferon- and a shift in the cytokine pro le from Th1 to Th2 as the infe
c- tion
proresses toward development of AIDS. 12,19,22 Other immunoloic a
normalities,
includin
defective antien presentation and oxidative
urst
y monocytes/macrophaes and
decreased natural
killer (NK) cell activity, have also
een o
served in AIDS patients. 19,21,22,25
CLINICAL
SYMPTOMS HIV infection causes a chronic infection that is character- ized
y a
proressive
decline in the immune system. Althouh the manifestations of the disease var
y in individ- ual
patients, the infection proresses throuh a clinical course that
eins
with primary, or
acute, infection, followed
y a period of clinical latency and eventually culmin
atin in AIDS.
19,21,23 The acute, or early, stae of infection is char- acterized
y a rapid

urst of viral
replication prior to the development of HIV-specific immune responses. In this s

tae, hih levels

of circulatin virus, or viremia, can
e seen in the
lood of infected individua
ls, and HIV

eins to dis- seminate to the lymphoid orans. As the immune system
ecomes ac
tivated, an
acute retroviral syndrome may develop. This syndrome, which has
een noted in
50 to 70
percent of patients with primary HIV infection, is characterized
y fluli
ke or infectious
mononucleosis-like symptoms, such as fever, lymphadenopathy, sore throat, arthra
lia, myalia,
fatiue, rash, and weiht loss. 19,21,23,27 Symptoms of the primary stae usuall
y appear 3 to 6
weeks after initial infection and resolve within a few days to a few weeks. Some
patients are
asymptomatic durin this stae. As HIV-speci c immune responses develop, they
ei
n to curtail
replication of the virus, and patients enter a period of clinical latency. This
stae is
characterized
y a decrease in viremia as the virus is cleared from the circulat
ion, and clinical
symptoms are su
tle or a
sent. 19,21,23,27 However, studies have demonstrated th
at the virus is
still present in the plasma, al
eit at lower levels, and more so in the lym- pho
id tissues, where
it causes a radual deterioration of the immune system. The period of clinical l
atency can vary
widely
ut has a median lenth of 10 years in untreated patients. 21 A small pro
portion of
HIV-infected individuals, termed lon-term nonproressors (LTNP), have normal or
mildly depressed
CD4 T cell counts, low viral loads, and remain asymptomatic for more than 10 years
in the a
sence
of antiretroviral therapy. 19,28 The factors that influence this slower rat
e of proression
are not completely under- stood
ut appear to
e associated with certain HLA typ
es, prevalence
of the R5 strain of HIV, and infection at a youner ae. 19 1814_Ch23_3
99-426.qxd
7/10/09 5:20 PM Pae 404 CHAPTER 23 La
oratory Dianosis of HIV Infection 405
Untreated
individuals will ultimately proress to the nal stae of HIV infection, AIDS, whi
ch is
characterized
y pro- found immunosuppression, a resurence of viremia, and life
-threatenin
infections and malinancies. The rate at which individuals proress to the devel
opment of AIDS
varies,
ut proression typically occurs within a median time of 10 years after
initial
infection. 23,25 The rate of prores- sion has
een dramatically decreased
with the use
of antiretroviral therapies (see the section on treatment). The CDC rst de ned AIDS
as a
disease, at least mod- erately predictive of a defect in cell mediated immunity,
occurrin in a
person with no known cause for diminished resistance to that disease. 29 The de nit
ion has
een
revised several times over the years as more information has
een acquired a
out
HIV and as
additional la
oratory tests for HIV have
een developed. In the 1993 case de nitio

n, the CDC
classified HIV-infected adults and adolescents into nine cateories (A1 throu
h C3),
ased
on CD4 T-cell counts, in association with clinical conditions found in HIV inf
ection (Ta
le
232). 30 Accordin to this de nition, HIV- infected individuals are classified as h
avin AIDS if
they have an a
solute CD4 T lymphocyte count of less than 200/L or certain opport
unistic
infections or malinancies indicative of AIDS (Ta
le 233). Individuals with AIDS
are classi ed
in cateories A3, B3, C1, C2, or C3. In addition to opportunistic infections and
malinancies,
HIV-infected individuals often demonstrate neuroloical symptoms resultin from
the a
ility of
HIV to infect cells in the
rain. In early HIV infection, these symptoms may man
ifest as
foretfulness, poor concentration, apathy, psy- chomotor retardation, and withdr
awal, while
proression to late disease may result in confusion, disorientation, seizures, d
ementia, ait
distur
ances, ataxia, or paraparesis. 21,31 The CDC has also pu
lished a separat
e case de nition
for AIDS in children. 32 Symptoms of AIDS in infants include failure to thrive,
persistent oral
candidiasis, hepatosplenomealy, lymphadenopathy, recurrent diarrhea, or recurre
nt
acterial
infections. 33,34 In addition, a
normal neuroloical ndins may
e present. The r
ate
y which
HIV infection proresses in chil- dren varies and may
e in uenced
y factors such
as maturity of
the immune system at the time of infection, the dose of virus to which the child
was exposed, and
the route of infection. 34 Since the advent of new antiretroviral therapies that
have delayed
proression to AIDS (see discussion in Treatment section), the CDC now tracks indi
viduals who
are HIV- positive
ut who have not developed AIDS in addition to followin peopl
e with AIDS. As a
result, they pu
lished a revised surveillance case definition for HIV infection
in 1999. 35
Accordin to this de nition, adults, adolescents, and children aed 18 months or o
lder are
considered to
e HIV- infected if they meet the previously pu
lished clin
ical criteria 30
or if they demonstrate positive test results on screenin and con rmatory te
sts for HIV
anti
ody or on an HIV viroloical test (i.e., HIV nucleic acid detection, HIV p2
4 antien test,
or HIV isolation in culture). The principles of these tests will
e discussed in
the La
oratory
Testin for HIV Infection section. TREATMENT Treatment of HIV infection involves
supportive care
of the infections and malinancies and administration of antiretro- viral drus
to suppress the
viruss replication. Several classes of antiretroviral drus have
een developed t
o treat HIV
infection: nucleoside analoue reverse transcriptase inhi
itors, nonnucleoside
reverse
transcriptase inhi
itors, protease inhi
itors, fusion inhi
itors, coreceptor a

ntaonists, and
inte- rase inhi
itors. 21,3638 These drus
lock various steps of the HIV replic
ation cycle.
Ta
le 23-2. 1993 Revised Classi cation System for HIV Infection and Expanded AIDS
Surveillance
Case De nition for Adolescents and Adults CLINICAL CATEGORIES (A) (B) (C) CD4 T-CEL
L
ASYMPTOMATIC, ACUTE SYMPTOMATIC, NOT AIDS-INDICATOR CATEGORIES CATEGORIES (PRI
MARY) HIV OR PGL*
(A) OR (C) CONDITIONS CONDITIONS (1) 500/ L A1 B1 C1 (2) 200499/ L A2 B2 C2 (3) 200/ L
(AIDS- A3 B3 C3 indicator T-cell count) *PGL = persistent generalized lymphadenopat
hy.
Clinical Category A includes acute (primary) HIV in
ection. These cells illustrat
e the expanded
AIDS surveillance case de nition. Persons with AIDS-indicator conditions (Category
C) as well as
those with CD4 T lymphocyte counts 200/ L (Categories A3 or B3) are reportable as AI
DS cases in
the United States and Territories, e

ective January 1, 1993. (For more in

ormat
ion, see Centers

or Disease Control and Prevention: 1993 revised classi cation system

or HIV in
e
ction and
expanded surveillance case de nition
or AIDS among adolescents and adults. MMWR 4
1 (RR-17):119,
1992.) 1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page 405 406 SECTION 4 Serologic
al Diagnosis o

In
ectious Disease The nucleoside analogue reverse transcriptase inhibitors are
similar in
structure to nucleosides and inhibit
urther action o
the reverse transcriptase
enzyme when they
incor- porate themselves into the viral DNA being generated. This class o
drugs
includes
zidovudine (also known as ZDV or azidothymidine [AZT]), lamivudine (deoxythiacyt
idine or 3TC),
didanosine (dideoxyinosine or ddI), abacavir (ABC), and teno
ovir (TDF). Another
class o
drugs,
the nonnucle- oside reverse transcriptase inhibitors, stops reverse transcr
iptase
rom
transcribing RNA into DNA by binding Table 23-3. Clinical Categories o
HIV In
e
ction Category A
One or more o
the conditions listed
ollowing in an adolescent (13 years) or adu
lt with
documented HIV in
ection. Conditions listed in Categories B and C must not have
occurred.
Asymptomatic HIV in
ection Persistent generalized lymphadenopathy Acute (primary
) HIV
in
ection with accompanying illness or history o
acute HIV in
ection Category B
Symptomatic
conditions in an HIV-in
ected adolescent or adult that are not included among co
nditions listed
in Category C and that meet at least one o
the
ollowing criteria: (1) conditio
ns are attributed
to HIV in
ection or are indicative o
a de
ect in cell-mediated immunity or (2)
conditions
considered by physicians to have a clinical course or require management that is
complicated by
HIV in
ection. Examples include: Bacillary angiomatosis Candidiasis, oropharynge
al (thrush)
Candidiasis, vulvovaginal; persistent,
requent, or poorly responsive to therapy
Cervix

dysplasia (moderate or severe)/cervix carcinoma in situ Constitutional symptoms,

such as
ever
(38.5C) or diarrhea lasting >1 month Hairy leukoplakia, oral Herpes zoster (shing
les),
involving at least two distinct episodes or more than one dermatome Idiopathic t
hrombocytopenic
purpura Listeriosis Pelvic in ammatory disease Peripheral neuropathy Category C
Candidiasis o
bronchi, trachea, or lungs Candidiasis, esophageal Cervical cance
r, invasive
Coccidiomycosis, disseminated or extrapulmonary Cryptococcosis, extrapulmonary
Cryptosporidiosis, chronic intestinal (>1 months duration) Cytomegalovirus (other
than liver,
spleen, or nodes) Cytomegalovirus retinitis (with loss o
vision) Encephalopathy
, HIV-related
Herpes simplex: chronic ulcer(s) (>1 months duration); or bronchitis, pneumonitis
, or
esophagitis Histoplasmosis, disseminated or extrapulmonary Isosporiasis, chronic
intestinal
(>1 months duration) Kaposis sarcoma Lymphoma, Burkitts Lymphoma, immunoblastic
Lymphoma, primary, o
brain Mycobacterium avium complex or M. kansasii, dissemin
ated or
extrapulmonary Mycobacterium tuberculosis, any site Mycobacterium, other species
or
unidenti ed species, disseminated or extrapulmonary Pneumocystis carinii pneumonia
Pneumonia,
recurrent Progressive multi
ocal leukoencephalopathy Salmonella septicemia, recu
rrent
Toxoplasmosis o
brain Wasting syndrome due to HIV For more in
ormation, see Re

erence 30.
1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page 406 CHAPTER 23 Laboratory Diagnosi
s o
HIV
In
ection 407 directly to the enzyme and rendering it inactive. This class o
dr
ugs includes
nevirapine (NVP), delavirdine (DLV), and e
avirenz (EFV) and etravirine. The pro
tease inhibitors
pre- vent the cleavage o
precursor proteins necessary
or the assembly and rele
ase o
HIV
virions during the last stage o
the viral reproductive cycle. These drugs inclu
de saquinavir
(SQV), indinavir (IDV), ritonavir (RTV), nel navir (NFV),
osamprenavir, and lopin
avir. Fusion
inhibitors, such as en
u- virtide (T20), block entry o
HIV into host ce
lls by
preventing
usion o
the HIV membrane with the target cell membrane. Coreceptor
antagonists like
maraviroc (MVC) block the binding o
HIV to the chemokine coreceptor nec- essary

or penetration
o
the host cell. Integrase inhibitors such as raltegravir inhibit integration o

