Clinically Significant

Antibody
Dr.M.Mohandoss
Pretransfusion testing
Upto late 1970s
To increase the sensitivity of pretransfusion testing
Attempt to detect all antibodies in pts sera
Identify the antibody specificity
Supply RBC that lacked cognate antigen
At that time, all antibodies were considered significant
Mollison in late 1950s demonstrated that
Antibodies which reacted at room temperature but
not at 37C were not clinically important
Blood group antibodies
Destruction of allogeneic red cells in HTR
Destruction of autologous red cells in AIHA
Destruction of fetal red cells in HDFN
Damage of transplanted tissue
Blood group antibodies
IgG, IgM and IgA are the most significant for blood bank
IgM
Activate complement
IgG and IgA
React with Fc receptors on macrophages
Activates complement sometimes


IgG subclass
IgG1, IgG2 or IgG3 react with Fc receptors on
macrophage
IgG3 most efficient in activating complement
IgG4 do not activate complement and no receptors on
macrophage
IgG2 rarely activate complement & varies in efficiency
in reacting with macrophages (85% of Japanese &
30% of Caucasians)

Intravascular hemolysis
IgM Ab which activate
classical complement
pathway
Formation of membrane
attack complex
Puncturing red cell
membrane
Ab commonly implicated are
Anti A, Anti B, Anti PP1Pk,
Vel, Lewis, Kidd antibodies
Extravascular hemolysis
The immunological mechanism of extracellular destruction of
antibody-sensitised red cells is by phagocytosis and/or lysis
by the mononuclear phagocytic cells of the spleen or the
Kupffer cells of the liver.
These cells have specific receptors for IgG1, IgG2, IgG3, and
the C3 component
They destroy red cells following attachment of the sensitized
red cells to the IgG(Fc) and C3 (CR1 and CR3) receptors on the
macrophage
Extravascular hemolysis
Extravascular hemolysis caused by IgG antibodies
Rh system are IgG1 and IgG3 by phagocytosis
Kell and Duffy are IgG1 and Kidd IgG3
Kidd and Duffy- C3 binding, but insufficient quantity for
significant intravascular hemolysis.
IgG and C3 will be sequestered by macrophages

Naturally occurring antibodies
In blood group serology, the term naturally occurring is
used for antibodies found in the serum of a subject who
has
Never been transfused (or)
Injected with red cells containing the relevant
antigen (or)
Been pregnant with a fetus carrying the relevant
antigen.
Naturally occurring antibodies
Landsteiner (1945) concluded that natural antibodies
had a dual origin



Antigen Induced (heteroagglutinis) Spontaneous
Eg: Anti-A & B, Anti-H, -PP1pk and -
Pk
These antibodies are believed to be
heteroagglutinins produced as a
response to substances in the envi-
ronment, which are antigenically
similar to red cell alloantigens
Eg: Anti-E and various others in the
Rh system
Generated without the intervention of
antigens
Most naturally occurring antibodies are IgM cold agglutinins, reacting
best at room temperature and activating complement
Naturally
occurring
alloantibodies to
red cell antigens
Immune antibodies
Immune antibodies are those antibodies found in the serum
of individuals who have been
Transfused (or)
Pregnant
Most immune antibodies are predominantly IgG that react
best at 37C
Eg: Antibodies of Rh, Kell, Duffy, Kidd, Ss blood group
systems
Expected Vs Unexpected
Expected antibodies
Anti A and anti B are often referred as expected antibodies,
Because in adults with a normal immune system these
antibodies are almost always present when the corresponding
antigens are absent on the red cells
Unexpected antibodies
All antibodies to red cell antigens, other than naturally
occurring anti A and anti B are considered irregular or
unexpected antibodies
Greatest concern to us are
the clinically significant
antibodies
AABB definition
An antibody is considered potentially clinically significant
if antibodies of its specificity have been previously
associated with
hemolytic disease of the fetus and new- born (HDFN)
Hemolytic transfusion reaction (HTR) or
with notably decreased survival of transfused red
cells.
Antibodies reactive at either 37 C or in the AHG test
phase are more likely to be clinically significant
Other Definitions
Shortened RBC survival
Laboratory evidence of hemolysis
(increased bilirubin levels, LDH)
Clinical signs of hemolytic transfusion reaction
(Jaundice)
Shortened RBC survival
Activating complement leading to intravascular or
Extravascular hemolysis by interacting with Fc receptor on
macrophages in spleen/liver
Affected by
Ig class, subclass
Thermal amplitude
Specificity
Antibody specificity
Many publications divide antibodies into
Clinically significant
Not clinically significant
For better understanding, described as
Usually
Sometimes
Not or rarely clinically significant
Specificity and potentially clinically
significance of 37C reactive antibodies
Methods to determine clinical
significance of antibody
Serology- Thermal amplitude
1hour
51
Cr RBC survival
In vitro cellular bioassays
Thermal amplitude
Only antibodies that react in vitro at 37C are considered
clinically significant
If does not react at 37C, it should cause
No significant red cell destruction and
No immediate clinical effects due to an immune reaction
Most naturally occurring antibodies are pre- dominantly
IgM, and are nonreactive at 37C (e.g., anti-I, - PI, -Lea, -Le
)

