Clinically Significant Antibody

8/11/2019 Clinically Significant Antibody

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Clinically SignificantAntibody

Dr.M.Mohandoss


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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


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Mollison in late 1950s demonstrated that

Antibodies which reacted at room temperature but

not at 37C were not clinically important


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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


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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


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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)


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Intravascular hemolysis

IgM Ab which activateclassical complementpathway

Formation of membraneattack complex

Puncturing red cellmembrane

Ab commonly implicated are

Anti A, Anti B, Anti PP1Pk,

Vel, Lewis, Kidd antibodies


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Extravascular hemolysis

The immunological mechanism of extracellular destruction of

antibody-sensitised red cells is by phagocytosisand/or lysis

by the mononuclear phagocytic cellsof 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 sensitizedred cells to the IgG(Fc) and C3 (CR1 and CR3) receptors on the

macrophage


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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


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Naturally occurring antibodies

In blood group serology, the term naturally occurring isused for antibodies found in the serum of a subject whohas

Never been transfused (or)

Injected with red cells containing the relevantantigen (or)

Been pregnant with a fetus carrying the relevantantigen.


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Naturally occurring antibodies

Landsteiner (1945) concluded that natural antibodieshad a dual origin

Antigen Induced (heteroagglutinis) SpontaneousEg: Anti-A & B, Anti-H, -PP1pk and -PkThese antibodies are believed to beheteroagglutininsproduced as aresponse to substances in the envi-

ronment, which are antigenicallysimilar to red cell alloantigens

Eg: Anti-E and various others in theRh systemGenerated without the intervention ofantigens

Most naturally occurring antibodies are IgM cold agglutinins, reactingbest at room temperatureand activating complement


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Naturallyoccurringalloantibodies to

red cell antigens


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Immune antibodies

Immune antibodies are those antibodies found in the serum

of individuals who have been

Transfused (or)

Pregnant

Most immune antibodies are predominantly IgGthat react

best at 37C

Eg: Antibodies of Rh, Kell, Duffy, Kidd, Ss blood group

systems


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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 correspondingantigens 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


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Greatest concern to us are

the clinically significantantibodies


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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


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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


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Antibody specificity

Many publications divide antibodies into

Clinically significant

Not clinically significant

For better understanding, described as

Usually

Sometimes

Not or rarely clinically significant


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Specificity and potentially clinically

significance of 37C reactive antibodies


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Methods to determine clinical

significance of antibody

Serology- Thermal amplitude

1hour 51Cr RBC survival

In vitro cellular bioassays


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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 predominantly

IgM, and are nonreactive at 37C (e.g., anti-I, - PI, -Lea, -Le)


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51Cr RBC survival

Giving pt with small amount (0.5ml) of 51Cr 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-minutesample should be, on average, about99% of that of the 3-minute sample

Donor RBCs may be transfused withminimal hazard

70% or greater survival at 60 minutes


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51Cr 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 reactionand requires further transfusion.


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Functional cellular assay

In-vitro assay were developed to simulate antibody- coated

cells interacting with peripheral blood Fc receptorbearing

cells, usually monocytes and their interaction

Monocyte monolayer assay (MMA)

Antibody dependent cellular cytotoxicity (ADCC)

Chemiluminescence


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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 cellsfrom 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 inculture media containing 5-percent fetal calf serum and added to 8-welltissue culture chamber slides

After a 1-hour incubation at 37C , the supernatant containing nonadherent

lymphocytes was removed via pipette and then by dipping the microscopeslides in phosphate-buffered saline.

Slides werestained with a Wright-Giemsa stain and observedmicroscopically.

200 to 600 monocytes were counted, and the percentage of reactive

monocytes (i.e., monocytes with RBCs adhering and/or phagocytized) was determined


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Disadv

Not easy to perform

Time consuming (>6hrs)

< 3% reactivity(similar to 1hr Cr RBC survival >70%)

Clinical insignificance & werenever associated with clinical illeffects

> 3% reactivity Clinically significant antibody &requiring compatible blood


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ADCC

ADCC assay:

RBCs are labelled with 51Cr

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


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Chemiluminescence (CLT)

Chemiluminescence:Metabolic response of monocytes during erythrophagocytosis ismeasured

Mononuclear cells are incubated at 37C with presensitized red cellsand luminolThe amount of luminescence produced by luminol, in the presenceof oxygen radicals (oxidative burst that accompanies phagocytosis)is measured


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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


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Patients requiring transfusion

Specificity of antibody- clinical significance

51Cr 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


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Correlation of MMA with transfusion

reactions

After incompatible transfusion reactions

Concluded negative result (


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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, -CobLeast incompatible red cells, butantigen-negative red cells for strongexamples of the antibody

Cromer, -Yta, -Gya, -Hy, -Joa, -Lan, -Ata, -Jra

Least incompatible red cells Gerbich, knops, anti-LWa, -LWb -JMH, -

Emm, -PEL, -ABTIIdeally antigen-negative red cells, but,due to their extreme rarity, leastincompatible red cells should be usedwith extreme caution.

Anti-Sc3, -Co3 -Oka, -MAM

The clinical significance of blood group antibodies. Transfus Med 12:287295,2002

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 andthe patient observed closely throughout


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Summary of Approaches for ProvidingBlood for the Alloimmunized Patient

Determine specificityof alloantibody. Ensure that antibody istruly active at 37C (e.g., use of prewarm technique)

If antibody is known to be usually clinically significant, selectRBCs lacking putative antigen(s) and crossmatch (includingantiglobulin test)

Provenclinically 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.


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Summary of Approaches for ProvidingBlood 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 51Cr-labeled incompatible

RBCs;

Functional cellular assay (e.g., MMA or CLA).


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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


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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 laboratoryevidence of hemolysis


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DHTR vs DSTR at Mayo Clinic (1980-

1988)

DHTR : 1/3 reactions

DSTR : 2/3 reactions


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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 werereported as No Adverse Effect

Robillard et al from Quebec Hemovigilance system

46% of 24 patients receiving ABO incompatible units werereported as Asymptomatic


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Conclusion

It is difficult to define clinically significant antibody because it hasdifferent 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 clinicallysignificant, whereas others not


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High Titer Low Avidity Antibodies

Titer 1:64

Avidity 1+ or 2+

Not clinically significant

Weak reactions at AHG phase

Variable reaction amongpanel cells

Inconsistent results,sometimes not reproducible

Reactions weaker on older

RBC

Not enhanced with PEG,LISS, Enzymes


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High Titer Low Avidity Antibodies


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Thank you


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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 antibodyagainst the high-prevalence antigen


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Recombinant Blood group antigens

Single recombinant bloodgroup proteins (rBGPs)

1. Reaction of an antibody with its

corresponding antigen directlyindicates the antibodyspecificity

2. Allows antibody detection andidentification in a single step

3. multiple antibodiessimultaneously present in aserum could be easily identified


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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

Documents

Clinically Significant Antibody