immunohematology basics

Deepa Babin @TMC Kollam 1

The study of immunologic reactions involving all components of

blood

Deals with antigens, antibodies and antigen-antibody reactions

IMMUNOHEMATOLOGY


Application in ....

transfusion of blood & its components diagnosis, prevention & management of

immunization asso with pregnancy

leukocyte testing for organ

transplantation laboratory resolution of parentage

problemsDeepa Babin @TMC Kollam 3

Agglutination / Hemolysis


Complete Antibodies

antibody

+

RBC w/ antigen


Incomplete Antibodies

+

RBC w/ antigenantibodies


1. ABO BLOOD GROUP2. Rh BLOOD GROUP SYSTEM3. OTHERS

A. MNSB. I/i C. DUFFYD. KELLE. KIDDF. PG. LUTHERANH. LEWIS

BLOOD GROUP SYSTEMS


ABO BLOOD GROUP

First blood groups discovered-Landsteiner(1900)

Most significant for transfusion practice

ABO compatibility is essential before other pretransfusion test is performed

ABO antigens are the only Ags for which reciprocal antibodies consistently and predictably exist in serum of normal individuals


ABO BLOOD GROUP

Anti-AAnti-BAnti-A,B

noneO

Blood Group RBC AgA AB B

AB A & BO none


GROUP A Express A antigen on RBC surface Genotypes AA or AO Have naturally occurring, clinically

significant, predominantly IgM (with a small amount of IgG) antibodies against type B (anti-B)

Subgroups A1 (80%) A2 (20%) Significance: some with A2 have antibodies

against the A1 subgroup (anti-A1)

ABO BLOOD GROUP…


ABO BLOOD GROUP…

GROUP B Express B Ag on RBC

surface Genotypes BB or BO Have naturally

occurring clinically significant, predominantly IgM (with a small amount of IgG) antibodies against type A cells


ABO BLOOD GROUP…

GROUP O Have neither A nor B antigens on their RBC Genotype OO (“universal donors”) Have naturally occurring, clinically

significant, very high titer, anti-A, anti-B and anti-A,B antibodies

Maternal anti-A,B can cross the placenta to cause hemolytic disease of the newborn

Group O cells have the most H antigen


ABO BLOOD GROUP…

GROUP AB Express A and B

Ag on RBC surface

Genotypes A1B or A2B

have no ABO antibodies (“universal recipients”)


H Antigen Seen on RBC of ABO-Universal

distributed

BOMBAY (Oh)PHENOTYPE Patients lack the H gene and

therefore cannot make H antigen, A or B antigen on their red cells

Have very strong anti-A, anti-B, and anti-H and can only receive cells from a Bombay donor


ABO BLOOD GROUPS

Serum AbSerum AbRBC AgRBC AgBlood Blood GroupGroup

Anti-BAA

Anti-ABBB

noneA & BAB

Anti-AAnti-BAnti-A,B

noneO


Blood being testedTyping Serum

Type AB (contains agglutinogenA and B )

Type B (contains agglutinogen B)

Type A (contains agglutinogen A)

Type O (contains no agglutinogen)

Anti – A Anti – B

red cells


ABO BLOOD GROUP… Testing

REVERSE GROUPING /TYPING Also called “serum” or “back” typing

Uses patient’s serum versus commercial A1 and B cells

Analyzes patient’s serum for the presence of anti-A and anti-B antibodies


ABO BLOOD GROUP… Testing

Testing for antibodies is not reliable until 6 months of age Newborn serum

may contain maternal IgG anti-a or anti-B


Rh BLOOD GROUP …

Complex blood group with >50 described antigens

Rhesus monkey D Ag is more potent No natural AB against Rh Nomenclature systems

Fisher-Race (English) Wiener (American)


Rh Groups: Fisher-Race

Anti-eAnti-e

Anti-CAnti-C

Anti-DAnti-D

genesgenes

linkedlinked

closely-closely-

ee

CC

DD

AbAbAgAgChromosomeChromosome

ee

CC

DD

5 major antigens: D, C, E, c and e Deepa Babin @TMC Kollam 22

Rh Groups: Weiner

RhRh11

hr”hr”

Anti- hr’Anti- hr’hr’hr’

Anti-rh”Anti-rh”rh”rh”

Anti-rh’Anti-rh’rh’rh’

Anti-RhoAnti-RhoRhoRho

AbAbAgAgCHROMOSOMECHROMOSOME

RR11 singlesingle genegene

Anti- hr”Anti- hr”

5 major antigens: Rho, rh’, rh”, hr’, hr”


Rh BLOOD GROUP… Testing

Testing for D(Rho) is the most common Rh test performed

D antigens are potent immunogens. Of D-negative patients, 80% will develop

an anti-D when transfused with a single unit of D-positive blood.