HIV DNA into

the host genome. New drugs continue to be developed as advances in this
area are made.
(Updated guidelines on the use o
these drugs are available in re
erence 38.) St
udies have shown
that treatment with multiple drugs is more e

ective in killing the virus and av

oiding viral
resist- ance than treatment with a single drug. Potent regimens involving a comb
ination o
drugs

rom at least two o

the drug classes mentioned earlier are the standard o
trea
tment and are
re
erred to as HAART, or highly active antiretro- viral therapy. 36,38 Currently

pre
erred
treatment protocols use combinations o
two nucleoside reverse transcriptase inh
ibitors and
either a nonnucleoside reverse transcriptase inhibitor or a protease inhibitor.
38 The goal o

this therapy is to reduce the patients HIV viral load, consequently boost- ing th
e patients
level o
immunocompetence. This is most likely to be achieved i
treatment is st
arted early in
the course o
in
ection and the patient can adhere to the treatment as prescribe
d. 23,36 HAART
has had a dramatic e

ect on the clinical course o

HIV in
ection, as evidenced
by a signi cant
decline in the incidence o
opportunistic in
ections, a delay in progression to
AIDS, and
decreased mortality in patients who have received this multidrug treatment.
3840 Retroviral
drugs have also had a signi cant impact in reducing perinatal transmis- sion o
HI
V, as discussed
previously in this chapter. 18 In 1994, investigators
rom the United States and
France published
the results o
a large clinical trial demonstrating that ZDV administered to HIV
-positive women
during pregnancy and labor and to the newborn during his or her rst
ew weeks o

li
e reduced
transmission o
HIV to the in
ant by two-thirds. 41 As a result o
this and subs
equent studies,
antiretroviral drugs and advice to avoid breast
eeding are recommended routinely

or pregnant
women who are HIV-positive. 18 Although antiretroviral drugs and HAART have sign
i - cantly
improved morbidity and mortality in HIV-in
ected patients, they cannot be co
nsidered a cure

or AIDS. Research has shown that while blood levels o

the virus are greatly
reduced in
patients treated with antiretroviral drugs, HIV is still harbored in lymphoid or
gans throughout
the body and still progressively destroys the immune system. 19,21 Viral resista
nce to the drugs
may also develop, and some patients may be unable to take the drugs because they
can- not
tolerate the side e

ects. There
ore, many approaches
or dealing with this viru
s have been
directed toward measures to prevent initial in
ec- tion
rom occurring. Communit
y-based education
aimed at high-risk groups such as homosexual males and intravenous drug users ha
s provided
bene cial in
ormation on reducing transmission o
the virus. In addition, th
e CDC and the
Occupational Sa
ety and Health Administration have published precautions to prev
ent transmission
o
HIV and other bloodborne pathogens in health-care workers. 42,43 Prophylactic
therapy with
antiretroviral drugs is also o

ered to health-care workers who may have been ex

posed to HIV
through percutaneous or mucous membrane contact with potentially in
ected blood
or body uids, in
hope that early treatment will prevent in
ection. 44 The ultimate means o
preve
nting HIV
in
ection is the development o
an e

ective vaccine. Some o

the strategies use

d in developing
an HIV vaccine include subunit vaccines, which consist o
recombinant HIV envelo
pe glycoproteins;
live vector-based vaccines, which use other viruses that have been genetically a
ltered to carry
HIV genes; and DNA vac- cines produced
rom DNA that codes
or HIV proteins. 4547
Much research
has been directed in this area, but the task has been very di
cult
or many reaso
ns, including
the ability o
HIV to rapidly mutate and escape immune recognition, the capabili
ty o
HIV to
persist despite vigorous immune responses o
the host, genetic variability in
HIV clades, the
need to induce mucosal immunity because HIV is usually transmitted through mucos
al sur
aces, the
need to induce potent CTL and antibody responses, and the lack o
an ideal anima
l model.
20,21,4547 I
research is unable to produce a vac- cine that can prevent HIV in
e
ction in the
traditional sense, it is possible that a less-than-per
ect vaccine may provide s
ome bene
its by
prolonging the disease-
ree period and reducing transmission o
this devastating
virus. 20
LABORATORY TESTING FOR HIV INFECTION Four types o
laboratory tests have been us
ed in diagnosing
and monitoring HIV in
ection: CD4 T-cell enumeration, antibody detection, antige
n detection, and
testing
or viral nucleic acid. Culturing
or the virus, although a de nitive meth
od o

demonstrating HIV in
ection, has been used pri- marily in research settings.
Principles o

each o
these methods are discussed along with their particular applica- bilit
y to diagnosis at
various stages o
the disease. CD4 T-Cell Enumeration Destruction o
the CD4 T l
ymphocytes is
central to the immunopathogenesis o
HIV in
ection, and CD4 lymphope- nia has lo
ng been
recognized as the hallmark
eature o
AIDS. There
ore, enumeration o
CD4 T
cells in the
peripheral blood has played a central role in evaluating the degree o
immune su
ppression in
HIV-in
ected patients
or several years. In untreated patients, there is a progr
essive decline in
the number o
CD4 T cells during the course o
1814_Ch23_399-426.qxd 7/10/09 5
:20 PM Page 407
408 SECTION 4 Serological Diagnosis o
In
ectious Disease in
ection (Fig. 234). A
s discussed
earlier, the CDC has used CD4 T-cell counts to classi
y patients into various st
ages o
HIV
in
ection, with those whose counts are below 200/L being classi ed as having AIDS.
30 In
addition, CD4 T-cell counts are used routinely to monitor the e

ective- ness o

antiretroviral
therapy. It is recommended that CD4 T-cell measurements be per
ormed every 3 to
6 months in
HIV-in
ected patients to guide physicians in determining when antiviral therapy
should be
initiated, whether a change in therapy is necessary, and i
prophylactic drugs

or certain
opportunistic in
ections should be administered. 21,4850 According to publish

ed guidelines,
antiretroviral therapy should be initiated in patients whose CD4 T-cell count is
less than
350/L; therapy should be changed i
CD4 T-cell counts decline more than 25 percen
t. 21,49 The
gold standard
or enumerating CD4 T cells is immunophenotyping with data
analysis by
low
cytome- try. The basic principle o
this method involves incubating whole periph
eral blood with a
panel o
uorescent-labeled monoclonal antibodies, removing the erythrocytes by ly
sis, and
stabilizing the leukocytes by xation with para
ormalde- hyde. 5052 The results are
analyzed via
histograms that display the patterns o
light scatter and uorescence emitted by i
ndi- vidual
cell populations. The CDC has published guidelines to standardize the per
ormanc
e o
CD4 T-cell
determina- tions by
low cytometry. 50,51 Early guidelines re
erred to a dual pl
at
orm technology
in which both a
low cytometer and a hematology analyzer are required to make CD
4 T- cell
measurements. According to this protocol, the percentage o
CD4 T cells in
a sample is
determined by dividing the number o
lymphocytes positive
or the CD4 marker by
the total number
o
lymphocytes counted by the ow cytometer, according to the
ollowing equation:
%
CD4 T cells
#
CD4 lymphocytes
100 total # lymphocytes In this determination, three- or
our-color immu
no u- orescence
assays are used in which the lymphocytes are di

erentiated
rom other cell type
s in the sample
by their side scatter and CD45 staining properties. Lymphocytes are identi ed on t
he basis o

their low side scatter (which rep- resents the amount o

cell complexity as dete
rmined by
properties such as granularity and membrane irregularity) and their ability to s
tain brightly
with
luorescent-labeled antibody to the CD45 marker, which is present on all wh
ite blood cells.
The percentage o
CD4 T cells obtained
or the patient sample is compared to a r
e
erence range
established by the laboratory per
orming the test. Absolute numbers o
CD4 T cel
ls are calculated
by multiplying the absolute number o
lymphocytes (deter- mined by the whole
blood cell
[WBC] count and di

erential
rom a hematology analyzer) by the percent- age
o
CD4 T cells
in the sample, according to the
ollowing equation: Absolute # CD4 T cells
WBC count
%
Lymphocytes
% CD4 T cells The absolute CD4 T-cell count is then compared to the
re
erence
range, which is typically
rom 500 to 1300 cells/L peripheral blood. 53 Newer tec
hnologies
employ three- or
our-color mon- oclonal antibody panels in a single-plat
o
rm approach,
which allows CD4 T-cell percentages and absolute num- bers to be obtained
rom o
ne tube using a
single instrument, the ow cytometer. 49,50 This is made possible by counting CD4 T
cells in a

precisely measured blood volume or by incubating the sample with a known number
o
commer- cially
available
luorescent microbeads, which
unction as an internal calibrator. The
counts can then
be determined by speci
ic
low cytometry so
tware, according to the
ol- lowing
equation: 50 C D
4
T
c e l l s
/
m m 3 P l a s m a
v i r e m i a
t i t e r 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 1:512 1:2
56 1:128 1:64 1:32
1:16 1:8 1:4 1:2 0 0 3 6 9 12 1 2 3 4 5 6 Years Weeks Clinic
al latency 7 8
9 10 11 Primary in
ection Opportunistic diseases Possible acute HIV syndrom
e Wide
dissemination o
virus Seeding o
lymphoid organs Constitutional symptoms Death
FIGURE 234.
Typical CD4 T cells numbers and plasma viremia during the natural course o
HIV
in
ection. (From
Pantaleo, G, Graziosi, C, and Fauci, AS. The immunopathogenesis o
human immunod
e ciency virus
in
ection. N Engl J Med 328:327335, 1993.) 1814_Ch23_399-426.qxd 7/10/09 5:20 P
M Page 408
CHAPTER 23 Laboratory Diagnosis o
HIV In
ection 409
#
o
events in the bright CD45 region o
events in the micro uorosphere region
Total # microspheres added Volume o
blood added As with the dual-plat
o
rm technology,
lymphocytes are selected
or analysis (or gated) on the basis o
their low side sc
atter and
ability to stain brightly with CD45 antibody. In addition to CD4 T-cell percentag
es and absolute
numbers, the ratio o
CD4 T cells to CD8 T cells may be reported to assess the d
ynamics between
the two T-cell populations. In HIV-in
ected patients, particularly those with AI
DS, the large
decrease in the number o
CD4 T cells, along with a possible increase in CD8
cyt
otoxic T cells,
results in an inverted ratio, or a ratio that is less than 1:1. While ow cytometr
y is the
accepted gold standard
or enumeration o
CD4 T cells, it is a costly method that
requires the
need
or highly skilled personnel. Less expen- sive methods o
lower complexity
have also been
developed to determine CD4 T-cell measurements. These include microcapillary ow c
ytometry, ow
cytometry dedicated
or CD4/CD8 measurements, a latex bead-based method read by
light microscopy,
magnetic bead cell isolation with u- orescent or light microscopy, CD4/CD8 enumer
ation with an
automated hematology analyzer, and an ELISA that measures CD4 protein in l
ysed whole blood.
49,5456 Some o
these methods may be suitable
or resource-limited labora- tories

