HCG (Human Chorionic Gonadotropin)

Glycoprotein hormone secreted by the developing

placenta during pregnancy

Nourishes the fertilized egg where this fertilized egg

becomes attached to the uterine wall

Levels can be detected by blood test about 11 days

after conception and about 12-14 days in the urine

Concentration in serum is equal to the concentration

in urine

Concentration in urine and serum doubles every 72

hours

Continue to rise during the first trimester of

pregnancy

Appears during early stages in pregnancy

Declines or even level off for the remainder of the

pregnancy

Makes an excellent indicator for the detection of

pregnancy

Prevents corpus luteum from degenerating

Continues to secrete progesterone and estrogen

Maintains the integrity of uterine wall and inhibits

subsequent ovulation due to lack of FSH or LH
Beta subunit hCG

An ectopic protein

Sensitive tumor marker with a metabolic half-life in

vivo of 16 hours

Serum level higher than 1 mg/mL is strongly

suggestive of pregnancy or malignant tumor as an
endodermal
sinus
tumor,
tetratocarcinoma,
choriocarcinoma,
molar
pregnancy,
testicular
embryonal carcinoma or oat cell carcinoma of the
lung
Weeks of Gestation
3 weeks
4 weeks
5 weeks
6 weeks
7-8 weeks
9-12 weeks
13-16 weeks
17-24 weeks
25-40 weeks
Non-pregnant females
Postmenopausal females

Levels of HCG in mIU/mL

5-50
5-426
18-7340
1080-56500
7650-229000
25700-288000
13300-254000
4060-165400
3640-117000
<5.0
<9.0

Materials/Equipment/Reagent

Sample collection cups or tubes (Flat bottom widemouth container is ideal)

Transfer pipettes

Timer

Urine sample

HCG cassette test device with desiccant

Positive and negative controls

Note: HCG test kit should be stored at temperature 2-8C in the
sealed pouch for the duration of the shelf-life.
Principle

Membrane of the test was coated with anti-HCG

antibodies on the test region and goat anti-mouse
IgG antibodies on the control region

Urine specimen is allowed to react with the HCG

monoclonal antibody-colloid gold conjugate (predried on the test trip)

Mixture then moves upward on the membrane

chromatographically by capillary action

Positive specimen: conjugate binds to the HCG

forming an antibody-antigen complex

The complex is captured by anti-hCG antibody

immobilized on the test region and produces a pink
color band when concentration is equal or greater
than 25mIU/mL

Absence of colored band in the test region suggests a

negative result

To serve a procedural control: colored band at the

control region will always appear regardless the
presence or absence of hCG
Specimen Collection

Urine specimens must be collected in a clean dry

container either plastic or glass without preservatives

No centrifugation or filtration of urine is required

Specimens collected at any time may be used

First
morning
urine
contains
the
highest
concentration of the hormone

Urine specimens maybe stored at room temperature

(15-30C) for up to 8 hours or refrigerated (2-8C) up
to 72 hours
If specimen is stored refrigerated, allow it to warm to
room temperature before use

Procedure

Bring all materials and specimens to room

temperature

Remove the test cassette from the sealed foil pouch

Label the test cassette with specimen identity on the

ID--- area of the cassette

Place the test cassette on a flat horizontal surface

Using the transfer pipette in a vertical position over

the sample well and dispense 2-3 drops (80-120uL)
of sample into the sample well

Read the result at 5 minutes after adding the sample

Interpretation of the Result

Positive: Two distinct pink-colored bands appear, one

in the Test Region (T) and the other in the Control
region (C)

Negative: Only one pink-colored band appears in the

control region

Invalid: total absence of pink-colored bands in both

regions is a n indication of procedural error and the
sample may be repeated with a new test device
Bence Jones Proteins

Single-peptide chains with a MW of 20kDa-22kDa

Dimerization occurs spontaneously to form 40-44kDa

Monoclonal kappa (K) or lambda ( ) immunoglobulin

free light chains (FLCs) not attached to the heavy
chain portion of the immunoglobulin molecule

Found in urine especially of persons affected with

multiple myeloma- 10% of MM- only BJP are produced
with no complete immunoglobulin mu (IgM),
immunoglobulin gamma (IgG) or immunoglobulin
alpha (IgA)

Proteinuria: common finding with over half of all MM

excreting abnormal amounts of BJP- much more likely
to have renal tubular defects

Two mechanisms by which deleterious effect on renal

function is exemplified:
o
It may result from the intratubular
precipitation of BJP and subsequent
intrarenal obstruction; where large casts
consisting of BJP obstructed collecting
tubules- may be referred to as myeloma of
the kidney
o
Direct
tubular
cell
injury
,
where
abnormalities in urine concentrating ability
and renal acidification are observed

Some patients may excrete large amounts of NJP for

years and maintain renal function although large
concentration of BJP is usually associated with some
degree of renal dysfunction

Can be detected in serum, urine, or both

Level of monoclonal light chains in serum or urine is

related to filtration, resorption, or catabolism of the
protein by the kidneys
Principle