* Rh-positive simply means “D positive”


Other blood group systems:

Kell (K,k,Kx)

Duffy (Fy)

Kidd (Jka, Jkb)

MNSs

P

Lewis (Le)

Lutheran (Lu)


Importance of other blood groups:

May cause transfusion reactions or

HDN

May interfere with crosshatching

For medico legal parenthood problems


COMMON TESTS USED IN IMMUNOHEMATOLOGY

Coombs Test (Antiglobulin Test) RBC typing Crossmatching Antibody Screening Compatibility Testing


I. ANTIGLOBULIN (COOMB’S) TEST

Remains the most important single test in Ab detection

Principle: Red blood cells sensitized by IgG or

complement can be made to agglutinate by adding antihuman globulin



DIRECT COOMB’S TEST (DAT)

Detects RBCs that have already been sensitized with IgG

Demonstrates that in vivo coating of RBC by Ab has occurred but does NOT identify the antibody


INDIRECT COOMB’S TEST (IAT) Detects antibodies to RBC antigens present in

the patient’s serum

Detects in vitro red cell sensitization if red cells contain antigen corresponding to serum antibody

Procedure: STEP 1: patient’s serum (with unknown Ab) + RBC (with known Ag)

STEP 2: product of step 1 + Coomb’s reagent



ANTIGLOBULIN TESTINGDIRECT ANTIGLOBULIN TEST (DAT)Patient’s red cells

INDIRECT ANTIGLOBULIN TEST (IAT)Patient’s serum

Detects in vivo antibody coating (sensitization) of red cells

Detects in vitro red cell sensitization if red cells contain antigen corresponding to serum antibody

Useful in:1.Detection of hemolytic disease of the newborn (employing infant’s red cells)2.Investigation of transfusion reactions3.Detection of autoimmune hemolytic anemia (AIHA)4.Detection of red cell sensitization by drugs (penicillin, cephalothin, alpha-methyldopa)

Useful in:1.Detection and identification of unexpected antibodies2.Compatibility testing (cross-matching)3.Red cell antigen phenotyping4.Investigation of transfusion reactions5.Detecting Du antigen (weak D)


II.RBC typing

Forward typing

Reverse typing

D antigenDeepa Babin @TMC Kollam 35

III.CROSSMATCHING

Absence of agglutination or hemolysis is essential to the safety of blood transfusions

Agglutination or hemolysis in any phase of the transfusion (ie incompatibility) = presence of Ab and its corresponding Ag


III.CROSSMATCHING

Uses

1.To detect antibodies in the donor or recipient

2.To detect ABO typing discrepancies


IV. ANTIBODY SCREEN

Use: to demonstrate unexpected antibodies in the serum of the recipient that may destroy donor RBCs that were thought to be compatible on the basis of the Rh and ABO typing

Has replaced minor cross matching


V. PRE-TRANSFUSION / COMPATIBILITY TESTING

1. Review of recipient’s blood bank history

2. ABO and Rh typing of recipient & donor

3. Antibody screening of recipient &

donor serum

4. Major crossmatching


HEMOLYTIC DISEASE OF THE NEWBORN (HDN)

Also referred to as erythroblastosis fetalis

Occurs when the mother is alloimmunized to antigen(s) found on the RBC of the fetus

Destruction of fetal RBCs by mother’s IgG antibodies


HEMOLYTIC DISEASE OF THE NEWBORN(HDN) Rh Incompatibility

Rh (-) mother & Rh (+) baby

ABO Incompatibility

“O” mother & “A” or “B” baby

Rh Incompatibility

Rh (-) mother & Rh (+) baby

ABO Incompatibility

“O” mother & “A” or “B” baby


HDN DUE TO RH INCOMPATIBILITY Set-up: Rh(-) mother + Rh(+) baby

Rh (-) person exposed to Rh(+) blood will develop reaction after 2 – 4 weeks

Mother develops antibody against the Rh(+) blood coming from the baby

First baby is not affected; HDN occurs during subsequent pregnancies


Rh(-) RBC

Rh(+) RBC


Antibody


anemia hemoglobindegradation

extramedullaryhematopoiesis

hydrops

hypoxic injury toliver & heart

bilirubin

jaundice kernicterus

hepato-splenomegaly


Kernicterus

Hydrops


HDN DUE TO RH INCOMPATIBILITY

May be prevented by giving anti-Rh

to Rh(-) mother in the ante-natal (28

weeks) & immediate postnatal period

(within 72 hours after delivery)


ABO Hemolytic disease

Maternofetal ABO incompatibility

A &B got IgM in blood which cannot cross placenta

O gp IgG-CROSS PLACENTA-HEMOLYTIC DISEASE


COMPLICATIONS OF BLOOD TRANSFUSION

Immunological/Non immunological

Infectious parameters(HIV,HBV,HCV,VDRL)

CMV,LEPTO ,MALARIA,TOXO,LEISH)

AUTOLOGUS BLOOD TRANSFUSION


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Immunohematology Deepa Babin *


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