, particularly
those in developing countries. Detection o
HIV Antibody Serological tests
or H
IV antibody have
many important pur- poses. 57 Since 1985, these tests have played a critical rol
e in screening
the donor blood supply to prevent transmission o
the virus through blood trans

usions or
administration o
blood products. In addition, serological tests are typically u
sed in the
initial diagnosis o
HIV in
ection, because most individuals will develop antibo
dy to the virus
within 1 to 2 months a
ter exposure. 57,58 Serological tests are also used in ep
idemiology
studies to provide health o
cials with in
ormation about the extent o
the in
ect
ion in
high-risk populations; these groups can then be targeted
or counseling, treatme
nt, vaccine
trials, and their medical or social concerns can be addressed. HIV antibody test
s can be divided
into two groups: (1) screening tests, whose goal is to detect all in
ected perso
ns, and (2)
con rmatory tests, per
ormed on samples giving a positive result on a screenin
g test, to
di

erentiate true- positive

rom
alse-positive results. The standard screenin
g method
or HIV
antibody has been the ELISA, and the stan- dard con rmatory test is the Western bl
ot. In addition
to the standard tests, rapid tests have been developed that can detect HIV antib
ody within
minutes, making them an attractive alternative to the ELISA in certain situation
s. Modi cations
o
these tests, which use saliva or urine or are available as home test kits, ha
ve also been
developed. The principles o
these methods are discussed below. Screening Tests
Using ELISA
Methodology Principles o
ELISA Using ELISAs (enzyme-linked immunosorbent assays
) to detect HIV
antibody was rst implemented in the United States in 1985 in response to the need
to screen
donated blood to curtail spread o
the in
ection. ELISAs have been the cornersto
ne o
screening
procedures
or HIV, because they are easy to per
orm, can be adapted to test a l
arge num- ber o

samples, and are highly sensitive and speci

ic. 21,57,58 Several manu
acturers h
ave developed
commercial kits that are use
ul in screening blood products and in diagnosing an
d monitoring
patients. 59 Over the years, technical advances have been made in these ELISAs,
resulting in
improved sen- sitivity and speci city. The rst-generation o
ELISAs was developed b
ased on a
solid-phase, indirect-assay system that detected antibod- ies to only HIV-1. 57,
58 (See Chapter
10
or general principles o
ELISA.) In these tests, HIV antibodies in patient s
erum were
detected a
ter binding to a solid support coated with viral lysate antigens
rom
HIV-1 cultured
in human T-cell lines,
ollowed by adding an enzyme-labeled conjugate and substr
ate. These
rst-generation assays were prone to
alse- positive results caused by reactions w

ith HLA
antigens or other components
rom the cells used to culture the virus, and they
were unable to
detect antibodies to HIV-2. 57,58 The second-generation ELISAs, introduced in th
e late 1980s,
were indirect binding assays that used highly puri- ed recombinant (i.e., genetic
ally
engineered) or synthetic antigens
rom both HIV-1 and HIV-2, rather than crude c
ell lysates.
57,58 These assays demonstrated improved speci-
icity and sensitivity overal
l and were
able to detect antibodies to both HIV-1 and HIV-2. However, decreased sensitiv
ity resulted when
samples containing antibodies to certain subtypes o
HIV that lacked the limited
antigens used in
the assays were tested. Third-generation assays use the sandwich technique,
based on the
ability o
antibody to bind with more than one antigen. 57,58 In this method, an
tibodies in
patient serum or plasma bind to recombinant HIV-1 and HIV-2 proteins coated onto
the solid phase.
A
ter washing, enzyme-labeled HIV-1 and HIV-2 antigens are added and bind to the
already bound
HIV-speci
ic patient antibodies. A
ter substrate is added, color development is
proportional to
the amount o
antibody in the test sample. This
ormat improved sensitiv- ity by
simultaneously
detecting HIV antibodies o
di

erent immunoglobulin classes, including IgM. Enh

ancements o
this
method have increased sensitivity
urther by detecting low a
nity antibodies and
antibodies to
group O HIV-1 and the more common group M. These enhancements resulted in a diag
nostic
sensitivity o
100 percent and diagnostic speci- city o
99.9 percent. 60 Most re
cently,

ourth-generation assays have been devel- oped that can simultaneously detec
t HIV-1
antibodies, HIV-2 antibodies, and p24 antigen. 57,58 These combination assays al
low
or slightly
earlier detection o
HIV in
ection 1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page
409 410 SECTION
4 Serological Diagnosis o
In
ectious Disease than the third-generation assay
s, because they
include the p24 antigen (see
ollowing). One study evaluating such a kit
ound
that the

ourth-generation assay could detect HIV in

ection 6.3 days earlier than an esta
blished thirdgeneration assay. 61 In addition, ELISAs with reduced sensitivity, known as detun
ed assays,
have been used to di

erentiate a recent HIV in

ection
rom a more established i
n
ection.
Individuals with a more recent in
ection (less than 4 months) will give a positi
ve result only
with a standard, highly sensitive ELISA test and a negative result with the less
sensitive,
detuned assay, while persons with a more established in
ection will give positiv
e results in both
assays. 21,57,58,62 Interpretation o
ELISA Results While ELISAs have a high lev
el o
sensitivity
and speci city, they may sometimes give erroneous results. False-negative results

occur
in
requently but may be due to the collection o
the test serum prior to the pat
ient developing
HIV anti- bodies (i.e., prior to seroconversion), to administration o
immunosup
pressive therapy
or replacement trans
usion, to conditions o
de
ective antibody synthesis such a
s hypogammaglobulinemia, or to technical errors attributed to improper handling o
kit re
agents. 58,63
False-negative results may also occur i
the patient harbors a genetically diver
se, recombi- nant
strain o
HIV, or an HIV-1 group O strain that is tested
or by an assay that do
es not detect
antibody to group O virus. The likelihood o

alse negatives occurring because t
he assay was
per
ormed prior to seroconversion has been reduced by the technical advances o

the newer
generation ELISAs, which can detect HIV antibodies 3 to 6 weeks a
ter in
ection.
63
False-positive results may also occur in HIV-antibody ELISA tests. These can res
ult
rom several

actors, includ- ing heat inactivation o

serum prior to testing, repeated
reez
ing/thawing o

specimens, presence o
autoreactive antibodies, history o
multiple pregnancies,
severe hepatic
disease, passive immunoglobulin administration, recent exposure to certain v
accines, and
certain malignancies. 58 The rate o

alse positives is substantially higher in
low-risk populations than in high-risk populations; in other words, in low-risk populations,
the ELISAs have
a low positive pre- dictive value, or probability that the patient truly has the
disease i
the
test result is positive. For example, the posi- tive predictive value o
a repea
tedly reactive
HIV ELISA was estimated to be only 13 percent in a large study done by the Ameri
can Red Cross on
low-risk blood donors, mean- ing that only 13 percent o
all positives were true
positives, and
87 percent were
alse positives in this population. 58,64 Any positive results o
btained by ELISA
must there
ore be con rmed by additional testing. According to a commonly accepted
testing
algorithm established by the CDC, when a sample screened
or HIV antibody by ELI
SA yields a
positive result, it should be retested in duplicate by the same ELISA test. I
t
wo out o
the
three specimens are reactive, then the results must be con rmed by a more speci c me
thod, usually
Western blot (see
ollowing). 58,65 Repeatedly reactive units o
blood are not u
sed
or
trans
usion, regardless o
results o
con rmatory testing. Rapid Screening Tests W
hile ELISAs are
ideal screening tests
or HIV antibodies in clinical laboratories that per
orm l
arge-volume batch
test- ing, they require complex instrumentation and skilled personnel with
technical
expertise, and typically have a turn- around time o
a
ew days. To overcome the
se limitations
and to encourage more patients to be tested, advances in technology have led to

the development
o
rapid and sim- pler methods to screen
or HIV antibody. Because these tests a
re highly
sensitive, simple to per
orm, and provide results in less than 30 minutes, they
are used
throughout the world. Rapid tests are ideal
or use in resource-limited settings
in developing
nations and in situations in which
ast noti cation o
test results is desired. 57
,63,66 For
example, rapid results are important in guiding decisions to begin prophy- lacti
c therapy with
antiretroviral drugs
ollowing occupational exposures, as this therapy appears t
o be most
e

ective when administered in the rst

ew hours
ollowing exposure. Other situat
ions in which
rapid tests are very bene cial include test- ing women whose HIV status is unknown
during labor
and delivery, and testing patients in sexually transmitted disease clinics or em
ergency
departments who are unlikely to make a return visit
or their test results. Stud
ies conducted by
the CDC and the World Health Organization have shown that the rapid tests
or HI
V anti- body
have a high level o
sensitivity and speci
icity, comparable to that o
t
he standard ELISA
tests. 67,68 At the time o
this writing, there are six rapid tests commercially
available in the
United States that are approved by the FDA. 69 These kits can be used to test se
rum, plasma,
whole blood, and oral uid (in one method). While each test has unique
eatures,
all are
lateral
low or
low-through immunoassays that produce a colorimetric reactio
n in the case o

a positive result. The

low-through assays require multiple steps in which the s
ample and
reagents are added to a solid support encased in a plastic device, while the lat
eral-
low assays
involve a one-step procedure in which the patient sample migrates along the test
strip by
capillary action. With either procedure, the patients sample is applied t
o a test
strip or membrane containing HIV antigens. The antigenantibody complexes
bind to an
enzyme-labeled conjugate or an antibody-binding (protein A) colloidal gold conju
gate and are
detected by a colori- metric reaction that produces a colored line or dot in the
case o
a
positive result. 57,58,66,69 Interpretation o
the results is made through visua
l observation o

the test device and does not require instrumentation. A reactive result i
s reported as a
preliminary positive and, like the standard ELISA, requires con
irmation by a mo
re speci
ic test
like the Western blot because o
the possibility o

alse-positive results. 63,6
6
1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page 410 CHAPTER 23 Laboratory Diagnosi
s o
HIV
In
ection 411 Other Screening Tests Methods that can test urine
or HIV antibody
have been
developed but are not as accurate as the blood or saliva tests and must be con rme

d with a
standard serological test. 58,63 In addition, the FDA has approved the use o
ho
me test kits
or
individuals who pre
er this
ormat to being tested by a clinic or private physic
ian. In this test
system, individuals are provided with written in
ormation about HIV and AIDS, ob
tain a small
blood sample by per
orming a ngerstick with a sterile lancet, and apply the sampl
e to a
designated area on a test card. 58,63 The card is then mailed to a certi ed lab- o
ratory, in
which traditional ELISA testing
or HIV antibody is per
ormed,
ollowed
with
con
irmation by Western blot. The results can be obtained by telephone using a
personal
identi cation number. Con rmatory Tests Because o
the possibility o
obtaining
als
e-positive
results, as discussed earlier, all positive samples
rom HIV screen- ing tests m
ust be re
erred

