Deepa Babin @TMC Kollam

Deepa Babin @TMC Kollam 1
IMMUNOHEMATOLOGY
The study of immunologic
reactions involving all
components of blood
Deals with antigens, antibodies
and antigen-antibody reactions

Application in ....
 transfusion of blood & its components
 diagnosis, prevention & management
of immunization asso with pregnancy
 leukocyte testing for organ
transplantation
 laboratory resolution of parentage
problems

Agglutination / Hemolysis

Complete Antibodies
antibody RBC w/ antigen

Incomplete Antibodies
antibodies RBC w/ antigen

BLOOD GROUP SYSTEMS
1. ABO BLOOD GROUP
2. Rh BLOOD GROUP SYSTEM
3. OTHERS
A. MNS
B. I/i
C. DUFFY
D. KELL
E. KIDD
F. P
G. LUTHERAN
H. LEWIS
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
Blood Group RBC Ag

A A
B B
AB A&B
O none
Anti-A
O none Anti-B
Anti-A,B
ABO BLOOD GROUP…
 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…
 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 Deepa Babin @TMC Kollam 12
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”) Deepa Babin @TMC Kollam 14
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
ABO BLOOD GROUPS
Blood Group RBC Ag Serum Ab
A A Anti-B
B B Anti-A
AB A & B none
Anti-A
O none Anti-B
Anti-A,B
Typing Serum
Blood being tested
Anti – A Anti – B
Type AB (contains agglutinogen

A and B )
red cells
Type B (contains agglutinogen B)
Type A (contains agglutinogen A)
Type O (contains no agglutinogen)

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
Chromosome Ag Ab
closely- D D Anti-D
linked C C Anti-C
genes e e Anti-e
5 major antigens: D, C, E, c and e

Rh Groups: Weiner
CHROMOSOME Ag Ab
Rho Anti-Rho
single gene R1 Rh1 rh’ Anti-rh’
rh” Anti-rh”
hr’ Anti- hr’
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
* Rh-positive simply means “D positive”
 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.

Other blood group systems:
 Kell (K,k,Kx)
 P
 Duffy (Fy)  Lewis (Le)
 Kidd (Jka, Jkb)  Lutheran (Lu)
 MNSs

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 TESTING
DIRECT ANTIGLOBULIN TEST (DAT) INDIRECT ANTIGLOBULIN TEST
Patient’s red cells (IAT)
Detects in vivo antibody coating Patient’s
Detects inserum
vitro red cell sensitization if
(sensitization) of red cells red cells contain antigen corresponding
to serum antibody
Useful in: Useful in:
2.Detection of hemolytic disease of the 2.Detection and identification of
newborn (employing infant’s red cells) unexpected antibodies
3.Investigation of transfusion reactions 3.Compatibility testing (cross-
4.Detection of autoimmune hemolytic matching)
anemia (AIHA) 4.Red cell antigen phenotyping
5.Detection of red cell sensitization by 5.Investigation of transfusion reactions
drugs (penicillin, cephalothin, alpha- 6.Detecting Du antigen (weak D)
methyldopa)

II.RBC typing
 Forward
typing
 Reverse
typing
 D antigen
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
3. To detect antibodies in
the donor or recipient
4. 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

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 hemoglobin
degradation
extramedullary hypoxic injury to

hematopoiesis liver & heart
↑ bilirubin
hepato-
splenomegaly
hydrops jaundice kernicterus

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

 Programme created for
Basic Vision on
Immunohematology
 Deepa Babin *

Deepa Babin @TMC Kollam

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Deepa Babin @TMC Kollam 1

Deepa Babin @TMC Kollam 2

Deepa Babin @TMC Kollam 3

Deepa Babin @TMC Kollam 4

antibody RBC w/ antigen

Deepa Babin @TMC Kollam 5

antibodies RBC w/ antigen

Deepa Babin @TMC Kollam 6

Blood Group RBC Ag

Blood Group RBC Ag Serum Ab

Type AB (contains agglutinogen

Type B (contains agglutinogen B)

Type A (contains agglutinogen A)

Type O (contains no agglutinogen)

Deepa Babin @TMC Kollam 17

 REVERSE GROUPING /TYPING

 Complex blood group with >50 described

5 major antigens: D, C, E, c and e

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

 Testing for D(Rho) is the most common Rh

* Rh-positive simply means “D positive”

 D antigens are potent immunogens.

Deepa Babin @TMC Kollam 24

Deepa Babin @TMC Kollam 25

 For medico legal parenthood problems

Deepa Babin @TMC Kollam 26

 Coombs Test (Antiglobulin Test)

Deepa Babin @TMC Kollam 28

 Remains the most important single test in Ab

Deepa Babin @TMC Kollam 29

 DIRECT COOMB’S TEST (DAT)

 Detects RBCs that have already been

 Demonstrates that in vivo coating of RBC by

 INDIRECT COOMB’S TEST (IAT)

 Detects in vitro red cell sensitization if red cells

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

Deepa Babin @TMC Kollam 34

 Agglutination or hemolysis in any

Deepa Babin @TMC Kollam 37

 Occurs when the mother is alloimmunized to

 Destruction of fetal RBCs by mother’s IgG

Deepa Babin @TMC Kollam 41

Rh (-) mother & Rh

“O” mother & “A” or

Deepa Babin @TMC Kollam 42

 Rh (-) person exposed to Rh(+) blood will

 Mother develops antibody against the Rh(+)

 First baby is not affected; HDN occurs

Deepa Babin @TMC Kollam 43

Deepa Babin @TMC Kollam 44

Deepa Babin @TMC Kollam 45

extramedullary hypoxic injury to

Deepa Babin @TMC Kollam 47

Deepa Babin @TMC Kollam 49

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

Deepa Babin @TMC Kollam 50

 AUTOLOGUS BLOOD TRANSFUSION

Deepa Babin @TMC Kollam 52