51
Cr RBC survival
Giving pt with small amount (0.5ml) of
51
Cr labelled RBC
Samples are then taken at 3, 10 and 60 minutes and the
radioactivity measured in the plasma and on the RBCs
If survival is 70% : HTR is unlikely
Compatible donor RBC Counting rate of the 60-minute
sample should be, on average, about
99% of that of the 3-minute sample
Donor RBCs may be transfused with
minimal hazard
70% or greater survival at 60 minutes
51
Cr RBC survival
International Committee for Standardization in
Hematology (ICSH) recommends red cell survival studies
utilizing radioisotopes as a test for compatibility
Main indications are
When serological tests suggest that all normal donors
are incompatible
When cold alloantibodies are present, active in vitro at
30C or higher, and a non-reacting donor cannot be found
When the recipient has had an unexplained hemolytic
transfusion reaction and requires further transfusion.
Functional cellular assay
In-vitro assay were developed to simulate antibody- coated
cells interacting with peripheral blood Fc receptor bearing
cells, usually monocytes and their interaction
Monocyte monolayer assay (MMA)
Antibody dependent cellular cytotoxicity (ADCC)
Chemiluminescence

Monocyte monolayer assay (MMA)
Sensitized RBCs (patients serum or plasma + antigen-positive or antigen-
negative RBCs fresh normal serum as a source of complement; incubated 60 min at
37C with no additive; washed)
Mononuclear cells from normal volunteer donors were separated by
centrifugation over a Ficoll-sodium diatrizoate density gradient (Ficoll-Paque).
The mononuclear cells were washed with phosphate-buffered saline, suspended in
culture media containing 5-percent fetal calf serum and added to 8-well
tissue culture chamber slides
After a 1-hour incubation at 37C , the supernatant containing nonadherent
lymphocytes was removed via pipette and then by dipping the microscope
slides in phosphate-buffered saline.
Slides were stained with a Wright-Giemsa stain and observed
microscopically.
200 to 600 monocytes were counted, and the percentage of reactive
monocytes (i.e., monocytes with RBCs adhering and/or phagocytized) was determined
Monocyte monolayer assay (MMA)




Disadv
Not easy to perform
Time consuming (>6hrs)

< 3% reactivity
(similar to 1hr Cr RBC survival >70%)
Clinical insignificance & were
never associated with clinical ill
effects
> 3% reactivity Clinically significant antibody &
requiring compatible blood
ADCC
ADCC assay:
RBCs are labelled with
51
Cr
Extracellular lysis: by the antibody-dependent cellular cytotoxicity
(ADCC) assay, using either lymphocytes (L) or monocytes (M)
The lysis being proportional to the amount of radioactivity
recovered in the supernatant
Chemiluminescence (CLT)
Chemiluminescence:
Metabolic response of monocytes during erythrophagocytosis is
measured
Mononuclear cells are incubated at 37C with presensitized red cells
and luminol
The amount of luminescence produced by luminol, in the presence
of oxygen radicals (oxidative burst that accompanies phagocytosis)
is measured
Peripheral blood mononuclear cells were isolated from blood pooled
from six donors by density gradient centrifugation
Washed four times in PBS containing BSA and resuspended at 2 106
cells mL in Hanks balanced salt solution (HBSS) {contain FCS}
Mononuclear cells (04mL) were then dispensed into polystyrene
cuvettes and incubated for 2 hr at 37C in 5% CO2 in air
Red cells (50 L) were sensitized with 100-L volumes of serum for 1 h
at 37 C
Cells were then washed three times and resuspended in 100 L HBSS.
Luminol and red cells were added to cuvettes containing monocytes
Chemiluminescent (CL) responses were monitored for 50min using a
luminometer
Clinical significance in HDFN
Measurement of antibody level in maternal circulation
Antibody titre
Critical titre 1:16 to 1:32 (except Kell)
Antibody quantification- by autoanalyser
(have a greater predictive value than titre)
Levels are 10-15IU/ml- aminocentesis/PUBS may be perfomed
Risks of transplacental hemorrhage