or testing with a more speci

ic con
irmatory method. A variety o
di

erent con

irmatory methods
have been developed
or this purpose. The most widely used con
irmatory test
or
HIV antibody,
and the standard test in use today, is the Western blot test. Western Blot Testi
ng Principles The
Western blot test, or immunoblot,
or HIV antibodies was introduced in 1984 and
has been used
or
systematic con rmation o
positive ELISA results since 1985. This technique is mor
e technically
demanding than ELISA but can provide an antibody pro
ile o
the patient sample t
hat reveals the
speci cities to individual HIV anti- gens present. Several commercial kits are ava
ilable
or this
type o
testing and can provide results within a
ew hours. 57,58,70 We
stern blot kits
are prepared commercially as nitrocel- lulose or nylon strips containing individ
ual HIV proteins
that have been separated by polyacrylamide gel elec- trophoresis and bl
otted onto the
test membrane. The protein antigens are derived
rom HIV virus grown in cell c
ulture. Antigens
with low molecular weight migrate most rapidly and are there
ore positioned towa
rd the bottom o

the test strip, while antigens o

high molecular weight remain toward th
e top o
the
membrane. The testing laboratory then reacts the test strip with patient serum.
During the
incubation period, any HIV anti- bodies present will bind to their corresponding
antigen on the
test membrane, and unbound antibody is removed by washing. Next, an antihuman im
munoglobulin with
an enzyme label (i.e., the conjugate) is added directly to the test strip and
binds to
speci
ic HIV antibodies
rom the patient sample. Unbound conjugate is r
emoved by
washing, and bound conjugate is detected a
ter adding the appropriate sub
strate, which
produces a chromogenic reaction. Colored bands appear in the positions wh
ere
antigen-speci
ic HIV antibodies are present. Separate HIV-1- and HIV-2-spec

i c Western blot
tests must be used to test
or antibodies to each virus. 57,58,70 In HIV-1 in
ec
tion, antibodies
to the gag proteins p24 and p55 antigens appear relatively early a
ter exposure
to the virus but
tend to decrease or become undetectable as clinical symptoms o
AIDS appear. 71
Antibodies to the
enve- lope proteins gp41, gp120, and gp160 appear slightly later but remain thro
ughout all
disease stages in an HIV-in
ected individual, making them a more reliable indica
tor o
the
presence o
HIV. 72 Other antibodies commonly detected by this method are those
directed against
pol proteins p51 and p66, while antibodies against the regulatory gene products
are usually not
detectable by conventional methods. 58,71 The bands produced by the test sample
are examined
visually
or the number and types o
antibodies present. Densitometry can also b
e per
ormed to
quantitate the intensity o
the bands, which would re
lect the amount o
each an
tibody produced.
Patients can be
ollowed over time to determine whether there is a change in the
antibody
pattern. Because Western blot testing is highly dependent on the laboratorians te
chnical skill
and subjective interpretation, it should be per
ormed only in laboratories that
have an adequate pro ciency testing program. The inclusion o
positive and negative control s
era in the test
run provides quality control
or the Western blot. For the test to be valid, the
negative control
should produce no bands, and the positive control should be reactive with p17, p
24, p31, gp41,
p51, p55, p66, and gp120/160. In addition, a vigorous external pro ciency program
should be in
place. Interpretation o
Western Blot Results A negative test result is reported
i
either no
bands are present or i
none o
the bands present correspond to the molecular we
ights o
any o

the known viral proteins. 58 Criteria

or determining a positive test result hav
e been published
by the Association o
State and Territorial Public Health Laboratory Directors a
nd CDC, the
Consortium
or Retrovirus Serology Standardization, the American Red Cross, and
the FDA. 58,7072
Although some controversy exists as to what banding pattern constitutes a positi
ve result, most
laboratories
ollow the criteria o
the Association o
State and Territorial Pub
lic Health
Laboratory Directors and CDC. 58 According to these criteria, a result sho
uld be reported
as positive i
at least two o
the
ollowing three bands are present: p2
4, gp41, and
gp120/gp160 71 (see Figure 235). Specimens that have some o
the characteristic b
ands present
but do not meet the criteria
or a positive test result are considered to be ind
eterminate. This
result may be pro- duced i
the test serum is collected in the early phase o
se
roconversion or
i
the serum contains antibodies that cross-react with some o
the immunoblot an

tigens, producing
alse-positive results. False positives may be caused by antibodies produced
to contaminants

rom the cells used to culture HIV to prepare the antigens

or the test; to auto
an- tibodies,
including those directed against HLA, nuclear, mitochondrial, or T-cell antigens
; or to
antibodies produced a
ter vaccinations. 57,58,63 1814_Ch23_399-426.qxd 7/10/09
5:20 PM Page
411 412 SECTION 4 Serological Diagnosis o
In
ectious Disease The use o
recombi
nant antigens
instead o
viral lysates has reduced the incidence o

alse-positive results. I

an indeterminate test result is obtained, it is recommended that the test be repeated w

ith the same or a

resh specimen; i
the test is still indeterminate, testing may be per
ormed wit
h a new specimen
obtained a
ew weeks later, and i
the pattern converts to positive, it can be c
oncluded that the
rst spec- imen was obtained during the early phase o
seroconversion. Failure o

an
indeterminate test pattern to convert to posi- tive a
ter a
ew weeks strongly s
uggests that the
pattern is due to a
alse-positive test rather than HIV in
ection. 21,58 During
this period,
additional tests that detect components o
the virus, such as HIV nucleic acid o
r p24 protein,
can be per
ormed to provide more conclusive results (see below). Because o
the
possibility o

alse-positive results, the level o

technical di
culty, and the time-consuming n
ature o
the
Western blot, this test is inappropriate
or use as an initial screen
or HIV in

ection. In the
standard testing algorithm described earlier, in which samples determined to be
repeat- edly
reactive by ELISA are then tested by Western blot, the Western blot has a positi
ve predictive
value greater than 99 percent
or both low-risk and high-risk populations. 58 Ot
her Con rmatory
Tests Other con
irmatory tests, including IFA, RIPA, line immunoassays, an
d rapid
con rmatory tests, have also been developed. 57,58 These assays may be used as an
alternative to
the Western blot in the initial con rmation o
HIV in
ec- tion or in cases where t
he Western blot
yields indeterminate results. 73 In the IFA (indirect immuno
luorescence assay),
antibodies are
detected to HIV antigens expressed on the sur
ace o
in
ected T-cell lines x
ed onto a glass
slide. This assay has the advantage o
being able to detect antibodies
to many HIV
antigens but requires a well- maintained uorescent microscope and subjective
interpretation o
the results. RIPA (radioimmunoprecipitation assay) uses minimally dena
tured antigens
but requires radioactive materials; its use is limited to research laboratories
that can maintain
HIV in cell cultures. Line immunoassays are sim- ilar to Western blots except th
at recombinant or
synthetic HIV proteins are applied to the test strips, rather than being electro

phoresed
rom
cultured virus preparations. These assays have allowed
or easier interpretation
because o

ewer
bands and
alse-positive results. In addition, several rapid or simple con rmatory
assays have
been developed, which may be suitable
or use in resource-limited countries that
do not have
sophisticated instrumentation or stable elec- tricity. 57 Such countries may als
o use con rmatory
strategies other than the traditional screening by ELISA/con
irma- tion by Weste
rn blot,
including an ELISA
ollowed by a rapid test, testing with two di

erent ELISAs,
and testing with
two di

erent rapid assays. 57 Detection o

HIV Antigen p24 Antigen Testing This
test detects p24
antigen
rom the core o
the HIV-1 virion. Levels o
this antigen in the circula
tion are thought
to correlate with the amount o
HIV replication, because they are high in the in
itial weeks o

in
ection during the early burst o
viral replication, then become undetectable
as antibody to
p24 develops, and then rise again during the later stages o
in
ection when impa
irment o
the
immune system allows the virus to replicate. Testing
or the p24 antigen played
an important role
in HIV testing in the United States prior to the development o
more sensitive t
ests that detect
nucleic acid o
the virus (see below). The rationale behind using the p24 test i
s that the
antigen appears about 1 week be
ore the appearance o
HIV antibody during the ac
ute stage o

in
ection, allowing
or slightly earlier detection o
the virus. 74 For this rea
son, the p24
antigen test was used in the United States
rom 1996 to 1999, along with the tes
ts
or HIV
antibody to screen blood and plasma donors, until nucleic acid testing was imple
mented. 70 This
marker has also been used to achieve earlier diag- nosis, detect HIV in
ectio
n in newborns,
and monitor patients on antiretroviral therapy. Today, p24 antigen test- ing is
used most
commonly in laboratories with limited resources, because it is less expensive an
d easier to
per
orm than the HIV nucleic acid tests, which have largely replaced tests
or p
24 antigen in
developed nations. 70 Test Principles The p24 antigen is detected by a solid-pha
se antigen capture enzyme immunoassay 58 (see Chapter 10
or details o
Positive gp160 gp120 p
66 p55 p51 gp41
gp31 p24 p17 Indeterminate p55 p24 Negative FIGURE 235. Western blot, showing res
ults
rom a
negative sample, an indeterminate sample, and a positive sample. 1814_Ch23_399-4
26.qxd 7/10/09
5:20 PM Page 412 CHAPTER 23 Laboratory Diagnosis o
HIV In
ection 413 capture a
ssays). In this
method, a solid support coated with monoclonal anti-HIV-1 antibody is incubated
with patient
serum or plasma. A
ter washing to remove any unbound antigens, a second anti-HIV
-1 antibody
conjugated with an enzyme label is added to the reaction. When substrate is adde

d, color
development indicates the presence o
cap- tured antigen. Optical density can be
measured against
a standard curve to make a quantitative determination o
anti- gen present. All
positive results
should be con rmed by a neutraliza- tion assay. This is accomplished by preincubat
ing the patient
specimen with human anti-HIV-1 antibody prior to per-
orming the antigen assay.
I
p24 antigen
is present, the neutralizing antibody will
orm immune complexes and will preven
t the antigen

rom binding to the HIV antibody on the solid support. In a positive test,
absorbance
should decreased by at least 50 percent. 58 Because p24 antigen becomes undetect
able as the host
pro- duces antibody and binds the antigen in immune complexes, the p24 antigen t
est cannot
replace the ELISA test
or HIV antibody as the primary screening test
or HIV-1
in
ection. The
overall sensitivity o
the p24 test is less than that o
the ELISA
or HIV an
tibody, with
only 20 to 30 percent o
asymptomatic HIV-in
ected individuals showing dete
ctable serum
levels o
p24 antigen. 58 This lack o
sensitivity is par- tially due to complex
ing o

ree
antigen with p24 antibody. Sensitivity may be improved by dissociating these imm
une complexes by
treatment with heat or acid prior to the assay. 58,70 More recently, combination
tests have been
developed that simultaneously detect p24 antigen and HIV antibody through a seri
es o
steps in an
enzyme immunoassay or a ran- dom access system. 58,61 A procedure combining p24
antigen detection
by EIA and HIV RNA by PCR has also been developed. 70 It is hoped that
combination
assays such as these will allow
or detection o
HIV in
ection in situations in
which antibody is
not detectable or yields indeterminate results. Nucleic Acid Testing Tests
or H
IV nucleic acid
have been incorporated into rou- tine clinical practice and have had an importan
t impact on the
management o
HIV-in
ected patients. These tests are used to determine the amoun
t o
virus
present in patients and to determine whether and what type o
drug resistance ha
s developed.
Quantitative tests
or HIV nucleic acid, also known as viral load tests, are use
d routinely to
help pre- dict disease progression, predict response to antiretroviral therapy,
and monitor
e