Heat stability is used t detect BJP, the urinary

protein characteristic of multiple myeloma

BJP is a soluble in the urine at room temperature

When urine is heated to 40C, a white cloud appears

A distinct precipitate forms at 60C

The precipitate dissolves again at around 100C

(boiling) but reappears on cooling

Minimal detectable concentration of BJ protein is

about 30mg/dL

Excessive amounts of acid and salt will prevent the

appearance of the precipitate
Specimen collection

Random or a 24-h urine specimen is collected in a

clean container

Specimen can be refrigerated to prevent bacterial

growth
Materials/Equipments/Reagents

Acetate buffer, 2 mol/mL, pH 4.9 or 10% aqueous

solution of acetic acid (for acetate buffer, add 4.1
mL of glacial acetic acid to 17.5 g sodium acetate
trihydrate, and then distilled water to a total volume
of 100 mL)
Procedure

Place 4 mL of clear urine into a test tube

Add 1mL of acetate buffer or 10% aqueous solution
of acetic acid and mix

Place the urine-buffer solution into a heat block or

boiling-water bath min at 56C for 15 minutes

If the solution develops turbidity (cloudness),

transfer the test tube to a 100C heat source, (eg.
Boiling water bath) for 3 minutes

Examine the urine solution for turbidity

Filter the urine solution immediately after removing

it from the boiling water bath

Examine the solution for turbidity as it cools; reexamine for turbidity as the solution reaches room
temperature
Note: if the precipitation is heavy, it is best to dilute the urine
(1:2) and repeat the procedure
Reporting Results

If BJP are present in the urine, the solution will

become cloudy as it cools and become clear as it
reaches room temperature
Qualitative and Semi- Quantitative Determination of
Infectious Mononucleosis Heterophile Antibodiesin the
Serum or Plasma (Monospot Latex)

Heterophile antibodies are composed of a broad class

of antibodies stimulated by one antigen and react
with an entirely unrelated surface antigen present on
cells from different mammalian species
Heterophile antibodies may be present in normal
individuals in low concentrations (titers), but a titer
of 1:56 or greater is clinically significant in patients
with suspected infectious mononucleosis
Immunoglobulin M (IgM) type of heterophile antibody
usually appears during acute phase of the infectious
mononucleosis but the antigen that stimulates its
production remains unknown
IgM heterophile antibody is characterized by the
following features:
o
Reacts
with
horse,
ox,
and
sheep
erythrocytes
o
Absorbed by beef erythrocytes
o
Not absorbed by guinea pig kidney cells
o
Does not react with Epstein Barr Virus
specific antigens

Heterophile Antigens and Antibodies

Forssman antigen is an example of a heterophile

antigen and is found on the RBCs of many species

Forssman anbodies formed against Forssman

antigens will agglutinate sheep RBCs

Infectious mononucleosis (IM) is a contagious viral

disease occurring worldwide caused by Eipstein Barr
Virus

Newborns from infected mothers have tendency to

be immune at birth because of the protection from
maternal antibody once passive protection
subsides, the infant becomes susceptible to EBV
infection

Childhood infections with EBV may as asymptomatic

or have mild symptoms

Adolescents and young adults become infected with

EBV may develop IM

EBV infection can cause more serious in severely

suppressed individual

Once infected, the virus remains present in cells of

the throat, blood, or immune system

Transmitted by an intimate contact with the mouth

and nasal discharges

Heterophile Antibody Titers

Determined with tube dilution

A titer of 1:40 after absorption with guinea pig cells is

considered
positive
for
acute
infectious
mononucleosis

60-90% of patients have test results that are positive

for heterophile antibodies in the second or third week

It begins to decline until less than 1:40 within 2-3

months

20% of patients still have positive titer results within

1-2 years

75% of patients have positive horse RBC agglutinin

findings at 1 year

10-30% of children younger than 2 years and 50-75%

of children aged 2-4 years develop heterophile
antibodies with primary EBV infection
Tests

Paul and Bunnell associated infectious mononucleosis

with sheep cell agglutination and developed a test
for IM heterophile
Davidsohn modified the original Paul- Bunnell test by
introducing a differential absorption to remove the
cross-reacting Forssman and serum sickness
heterophile antbodies
Rapid agglutination tests are also available

Paul- Bunnell

Original test is simple titration of sheep cell

agglutinins

Tissues rich in Forssman antigen (guinea pig kidney)

absorb Forssman antibodies but do not affect the
heterophil antibodies in IM

Forssman hapten s a glycolipid usually associated

with a protein, the determinant being largely
carbohydrate and therefore heat stable

Heterophile antibodies are absorbed by beef cells

Davidsohn Differential Test

Principle is that the two types of sheep agglutinins

are distinguished by titrating the before and after
absorption with guinea pig kidney and ox cells

Patients serum containing antibodies due IM is added

to guinea pig kidney cells
o
Antibodies are not absorbed by the kidney
cells but react with beef (ox) red blood cells
which causes agglutination and is positive
for IM

Patients serum containing Forssman antibodies are

added to guinea pig kidney cells
o
Antibodies are absorbed by the kidney cells
then allowed to react with beef red blood
cells which does not cause agglutination
o
Positive for Forssman antigens
Agglutination Test

Monospot Latex reagent is a suspension of

polysterene latex particles of uniform size coated
with highly purified Paul-Bunnell antigen from bovine
red cell membranes

Degree of purity of the antigen is it only reacts with

infectious mononucleosis heterophile antibodies

Latex particles allow visual observation of the

antigen-antibody reaction

If IM antibodies are present in either serum or

plasma, the latex suspension changes its uniform
appearance and a clear agglutination becomes
evident