Clinical significance in HDFN
Cellular bioassay: better predictor of severity than
quantification or titre
Studies showing that ADCC (M) has better predictive value
than autoanalyser and titre


CLT >30% were always associated with elevated bilirubin
levels, and indicative of significant hemolysis
44 D neg patient with D pos infant, ADCC predicted severity in 39
cases, autoanalyzer in 35 and MMA in 32 cases
Patients requiring transfusion
Specificity of antibody- clinical significance

51
Cr labelled RBC aliquot
Among cellular assay, MMA is used widely ( CLT recently)
Ab against high incidence Ag
Safe and preferable to Ag neg unit
Caution with Ab of unidentified specificity
Correlation of MMA with transfusion
reactions
After incompatible transfusion reactions
Concluded negative result (<5%) indicated, incompatible blood could be
given without risk of overt HTR, but normal survival could not be guaranteed

Recommendations for Red Cells to be Selected for Transfusion
Antigen-negative red cells ABO, Rh, Kell, Duffy Kidd antibodies, anti-
Coa, -Vel
Red cells compatible by IAT at 37C Lewis antibodies, anti-A1, -P1, -Lua, -Doa,
-Dob, -Cob
Least incompatible red cells, but
antigen-negative red cells for strong
examples of the antibody
Cromer, -Yta, -Gya, -Hy, -Joa, -Lan, -Ata, -
Jra
Least incompatible red cells Gerbich, knops, anti-LWa, -LWb -JMH, -
Emm, -PEL, -ABTI
Ideally antigen-negative red cells, but,
due to their extreme rarity, least
incompatible red cells should be used
with extreme caution.
Anti-Sc3, -Co3 -Oka, -MAM
The clinical significance of blood group antibodies. Transfus Med 12:287 295,2002
According to the NBS guidelines in England, if incompatible blood is to be transfused,
where possible, serologically least incompatible units should be selected.
Transfusion should be given at the slowest rate consistent with clinical condition and
the patient observed closely throughout
Summary of Approaches for Providing
Blood for the Alloimmunized Patient
Determine specificity of alloantibody. Ensure that antibody is
truly active at 37C (e.g., use of prewarm technique)
If antibody is known to be usually clinically significant, select
RBCs lacking putative antigen(s) and crossmatch (including
antiglobulin test)
Proven clinically insignificant antibodies (e.g., anti-Ch, Rg,
Bg, HTLA), then random units of the appropriate ABO/Rh type can be
crossmatched and the units that are compatible, or the least incom-
patible, can be issued.