ects o
the therapy. 70,7578 Viral load testing is per
ormed by nucleic acid a
mpli cation
methods. Tests
or drug resistance can be per
ormed by genotypic or phenotypic a
ssays. Viral Load
Assays Viral load assays are based on ampli
ication methods that increas
e the number
o
HIV RNA copies (or their derivatives) in test samples to detectable
levels.
Several ampli
ication methods have been developed
or this pur- pose, including
the reverse
transcriptase polymerase chain reaction (RT-PCR) and real-time PCR, which ampli

y com- plementary
DNA generated
rom HIV RNA; the branched chain DNA assay (bDNA), which ampli es a
labeled signal bound to the test plate; and nucleic acid sequence-based ampli
ication (NASB
A), which
ampli
ies HIV RNA. The basic principles o
each o
these methods are dis
cussed brie y
here and are covered in more detail in Chapter 11. Polymerase Chain Reaction (PC
R) Two kinds o

polymerase chain reaction methods have been developed to detect HIV nucleic acid
: the
reverse-transcriptase polymerase chain reaction (RT-PCR) and more recently, real
-time PCR. A
commercial RT-PCR was the rst assay to be licensed by the FDA
or quantitative me
asurement o

circulating HIV nucleic acid. The basic principle o

this test is to a
mpli
y a DNA
sequence that is complementary to a por- tion o
the HIV RNA genome. 70,75,77,79
In this assay,
HIV RNA is isolated
rom patient plasma by lysis o
the virions and precipitatio
n with alcohol.
The RNA is treated with a thermostable DNA polymerase enzyme that has both
reverse
transcriptase activity and the ability to initiate DNA synthesis in the presence
o
the
appropriate reagents; the enzyme is also stable at high temperatures. The revers
e tran- scriptase
activity o
the enzyme transcribes the HIV RNA into complementary DNA (cDNA). Th
e cDNA is then
ampli
ied by standard polymerase chain reaction (PCR) methodology. In this proce
ss,
double-stranded cDNA mol- ecules are separated into single strands by heating, a
nd each can then
serve as a template
or synthesis o
a new DNA strand. The separated strands are
incubated with
primers, or short pieces o
DNA that are complementary to the ends o
a highly c
onserved region
o
the HIV-1 gag gene. Cooling o
the reaction allows the primers to bind to the
cDNA at the
appropriate sites. The reaction is heated again, and the region in between the p
rimers is
synthesized in the pres- ence o
the DNA polymerase enzyme and the
our
deoxynucleoside
triphosphates. This series o
steps, re
erred to as one cycle, generates a copy
o
the selected
portion o
the cDNA, called an amplicon. This process is repeated
or several cy
cles in an
automated thermocycler, resulting in exponential growth in the number o
amplico
ns produced.
A
ter the ampli cation process is complete, the amplicons are chemically denatur
ed into
single strands, bound to microtiter plates, and quantitated by the addition o

an
enzyme-labeled probe,
ollowed by a substrate. The amount o
color change produc
ed in each well
(i.e., the optical den- sity o
the sample) is proportional to the amount o
HIV
RNA contained in
the specimen. The standard RT-PCR method can detect 400 to 750,000 copies
o
HIV-1 RNA per
mL o
plasma, while an ultrasensitive version o
the test quantitates HIV-1 RNA

over a range
o
50 to 100,000 copies/mL. 70,76,77 Both 1814_Ch23_399-426.qxd 7/10/09 5:
20 PM Page 413
414 SECTION 4 Serological Diagnosis o
In
ectious Disease versions o
the test m
ust be per
ormed

or samples whose values

all outside o
the dynamic range. Technical care must
be taken when
per
orming the RT-PCR, because this assay is susceptible to cross-contamination
o
test samples
with amplicon, target DNA, or target RNA. 77 To minimize cross- contamination, p
hysically
separate work areas should be designated
or reagent preparation, sample prepara
tion,
ampli
ication, and detection, and the work should
low in one direction only. Mo
re recently,
real-time RT-PCR assays have been devel- oped to quantitate HIV RNA. 70,76,79,80
In these assays,
the amplicons are analyzed at the same time they are produced (i.e., in real tim
e). This is
accomplished by adding a
luo- rescent marker that is incorporated into the ampl
icon during the
reaction. The
luorescent signal produced is propor- tional to the amount o
amp
licon generated
and is compared to a standard curve to produce a quantitative result. The reacti
on is carried out
in a closed tube, minimizing the risk o
carryover contamination with other ampl
icons. Real-time
PCR is highly sensitive and is capable o
detecting a broad range o
RNA copies,

rom 40 to 10
million copies/mL. 70,76,80 The test is also capable o
detecting all subtypes o

groups M, N, O,
and recombinant strains o
HIV. 76,80 Because o
these advantages, real-time RTPCR is likely to
replace stan- dard RT-PCR methods to detect HIV RNA in many laboratories
in the
uture.
70 Branched-Chain DNA (bDNA) Assay In contrast to RT-PCR, the bDNA method is bas
ed on ampli
ying
the detection signal generated rather than ampli-
ying the HIV target sequence.
This is
accomplished by using a solid-phase sandwich hybridization assay that incor- por
ates multiple
sets o
oligonucleotide probes and hybridization steps to create a series o

branched molecules. 70,75,77,79 First, RNA isolated

rom lysed virions in patient plasm
a is captured on
wells o
a microtiter plate coated with a number o
probes. The captured
RNA is then
hybridized with branched ampli er probes and incu- bated with an enzyme-labeled pr
obe that will
bind to the DNA branches. Finally, a chemiluminescent substrate is added, and co
lor change is
measured with a luminometer. Quantitative results are generated
rom a standard
curve. The bDNA
test can detect 50 to 500,000 copies o
HIV RNA/mL o
plasma. 76,77 As compared
to the RT-PCR,
the bDNA has higher upper limits in its detection ranges, does not require separ
ate rooms
or its
various steps because ampli cation o
the target is not involved, and is better at
detecting all
known subtypes o
HIV; however, it is more conducive
or laboratories with highe

r testing volumes
and requires a larger sample volume. 70,77 Nucleic Acid Sequence-Based Ampli catio
n (NASBA) NASBA
is a target ampli cation assay based on ampli ca- tion o
HIV RNA. 70,75,79 In this
procedure,
HIV viral RNA is isolated
rom the clinical specimen to be tested and is ini- ti
ally treated with
reverse transcriptase to
orm cDNA. An RNase H enzyme is used to remove R
NA
rom the
RNA:cDNA hybrid
ormed, and the cDNA generated serves as a template to generate
new RNA molecules
in the pres- ence o
RNA polymerase, primers speci
ic
or a region o
the gag ge
ne, and the
ribonucleotide triphosphates. All steps o
the reaction are per
ormed at a const
ant temperature;
thus, a thermocycler is not required. The amplicons pro- duced are captured onto
probes bound to
magnetic beads, which are in turn hybridized with chemiluminescent probes and at
tached to the
sur
ace o
an electrode, where an elec- trochemiluminescent reaction occurs. The
amount o
light
generated is proportional to the amount o
amplicon in the sample, and quantitat
ive results are
obtained via internal calibrators that are used to produce a standard curve. The
NASBA method has
a wide detection range: 176 to 3.4 million copies o
HIV-1 RNA/mL o
specimen. 7
0,76 It requires
a small volume o
specimen, making it conducive to testing neonates and children
, and it can be
used
or a variety o
specimens in addition to plasma, including cere- brospinal
uid, whole
blood, semen, cervical washings, and sputum. 75,76 However, NASBA is a more tech
nically com- plex
method to per
orm than RT-PCR or bDNA and may be best suited
or research labora
tories. Clinical
Utility o
Viral Load Tests Viral load tests play an essential role in helping p
hysicians
determine when to initiate antiretroviral therapy, monitor- ing patient response
s to therapy, and
predicting time o
progression to AIDS. 70,78 They may also be help
ul in establishing a
diagnosis o
HIV in
ection in cases where the HIV antibody tests are negative or
indeterminate.
70,78 The amount o
HIV RNA, or the viral load, in a patients plasma re ects the na
tural history
o
HIV in
ection in that individual. 12,81 HIV RNA levels become detectable abou
t 11 days a
ter
in
ection and rise to very high levels shortly therea
ter, during the initial
burst o

viral replication. Typically, over a period o

a
ew months, the viral load dro
ps as the
individuals immune system clears viral particles
rom the circulation, and a stab
le level o

plasma HIV RNA, known as the set point is achieved (see Fig. 234). In unt
reated
individuals, this level can persist
or a long time and then rise again later as
the immune
system deteriorates and the patient progresses to AIDS. Success
ul therapy with
antiretroviral
drugs will result in a drop in the viral load level. There
ore, viral load tests

are used
routinely to monitor patients during the course o
HIV in
ection and play a crit
- ical role in
the clinical management o
these individuals. Studies per
ormed by the Multicent
er AIDS Cohort
and other groups have demonstrated that in
ormation obtained
rom viral load tes
ts has prognostic
value. 48,8183 These stud- ies have shown that baseline plasma viral load
values
obtained in patients prior to the start o
antiretroviral ther- apy are an im
portant
predictor o
disease progression, in that a higher number o
HIV RNA copies/m
L o
plasma is
associated with more rapid development o
an AIDS- de ning illness or AIDS-related
death.
1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page 414 CHAPTER 23 Laboratory Diagnosi
s o
HIV
In
ection 415 In addition, viral load values have been instrumental in monitorin
g patients during
the course o
antiretroviral ther- apy. Patients who attain a lower number
o
HIV-RNA
copies/mL o
plasma are more likely to achieve a longer treatment response. 38,4
8,81,82 The
optimal goal o
therapy is to reach undetectable levels o
HIV RNA (i.e.,
50 to 80
copies/mL, depending on the assay). 38,70 Patients who
ail to achieve a signi can
t decrease in
viral load a
ter receiving antiretroviral drugs may not be adhering to the appro
pri- ate drug
administration schedule or may have problems absorbing the drugs, while pati
ents whose
viral loads increase over a period may have developed viral resistance to the d
rugs. 48 The U.S.
Department o
Health and Human Services recommends that plasma HIV RNA testing b
e per
ormed
be
ore antiretroviral therapy begins, to obtain a baseline value; testing should
then be done 2
to 8 weeks a
ter the ini- tiation o
therapy and every 3 to 4 months therea
ter
to determine the
e

ectiveness o
the therapy. 38,70,78 In order to obtain an accurate assessment
o
viral load
dynamics in a single patient, it is recommended that the same assay be used
or
these viral load
measurements, because values may di
-
er between di

erent molecular tests. 70

A change in viral
load is considered to be signi cant i
there is at least a three-
old or 0.5 l
og increase
or decrease in the number o
copies/mL. 38,70 Initiation o
antiretrovira
l therapy or a
change in the therapy protocol is recommended
or patients whose HIV RNA levels
and CD4 T-cell
counts reach crit- ical values (200 to 350 cells/mm 3 ). 38,81 Viral load testin
g is also
recommended
or individuals who develop symptoms consistent with primary HIV in

ection, who
develop a clin- ically signi
icant opportunistic in
ection, or whose CD4 T-cell
counts drop
unexpectedly. Although molecular methods are not approved by the FDA
or the dia
gnosis o
HIV
in
ection, these tests can be help
ul in identi
ying the in
ection in certain in

dividuals, such
as those who are HIV-antibody-negative or who have equivocal antibody test resul
ts (e.g., those
tested prior to seroconversion, in
ants). 70,78 Drug-Resistance Testing Because
HIV has high
replication and mutation rates and because no treatment completely eradicates th
e virus, it is
possible
or drug-resistant subpopulations to emerge dur- ing the course o

antiretroviral
therapy. Two types o
laboratory methods can be used to test
or drug resista
nce: genotype
resistance assays and phenotype resistance assays. 38,48,70,77,84,85 Genotyp
e resistance
assays are per
ormed more
requently than phenotype resistance assays, because t
hey are less
expensive, are more widely available, and have a shorter turnaround time. 70 Gen
otype resistance
assays detect mutations in the reverse transcriptase and protease genes o

HIV. In these
tests, RNA is isolated
rom patient plasma, the desired genes are ampli
ied by R
T-PCR, and the
products are analyzed
or mutations associated with drug resistance by automated
DNA sequencing
or hybridization. 38,48,70,77,84 Commercial kits
or genotyping assays are avail
able, and there
are online services to assist in interpreting the results. 38,70,77,84 The tests
may also be
per
ormed and interpreted by re
erence labo- ratories. While genotyping tests ar
e less expensive
and have a shorter turnaround time than phenotypic methods, they can identi
y on
ly known
mutations and cannot assess the e

ects o
combinations o
individual mutations
on drug
resistance. 84 Phenotype resistance assays determine the ability o
clin- ical i
solates o
HIV to
grow in the presence o
antiretroviral drugs. 38,48,70,77,84,85 These assays are
technically
complex and are per
ormed only by highly specialized re
erence labora- tories. I
n these assays,
recombinant viruses are created by inserting the reverse transcriptase and
protease gene
sequences
rom HIV RNA in the patients plasma into a lab- oratory re
erence str
ain o
HIV
and trans
ecting the recombinant virus into mammalian cells. Varying concen- t
rations o

antiviral drugs are incubated with the trans

ected cells, and the IC 50 values,
or drug
concentrations needed to suppress the replication o
the patients viral is
olate by 50
percent, are calculated. These values are then compared to the IC 50 values o
c
ells trans
ected
with a re
erence strain o
HIV in order to determine drug resistance. 38,48,84 T
he major
advantage o
phenotypic assays is that they measure drug susceptibility directly
, on the basis o

all mutations present in the patients isolate. However, they involve sophis- tica
ted
technologies that are not widely available, are expensive, and have a long
turnaround time.
84 Both genotypic and phenotypic assays require that a viral load o
at least 50

0 to 1000 copies
o
HIV RNA/mL be pres- ent in the test sample and
or the resistant virus to con
stitute more than
20 percent o
the total viral population in the patient to be detected. 48,70,84
Despite these
limitations, stud- ies have shown that patients undergoing drug-resistance testi
ng, particularly
by genotyping methods, have a better chance o
receiving antiretroviral therapy
regimens that are
more likely to result in greater reductions in viral load. 48,8588 There
ore, the
U.S.
Department o
Health and Human Services recommends that drug-resistance testing
be per-
ormed
in individuals prior to initiating antiretroviral therapy, in patients in w
hom HAART has

ailed or viral load values have not been optimally reduced by antiretroviral th
erapy, and in all
HIV-positive pregnant women. 38,70 Virus Isolation De
initive evidence o
HIV
in
ection can
be provided through the use o
cell culture techniques to isolate viral particl
es
rom patient
cells and tissues. A cocultivation sys- tem has been used in which cells
rom pa
tients are mixed
with T-cell lines or peripheral blood mononuclear cells
rom HIV-seronegative do
nors that have
been stimulated with phytohemagglutinin and interleukin-2. 70,89 The best patien
t sample is
peripheral blood, but virus can be isolated
rom other body uids, such as cerebro
spinal uid,
saliva, cervi- cal secretions, semen, and tears, and
rom organ biopsies. 88
1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page 415 con rmed by serological tests at
12 to 18
months o
age. 91,92 The HIV status o
breast
ed in
ants, who are continually ex
posed to the
virus, cannot be determined accurately until breast
eeding is stopped. 92 Increa
sed emphasis on
screening pregnant women
or HIV in
ection should also help in the identi cation o

HIV- positive
in
ants. 95 Rapid tests
or HIV antibody should be per
ormed on women whose HIV
status is unknown
and on their newborn in
ants soon a
ter birth. 92 Prompt detection o
HIV in
ect
ion in newborns
is important, because in
ected in
ants have a better prognosis when HAART is sta
rted early and
can bene t
rom treatment with prophylactic drugs
or opportunistic in
ections. 91
,94 SUMMARY
Human immunode ciency virus type 1 (HIV-1) is respon- sible
or the majority o
AI
DS cases
throughout the world. A related virus, HIV-2, may also cause AIDS but is generally less
pathogenic. These viruses belong to the retrovirus
amily, which contains RNA as
the genetic
material
rom which DNA is transcribed. Transmission o
HIV occurs by three majo
r routes: through
intimate sexual contact, through contact with contaminated blood or body uids, or
through
vertical transmission
rom in
ected mother to her
etus or in
ant. The three mai
n structural
genes o
HIV are gag, env, and pol. The gag gene codes
or the core proteins o

the virus,

including p24 antigen, the rst to appear during in

ection. Products o
the pol ge
ne are the
enzymes reverse transcrip- tase, integrase, and protease. The env gene codes
or
the envelope
proteins gp120 and gp41, which
acilitate attach- ment o
the virus to the
CD4 receptor
and chemokine coreceptors on susceptible host cells. Several other genes are pr
esent that

unction to regulate replication o

the virus. A
ter HIV binds to a host cell,

usion occurs, and

the viral particle is taken inside the cell. Reverse transcriptase pro- duces DN
A
rom the viral
RNA, and HIV is incorporated into the host-cell DNA as a provirus. A burst o
vi
ral repli- cation
occurs a
ter initial in
ection. This is
ollowed by a period o
latency that beg
ins as an immune
response to the virus develops and can last
or 10 years or more as viral replication is held
in check. The prime targets o
HIV in
ection are the T helper cells, which have
large numbers o

CD4 molecules on their cell sur

ace. Untreated HIV in
ection results in a pro
gressive
destruction o
these cells, with resumption o
high rates o
viral replicati
on and development o
pro
ound immunosuppression in the host. Clinical symptoms o
HIV in
ecti
on are associated
with the levels o
viral replication and helper T cell destruction in the host.
The in
ection
progresses through an acute stage,
ollowed by a latent stage, and eventual prog
ression to AIDS.
During the latent period, the virus is harbored in the lym- phoid tissues, where
it causes
gradual destruction o
the immune system. In untreated patients, this will event
ually result in
AIDS, which is characterized by an increase in viral 416 SECTION 4 Serological D
iagnosis o

In
ectious Disease Samples other than peripheral blood, however, have vary- ing
levels o
virus,
which makes culture less e
cient. In
ection can be con
irmed by detecting reverse
transcriptase activity or p24 antigen in the culture supernatant, detecting the HI
V genome by
molecular methods, or observing characteristic cytopathic e

ects. 90 Presence
o
the virus can
also be demonstrated by using uorescent-labeled monoclonal anti-HIV antibody. Typ
ically, culture
results become positive within 2 weeks o
incubation, but periods o
up to 60 da
ys may be
required
or some samples. 89 Viral culture can also be used in drug-resistance
assays and to
determine the viral replicative capacity in isolates
rom patients undergoing an
tiretroviral
therapy. 70 Viral culture is laborious, time-consuming, and costly, and it may b
e hazardous to
laboratory personnel involved in the testing process. Because o
these reasons,
it is not
suitable
or implementation in the clinical laboratory and is employed primarily
in research. In
most cases, in
orma- tion regarding HIV in
ection can be obtained in a more prac
tical manner by

per
orming tests that detect HIV com- ponents, such as RT-PCR and p24 antigen. T
esting o
In
ants
and Young Children The traditional algorithm
or laboratory diagnosis o
HIV in

ection in adults,
as discussed above, involves screening individuals using an ELISA or rapid test

or HIV antibody
and con rming positive results by Western blot. However, serological tests are not
reliable in
detecting HIV in
ection in children younger than 18 months o
age because o
pla
- cental passage
o
IgG antibodies
rom an in
ected mother to her child. These maternal antibodie
s persist in the
blood- stream o
the in
ant during the rst year o
li
e (or longer in a small pro
portion o

in
ants) and can con
use the inter- pretation o
serological results
rom in
ant
samples. 91,92
Thus, a child born to an HIV-positive mother may test positive
or HIV antibody
during the
irst
18 months o
li
e even though the child is not in
ected. Because o
the di
cultie
s with
serological testing, HIV in
ection in in
ants is best diagnosed using molecular
meth- ods. A
qualitative HIV-1 DNA PCR test is the pre
erred method
or this purpose. This te
st, which detects
proviral DNA within the in
ants peripheral blood mononuclear cells, has a sensiti
vity o
90 to
100 percent and a speci city o
95 to 100 percent in in
ants older than 1 month o

age. 91,92
Alternatively, quantitative HIV RNA assays may be used to diagnose HIV in
ection
in in
ants and
young children and are more likely to detect in
ections with strains other than
subtype B. 91,92
Testing
or p24 antigen is not recommended, as it has a low sensitivity in in
an
ts. 91,93 It is
recommended that nucleic acid tests
or HIV be per-
ormed in in
ants with known
perinatal
exposure at the ages o
14 to 21 days, 1 to 2 months, 4 to 6 months, and possibly at birth. 94
A positive test result should be con rmed by repeat testing. Negative test results
provide
presumptive evidence
or the absence o
HIV in
ection and should be 1814_Ch23_39
9-426.qxd
7/10/09 5:20 PM Page 416 CHAPTER 23 Laboratory Diagnosis o
HIV In
ection 417
reproduction,
with pro
ound immunosuppression and host susceptibility to several opportunistic
in
ections and
malig- nancies. The use o
HAART, a combination o
antiretroviral drugs that inh
ibit HIV
replication, has resulted in improved immune
unction in in
ected individuals, w
ith a decline in
the incidence o
opportunistic in
ections and a delay in pro- gression to AIDS.
Antiretroviral
drugs have also played an important role in decreasing perinatal transmission. T
he main
laboratory tests
or HIV in
ection are CD4 T-cell enumeration, HIV antibody dete
ction, p24
antigen detection, and testing
or HIV nucleic acid. The standard method
or CD4
T-cell
enumeration is multicolor immuno-
luorescence staining,
ollowed by analysi
s with
low

cytometry. CD4 T-cell counts are routinely monitored in HIV-in

ected individuals
and provide
important in
orma- tion regarding likelihood o
disease progression and de
velopment o

opportunistic in
ections, and e

ectiveness o
antiretroviral therapy. These cou
nts are also used
in the CDC classi cation system
or HIV in
ection and AIDS. Detection o
HIV antib
odies is
per
ormed with an initial ELISA screening test. Third-generation ELISAs employ a
sandwich
technique in which HIV antibodies in patient serum or plasma are sandwiche
d between two
sets o
HIV antigens, one bound to a solid phase and a second contain- ing an en
zyme label. ELISA
testing is sensitive but is subject to
alse-positive reactions created by cross
-reactivity o