Summary of Approaches for Providing
Blood for the Alloimmunized Patient
Specificity is known to be associated with both clinically
significant and insignificant antibodies (e.g., anti-Yt , -Ge,
Lan, -Lub), or
Specificity cannot be determined, and
Donors lacking the putative antigens are not freely available,
Tests performed to determine the clinical significance of the
antibody:
1 hr/24 hr RBC survival studies using
51
Cr-labeled incompatible
RBCs;
Functional cellular assay (e.g., MMA or CLA).
Summary of Approaches for Providing
Blood for the Alloimmunized Patient
If not possible to do the tests,
A biological compatibility test can be performed
10-50 ml of incompatible blood can be given slowly and the
patient monitored closely
Blood sample should be taken and the plasma examined for
hemoglobin.
Limited value and it only tests for the presence of antibody
capable of causing complement-mediated intravascular
hemolysis
Will not measure the clinical significance of even IgG antibody
(e.g., Rh) capable of extravascular destruction
Definition
AABB defines in Standards for Blood Bank and Transfusion
Services as
Antibodies that is capable of causing shortened cell survival
UK definition says
Antibodies that are capable of causing patient morbidity due to
accelerated destruction of significant proportion of transfused
RBCs
Sometimes significant
Ab of all specificities including Kidd, Duffy, Kell, and MNS
systems, may be clinically significant sometimes & not at
other times
Delayed Hemolytic Transfusion Reaction (DHTR)
Reactions occur days to months or even years after transfusion
Delayed Serological Transfusion Reaction (DSTR)
Rapid development of alloantibody in the absence of laboratory
evidence of hemolysis
DHTR vs DSTR at Mayo Clinic (1980-
1988)
DHTR : 1/3 reactions
DSTR : 2/3 reactions
ABO antibodies
Studies shows that even ABO Ab are not always clinically
significant
Linden et al 2000, report on 10 years of transfusion errors in
New York state
47% of 237 patients receiving ABO incompatible units were
reported as No Adverse Effect
Robillard et al from Quebec Hemovigilance system
46% of 24 patients receiving ABO incompatible units were
reported as Asymptomatic
Conclusion
It is difficult to define clinically significant antibody because it has
different meaning to different people and in different applications
In sickle cell disease (Hematology pt) : RBC survival is optimal
In Surgical pt (non hematology) : normal RBC survival is not so
important as pt will be producing their own RBCs with normal survival
We may need to use different definitions for different patients
Difficult to understand why some antibodies are hemolytic and clinically
significant, whereas others not
Rh MNSs P Lewis Lutheran
Kell Duffy Kidd Xg
a
D C E c e f V VS Cw M N S s P
1
Le
a
Le
b
Lu
a
Lu
b
K k Kp
a
Js
a
Fy
a
Fy
b
Jk
a
Jk
b
Xg
a
IS RT 37 AHG CC
1 0 + 0 + + + 0 0 0 + + + 0 0 + 0 + + 0 + 0 0 + + + + + 0 0 0 +/-
2 + + 0 0 + 0 0 0 + 0 + 0 + + + 0 0 + 0 + 0 0 + 0 + + + 0 0 0 m+
3 + + 0 0 + 0 0 0 0 + + 0 + + 0 + 0 + + + 0 0 0 + 0 + + 0 0 0 1+
W
4 + 0 + + O 0 0 / 0 + 0 0 + + 0 0 0 + 0 + 0 0 0 0 + 0 + 0 0 0 1+
W
5 0 0 + + + + 0 / 0 + 0 + + + 0 + 0 + 0 + 0 0 + + + + + 0 0 0 1+
W
6 0 0 0 + + + + + 0 0 + 0 + + 0 + 0 + 0 + 0 0 + 0 + 0 0 0 0 0 1+
W
7 0 0 0 + + + 0 0 0 + 0 0 + W 0 + 0 + + 0 0 0 0 + + + 0 0 0 0 1+
W
8 0 0 0 + + + 0 0 0 + + 0 + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 0 0 m+
9 0 0 0 + + + 0 / 0 + + + + 0 0 + 0 + 0 W + 0 + 0 + 0 + 0 0 0 m+
10 0 0 0 + + + 0 0 0 0 + 0 + + 0 + 0 + 0 + 0 0 + + 0 + + 0 0 0 m+
11 + + 0 0 + / 0 / 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + + 0 0 0 m+
Auto
Control
0 0 0 0 2+
High Titer Low Avidity Antibodies
Titer 1:64
Avidity 1+ or 2+
Not clinically significant
Weak reactions at AHG phase
Variable reaction among
panel cells
Inconsistent results,
sometimes not reproducible
Reactions weaker on older
RBC
Not enhanced with PEG,
LISS, Enzymes
High Titer Low Avidity Antibodies
Thank you
High prevalence antibodies
Cocktails of recombinant proteins
to neutralize antibodies against
high-prevalence antigens in pre-
transfusion antibody diagnostics
One protein cocktail (Lub, Yta,
Kpb k, Ge2, LWa) being specific
for clinically relevant and
Other cocktail (Cha, Rga, JMH,
Sc1, Kna McCa, Cra) being
specific for clinically insignificant
antibody specificities
A positive inhibition test directly indicates the clinical significance of the antibody
against the high-prevalence antigen
Recombinant Blood group antigens
Single recombinant blood
group proteins (rBGPs)
1. Reaction of an antibody with its
corresponding antigen directly
indicates the antibody
specificity
2. Allows antibody detection and
identification in a single step
3. multiple antibodies
simultaneously present in a
serum could be easily identified

Antibodies
Antibodies (or) immunoglobulins (Ig) are
Complex proteins produced by plasma cells
With specificity to antigen or immunogens that stimulate their
production
Antibodies have multiple functions
They bind antigens
Fix complement
Facilitate phagocytosis and
Neutralize toxic substances in the circulation