anti- bodies produced in a number o

other conditions. There
ore, con rmatory test
ing o
positive
specimens is necessary. The Western blot is the most commonly used con rmatory tes
t. In this
test, patient serum is reacted with a nitrocellulose strip containing separate a
reas o
distinct
HIV antigens. HIV antibodies to speci c antigens on the strip are detected a
ter a
ddition o
an
enzyme-labeled antihuman immunoglobulin and substrate. Colored bands develop in
the areas on the
strip where HIV antibodies have bound. A positive test result is indicated by th
e presence o
two
o
the
ollowing three major bands: p24, gp41, or gp120/160. Detection o
p24 an
tigen was
designed to identi
y HIV in
ection during the window period be
ore antibod
y is detectable.
This test may be used to detect in
ection in patients whose antibody status is n
egative or
indeterminate. An antigen capture or sandwich assay is employed, which uses a so
lid-phase
antibody to capture p24 in the patient specimen. A second labeled antibody is ad
ded and adheres
only to antigen caught on the solid phase. Nucleic acid testing
or HIV is used
to determine the
amount o
virus harbored by patients and the development o
drug-resistant strai
ns o
virus.
Quantitative tests
or HIV nucleic acid are known as viral load tests. These tes
ts have had an
important impact on the clinical management o
HIV-in
ected patients by allowing
physicians to
predict dis- ease progression, to predict response to antiretroviral thera
py, and to
monitor e

ects o
the therapy. Viral load tests are per
ormed by one o
three m
olecular methods:
reverse transcriptase polymerase chain reaction (RT-PCR), a method that converts
HIV RNA into
cDNA and then ampli es the cDNA generated; branched chain DNA assay (bDNA), which
ampli
ies a
labeled signal bound to a test plate; and nucleic acid sequence-based ampli

ication (NASBA),
which ampli es HIV RNA. Drug-resistance test- ing can be per
ormed by genotypic as
says that use
molecular methods or by phenotypic assays in which HIV replication in clinical i
solates is

assessed in the presence o

varying con- centrations o
antiretroviral drugs. Is
olation o
HIV in
culture represents another technique
or identi
ying HIV in
ection. Because it i
s time-consuming,
costly, and hazardous to workers, it is not used to a great extent. Diagnosis o

HIV in neonates
is more complex than test- ing in adults. The presence o
maternally acquired an
tibody in
newborns makes ELISA tests
or HIV antibody unreli- able until a child is o
ver 18 months
old. The pre
erred method
or testing in
ants and children younger than
18 months is a
PCR that detects HIV proviral DNA in the patients peripheral blood mononuclear ce
lls. Care
ul
mon- itoring o
HIV-in
ected mothers and early testing o
in
ants at risk is bei
ng implemented to

acilitate prompt medical intervention. 1814_Ch23_399-426.qxd 7/10/09 5:20 PM

Page 417 418
SECTION 4 Serological Diagnosis o
In
ectious Disease 1. A young woman recently
discovered that
her boy
riend tested HIV-positive. She was concerned that she may have also co
ntracted the
in
ection, because she had experienced ulike symptoms 1 month ago. She decided
to visit her
physician
or a medical evaluation. Questions a. What initial laboratory tests s
hould be
per
ormed on the young woman to determine i
she has been exposed to HIV? b. I

the woman tests

positive in the initial evaluation, how can it be determined whether her test re
sults are truly
positive because o
HIV in
ection or i
they represent a
alse-positive result?
c. I
the womans
test results are truly positive, what tests can be done to monitor her over time
? CASE STUDY
1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page 418 CHAPTER 23 Laboratory Diagnosi
s o
HIV
In
ection 419 SIMULATION OF HIV-1 DETECTION Principle: An HIV Screening Simulati
on Enzyme linked
immunosorbent assay (ELISA) tests were originally developed
or antibody mea
surement. These
immunoassays have also been adapted to success
ully detect samples that contain
antigens. This
HIV ELISA simulation experiment has been designed to detect a hypothetica
l patients
circulating IgG directed toward the viral (HIV) antigen. ELISAs are done in micr
otiter plates
that are gen- erally made o
polystyrene or polyvinyl chloride. The plates are s
omewhat
transparent and contain many small wells, in which liquid samples are deposited.
The
ollowing
are the basic steps o
the ELISA reaction. 1. Antigens are added to the wells, w
here some remain
adsorbed to the wells by hydrophobic bonds. Antigens can be
rom whole HIV lysat
es, speci c HIV
proteins, or a mixture o
the two. There is no speci city involved with the adsorp
tion process to
the wells, although some substances may exhibit di

erential binding. In certain

cases, the
antigens can be covalently cross-linked to the plastic using UV light. 2. Wells
are washed to

remove unadsorbed antigens. 3. Block the unoccupied sites on the walls o

the pl
astic wells with
proteins, typically gelatin or bovine serum albumin. 4. In
ection by HIV-1 cause
s the individual
to mount an anti- body response that eventually results in plasma IgG mol
ecules that bind
to di

erent HIV proteins (or di

er- ent areas or the same protein). I

these antibodies
are present, as in the plasma sample o
an HIV-positive patient, they will bind
to the adsorbed
antigens in the well and will remain there a
ter washing. I
the antibody (
rom
an HIV- positive
patient) remains bound to the antigen in the well, then the secondary antibody w
ill bind to it
and remain attached a
ter washing. I
the patient is negative
or HIV, there wil
l be no primary
antibody to bind to the antigen and, in turn, no secondary antibody binding. Sec
ondary antibodies
are usually raised in rabbits and goats immu- nized with human IgG tractions. Se
condary
antibodies (anti-HIV-IgG) are puri ed and covalently cross-linked to horseradish p
eroxidase. This
modi cation does not usu- ally a

ect the binding speci city and a

nity o
the antibo
dy or the
enzymatic activity o
the peroxidase. 5. Wells are washed to remove unbound
secondary
antibody. 6. A
ter washing, a solution containing hydrogen peroxide and aminosal
icylate is added
to each well. Peroxidase pos- sesses a high catalytic activity and can exceed tu
rnover rates o

106 per second. Consequently, ampli cation o

an HIV-positive sample can occur ove
r several
orders o
magnitude. Many hydrogen donor cosubstrates can be used by peroxidase.
These
cosubstrates include o-dianisidine, aminoantipyrine, aminosalicylic acid, and nu
merous phe- nolic
compounds that develop color upon oxidation. The substrate solution used
or the
ELISA reaction
is nearly colorless. Peroxidase converts the peroxide to H 2 O O 2 using the sali
cylate as the
hydrogen donor. The oxidized salicylate is brown and can be easily observed in w
ells con- taining
anti-HIV-1 IgG (positive plasma). It should be noted that polyclonal antibody pr
eparations to a
given anti- gen can have variable binding a
nities due to di

erences in the immu

nologic
responses between animals. Di

erent immunizations with the same antigen in the

same animal can
also produce variable binding a
nities. The use o
monoclonal antibodies directed
against a
single epitope eliminates this variability. Western blot analysis o
positive EL
ISA samples is
used to con rm the presence o
HIV in a patient. Reagents, Materials, and Equipmen
t EDVOTEK Kit
#271: AIDS Kit 1Simulation o
HIV-1 Detection, containing the
ollowing: HIV anti
gens
(simulated) Positive control (primary antibody) Donor 1 serum (simulated) Donor
2 serum
(simulated) Anti-IgG-peroxidase conjugate (secondary antibody) Hydrogen peroxide
, stabilized

Aminosalicylic acid (peroxide cosubstrate) Phosphate bu

ered saline concentrate

Microtiter
plates Trans
er pipettes Microtest tubes with attached caps 1 mL pipettes Plasti
c tubes, 50 mL
This experiment does not contain HIV virus or its com- ponents. None o
the comp
onents have been
prepared
rom human sources. Additional Materials Required Distilled or deionize
d water Beakers
37C incubation oven Disposable lab gloves Sa
ety goggles Automatic micropipettes
and tips
recommended Make sure glassware is clean, dry, and
ree o
soap residue. EXERCIS
E
1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page 419 420 SECTION 4 Serological Diag
nosis o

In
ectious Disease Experiment Objective The objective o
this experiment is to u
nderstand the
molec- ular biology o
HIV and the pathogenesis o
AIDS. The experimental conce
pts and
methodology involved with enzyme linked immunosorbent (ELISA) assays will be i
ntro- duced in
the context o
the clinical screening o
serum samples
or antibodies to
the virus.
Laboratory Sa
ety 1. Exercise extreme caution when working with equipment that i
s used in
conjunction with the heating or melting o
reagents. 2. Do not mouth pipette rea
gentsuse pipette
pumps or bulbs. 3. Gloves and goggles should be worn routinely as good laborator
y practice.
Procedure* General Instructions and Procedures Labeling The Microtiter Plate Mar
k the
microtiter plate with your initials or lab group number and number the rows 1 to
4 down the side.
Label 5 trans
er pipets as
ollows:
(negative) (positive) DS 1 (donor serum 1) D
S 2
(donor serum 2) PBS (phosphate bu

ered saline) Use the appropriately labeled pl

astic trans
er
pipette
or liquid removals and washes. Instructions
or Adding Liquids and Wash
ing Wells Adding
reagents to wells: For adding reagents to the wells, use the same 1 ml pipette.
Rinse the
pipette thoroughly with distilled water be
ore using it
or adding the next reag
ent. Liquid
Removal and Washes When instructed in the experimental procedures, remove liquid
s with the
appropriately labeled trans
er pipette, and then wash the wells as
ollows: 1. U
se the trans
er
pipette labeled PBS to add PBS bu

er to the wells in all rows. Add PBS bu

er unt
il each well
is almost
ull. The capacity o
each well is approximately 0.2 ml. Do not allow
the liquids to
spill over into adja- cent wells. 2. With the appropriately labeled trans
er pip
ette, remove all
the liquid (PBS bu

er)
rom the wells in each row. Dispose the liquid in the be
aker labeled
waste. Experimental Steps
or the Elisa 1. To all 12 wells, add 100 l o
HIV (viral
antigens). 2. Incubate
or 5 minutes at room temperature. 3. Remove all the liqu
id (viral
antigens) with a trans
er pipette. 4. Wash each well once with PBS bu

er as des
cribed in Liquid
Removal and Washes. Wear sa
ety goggles and gloves. In research labs,
ollowing t

his step, all

sites on the microtiter plate are saturated with a blocking solution consisting
o
a protein
mixture, such as BSA. We have designed this experiment to eliminate this step to
save time. 5.
Add reagents as outlined below: Remember to rinse the 1 ml pipette thoroughly wi
th dis- tilled
water be
ore adding a new reagent. I
you are using automatic micropipettes, use
a clean
micropipette tip
or each reagent. Add 100 l o
PBS bu

er to the three wells in

row 1. (This
is the negative control.) Add 100 l o

(positive) to the three wells in row 2. (T

his is the
positive control.) Add 100 l o
donor serum DS1 to the three wells in row 3. Add 10
0 l o

donor serum DS2 to the three wells in row 4. 6. Incubate at 37C

or 15 minutes. 7.
Remove all
the liquid
rom each well with the appro- priately labeled trans
er pipette. 8.
Wash each well
once with PBS bu

er (as described under Liquid Removal and Washes). 9. Add 100 l o

the
anti-IgG peroxidase conjugate (2Ab) to all 12 wells. 10. Incubate at 37C
or 15 mi
nutes. At
this time you can obtain the substrate to be used in step 13. Since the substrat
e must be
prepared just prior to use, your instructor will prepare it toward the end o
th
e incubation in
step 10. 11. Remove all the liquid
rom each well with the appro- priately label
ed trans
er
pipette. Quick Re
erence: The positive control, which contains IgG directed agai
nst HIV antigens,
is the primary anti- body. Positive serum samples will also contain anti-HIV IgG
, while negative
serum samples will not contain anti- HIV IgG. * Permission granted to reproduce
the
ollowing
in
ormation by EDVOTEKThe Biotechnology Education Company (edvotek@aol.com).
1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page 420 CHAPTER 23 Laboratory Diagnosi
s o
HIV
In
ection 421 12. Wash each well once with PBS bu

er (as described under Liquid

Removal and
Washes). 13. Add 100 l o
the substrate to all 12 wells. 14. Incubate at 37C
or 5
minutes.
15. Remove the plate
or analysis. 16. I
color is not
ully developed a
ter 5 m
inutes, incubate
at 37C
or a longer period o
time. Interpretation o
Results The positive contro
l, which
contains IgG directed against HIV antigens, is the primary antibody. Positive se
rum sam- ples
will also contain anti-HIV IgG. Wells containing positive control or posit
ive serum samples
will develop a brown color. Negative serum samples will not contain anti- HIV Ig
G and will remain
colorless. 1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page 421 422 SECTION 4 Serol
ogical Diagnosis
o
In
ectious Disease 1. All o
the
ollowing apply to HIV except a. it possesse
s an outer
envelope. b. it contains an inner core with p24 antigen. c. it contains DNA as i
ts nucleic acid.
d. it is a member o
the retrovirus
amily. 2. Which o
the
ollowing genes is r
esponsible
or

the cod- ing o

reverse transcriptase? a. Env b. Pol c. Gag d. Tat 3. HIV virio
ns bind to
host T cells through which receptors? a. CD4 and CD8 b. CD4 and the IL-2 rec
eptor c. CD4 and
CXCR4 d. CD8 and CCR2 4. Antibodies to which o
the
ollowing viral antigens are
usually the rst
to be detected in HIV in
ection? a. gp120 b. gp160 c. gp41 d. p24 5. Which o
th
e
ollowing is
typical o
the latent stage o
HIV in
ection? a. Proviral DNA is attached to cel
lular DNA. b.
Large numbers o
viral particles are synthesized. c. A large amount o
viral RNA
is synthesized.
d. Viral particles with no envelope are produced. 6. The decrease in T-cell
numbers in
HIV-in
ected individuals is due to a. lysis o
host T cells by replicating virus
. b.
usion o

the T cells to
orm syncytia. c. killing o
the T cells by HIV-speci c cytotoxic T
cells. d. all
o
the above. 7. The most common means o
HIV transmission world- wide is throug
h a. blood
trans
usions. b. intimate sexual contact. c. sharing o
needles in intravenous d
rug use. d.
transplacental passage o
the virus. 8. All o
the
ollowing are likely immunolo
gic mani
estations o
HIV in
ection except a. decreased CD4 T-cell count. b. increased CD8 Tcell count. c.
increased response to vaccine antigens. d. increased serum immunoglobulins. 9. T
he drug
zidovudine is an example o
a a. nucleoside analogue reverse transcriptase inhib
itor. b.
nonnucleoside reverse transcriptase inhibitor. c. protease inhibitor. d.
usion
inhibitor. 10.
Which o
the
ollowing methods is used in third- generation ELISA tests

or HIV antibody?
a. Binding o
patient antibody to solid-phase recom- binant HIV antigens
ollowe
d by addition o

enzyme-labeled antihuman immunoglobulin b. Binding o

patient antibody to solidphase recombinant HIV antigens,
ollowed by addition o
enzyme-labeled HIV-speci c antibodies
c. Binding o

patient antibody to solid-phase recom- binant HIV antigens,

ollowed by addition
o

enzyme-labeled HIV antigens d. Binding o

patient antibody to a solid-phase coat
ed with antigens
puri ed
rom HIV viral lysates,
ol- lowed by addition o
enzyme-labeled antihuman
immunoglobulin
11. I
a test has a high positive predictive value, which o
the
ollowing is tr
ue? a. There will
be no
alse negatives. b. Most positives are true positives. c. It is not a good
screening test.
d. The number o
true positives will vary with the population. 12. False-negativ
e test results in
the ELISA test
or HIV antibody may occur because o
a. heat inactivation o
the
serum prior to
testing. b. collection o
the test sample prior to seroconversion. c. inter
eren
ce by
autoantibodies. d. recent exposure to certain vaccines. 13. Which o
the
ollowi
ng combinations
o
bands would represent a positive Western blot
or HIV antibody? a. p24 and p5
5 b. p24 and p31

c. gp41 and gp120 d. p31 and p55 REVIEW QUESTIONS 1814_Ch23_399-426.qxd 7/10/09
5:20 PM Page
422 CHAPTER 23 Laboratory Diagnosis o
HIV In
ection 423 14. Which o
the
ollow
ing tests would
give the least reli- able results in a 2-month-old in
ant? a. CD4 T-cell count b
. ELISA
or HIV
antibody c. RT-PCR
or HIV nucleic acid d. NASBA
or HIV nucleic acid 15. The RT
-PCR is a highly
sensitive method that involves a. direct ampli cation o
HIV RNA. b. ampli cation o

a label
attached to HIV RNA. c. ampli cation o
a complementary DNA sequence to a portion
o
the HIV RNA.
d. DNA sequencing o
a portion o
HIV RNA. 1814_Ch23_399-426.qxd 7/10/09 5:20
PM Page 423 424
SECTION 4 Serological Diagnosis o
In
ectious Disease Re
erences 1. World Healt
h Organization.
Global HIV prevalence has leveled o

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entre/news/
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or Dis
ease Control and
Prevention. HIV/AIDS sur- veillance report, 2006, vol. 18, rev. ed. U.S. Departm
ent o
Health and
Human Services, Centers
or Disease Control and Prevention, Atlanta, 2007, pp. 15
5. 3.
Barre-Sinoussi, F, et al. Isolation o
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rom a pati
ent at risk
or
acquired immunode
iciency syndrome (AIDS). Science 220:868870, 1983. 4. Gallo, RC
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T-lymphotropic retrovirus, HTLV- III isolated
rom AIDS patients and donors at r
isk
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rom
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55(RR-14): 117, 2006. 1814_Ch23_399-426.qxd 7/10/09 5:20 PM Page 426 Accelerat
ed rejection: A

orm o
rejection that occurs within 1 to 5 days a
ter second exposure to tissue
antigens based
on reactivation o
B- and T-cell responses. Accuracy: The ability o
a test to a
ctually measure
what it claims to measure. Acquired immunity: See adaptive immune response. Act
ivation unit: The
combination o
complement components C1, C4b, and C2b that
orm the enzyme C3 co
nvertase, whose
substrate is C3. Acute cellular rejection: A type o
rejection that occurs days
to weeks a
ter
transplantation due to cellular mechanisms and antibody
ormation. Acute GVHD: G
ra
t-versus-host
disease, which occurs shortly a
ter immunocompetent cells are transplanted into
a recipient. It
is characterized by skin rashes, diarrhea, and increased suscep- tibility to in

ection. Acute
phase reactants: Normal serum proteins that increase rapidly as a result o
in
e
ction, injury, or
trauma to the tissues. Acute phase response: Proteins and cells in the bl
ood that
increase rapidly in response to an in
ectious agent. It is consid- ered part o

natural immunity.
Acute rheumatic
ever: A disease that develops as a sequel to group A streptococ
cal pharyngitis,
characterized by the pres- ence o
antibodies that cross-react with heart tissue
. Adaptive immune
response: Host response to
oreign agents that depends on T and B lymphocytes an
d is
characterized by speci city, memory, and recognition o
sel
versus nonsel
. Adapt
ive T
regulatory 1 cells (TR1): CD4 T cells induced
rom antigen-activated nave T cells
under the
in
luence o
interleukin-10. They exert suppressive activities. Adjuvant: A subs
tance
administered with an immunogen that enhances and potentiates the immune response
. A
nity: The
initial
orce o
attraction that exists between a Fab site on an antibody and on
e epitope or a
determinant site on the corresponding antigen. Agglutination: The process by whi
ch particulate
antigens such as cells aggregate to
orm large complexes when speci c anti- body i
s present.
Agglutination inhibition reaction: An agglutination reaction based on competit
ion between
antigen-coated particles and soluble patient antigens
or a limited number o
a
nti-

body-combining sites. Lack o

agglutination is a positive test result. Agglutini
n: An antibody
that causes clumping or agglutination o
the cells that triggered its
ormation.
AIDS: Acquired
immunode ciency syndrome, a disease a

ecting the immune system caused by the huma

n
immunode
iciency virus (HIV). Allele: A di

erent
orm o
a gene that codes
or
a slightly
di

er- ent
orm o
the same product. Alloantigen: An antigen that is
ound in a
nother member o

the hosts species and that is capable o

eliciting an immune response in the host
. Allogra
t:
Tissue trans
erred
rom an individual o
one species into another individual o

the same species.

Allotype: A minor variation in amino acid sequence in a particu- lar class o

immunoglobulin
molecule that is inherited in Mendelian
ashion. Alternative pathway: A mean
s o
activating
complement pro- teins without antigenantibody combination. This pathway is trigge
red by
constituents o
microorganisms. Amplicon: A copy o
a select portion o
DNA that
is obtained by
the polymerase chain reaction. Analyte: The substance being measured in an immun
oassay.
Anaphylatoxin: A small peptide
ormed during complement acti- vation that causes
increased
vascular permeability, contraction o
smooth muscle, and release o
histamine
r
om basophils and
mast cells. Anaphylaxis: A li
e-threatening response to an allergen charac- teri
zed by the
systemic release o
histamine. Anaplastic: Tumors that are poorly di

erentiated
and are similar to
etal or embryonic tissue. Aneuploidy: Any deviation
rom the normal numb
er o
chromosomes. Antibodies: Serum
actors in the blood
ormed in response to
oreign sub
stance exposure.
Antibodies are also known as immunoglobulins. Antibody conjugates: Antibody
that is attached
to toxins or radioisotopes to help speci cally destroy cancer cells. Antibody-depe
ndent cell
cytotoxicity: The process o
destroy- ing antibody-coated target cells by n
atural killer
cells, monocytes, macrophages, and neutrophils, all o
which have speci c receptor
s
or antibody.
Antibody screen: The process o
testing recipient serum
or the presence o
anti
bodies to HLA
antigens on potential donor transplant cells. Anti-DNase B: An antibody directed
against DNase B,
which is secreted by group A streptococci. Antigenic variation: Result o
the pr
ocess o
antigen
switching. Antigens: Macromolecules that are capable o
eliciting
ormation o

immunoglobulins
(antibodies) or sensitized cells in an immunocompetent host. Antigen switch
ing: A protecting
mechanism used by parasites that involves varying synthesis o
sur
ace antigens
to evade an
immune response by the host. Anti-HBc: Antibody to hepatitis B core antigen. Ant
i-Hbe: Antibody
to hepatitis B capsid antigen. Anti-HBs: Antibody to hepatitis B sur
ace antigen
. Antinuclear

antibody (ANA): Antibody produced to di

erent com- ponents o

the nucleus durin
g the course o

several autoimmune diseases. Examples include anti-DNA, antideoxyribonucleoprotein, and

antiribonuclear protein antibodies, all o
which occur in systemic lupus erythem
atosus.
Apoptosis: Programmed cell death. Arthus reaction: A type III hypersensitivity r
eaction that
occurs when an animal has a large amount o
circulating antibody and 427