Abo Blood

HE M AT O L O G Y TO P IC P R E S E N TAT IO N
BL O O D GR O U P IN G
Dr. Ramesh Paudel

1st year Resident
Department of Pathology
HU M A N BL O O D GR O U P S
Discovered by Landsteiner in 1901
Blood group antigens are structures on the outer surface of

human red blood cells (RBCs) that can be recognized by the
immune system of individuals who lack that particular
structure.
Presence or absence of these antigens is used to classify

blood groups.
The International Society of Blood Transfusion recognizes 35

blood group systems and 6 antigen collections
Each blood group system consists of a group of antigens

encoded by alleles at a single gene locus or very closely linked
loci.
Source : Williams Hematology Ninth Edition,

Copyright © 2016,
Major blood groups – ABO & Rh

Minor blood groups –
MNS system
P
Lutheran
Kell
Lewis
Duffy
Kidd
Diego
Hh/Bombay
Kx ……..
Most blood group genes have been assigned to specific

chromosomes (e.g. ABO system on chromosome 9, Rh
system on chromosome 1).
The term genotype is used for the sum of the inherited alleles
of a particular gene (e.g. AA, AO) and most red cell genes are
expressed as codominant antigens (i.e. both alleles are
expressed in the heterozygote).
RBC antigens are inherited carbohydrate or protein structures

located on the outside surface of the RBC membrane.
Although most of the protein blood group antigens are carried

on integral transmembrane proteins (either single-pass type I
or type II, or multipass; a few are carried on
glycosylphosphatidylinositol (GPI)-linked proteins or adsorbed
from plasma.
The main clinical importance of a blood group system:
depends on the capacity of alloantibodies (directed against

the antigens not possessed by the individual) to cause
destruction of transfused red cells.
To cross the placenta and give rise to haemolytic disease in

the fetus or newborn.
On these criteria, the ABO and Rh systems are of major

clinical importance.
ABO BL O O D GR O U P
Discovery of the ABO system by Landsteiner marked the

beginning of safe blood transfusion.
Discovered by Karl Landsteiner in 1901
When he noticed that the red cells of some individuals could be

agglutinated by the serum of others.
Landsteiner’s Rule
If an antigen (Ag) is present on a patients red blood cells the

corresponding antibody (Ab) will NOT be present in the patients
plasma, under ‘normal conditions’.
ABO blood group consist of

two antigens (A & B) on the surface of the RBCs
two antibodies in the plasma (anti-A & anti-B)
BLOOD GROUP =B
Expressed on a wide variety of human tissues and are

present on most epithelial and endothelial cells.
Important histocompatibility antigens.
Transplantation of ABO-incompatible solid organs increases

the potential for hyperacute graft rejection.
There are four main blood groups: A, B, AB and O
In Caucasian population, the frequency of group A is 42%,

B 9%, AB 3% and O 46%.
“GE N E T IC S ”
The expression of ABO antigens is controlled by three
separate genetic loci:
ABO located on chromosome 9
FUT1 (H) and
FUT2 (Se), both of which are located on chromosome 19.
The genes from each locus are inherited in pairs as

Mendelian codominants.
Each gene codes for a different enzyme (glycosyltransferase), which
attaches specific monosaccharides onto precursor disaccharide
chains.
Presence or absence of the ABH antigens on the red cell membrane

is controlled by the H gene(FUT1).
Presence or absence of the ABH antigens in secretions is indirectly

controlled by the Se gene(FUT2)
H gene – H and h alleles

Se gene – Se and se alleles
ABO genes – A, B and O alleles
Three alleles of the ABO gene (A, B, and O)
A and B are co-dominant alleles.

O is recessive allele.
Four different phenotypes: A, B, AB and O

Six possible genotypes: AA, AO, BB, BO, AB and OO
H Antigen
The H gene codes for an enzyme that adds the sugar fucose to
the terminal sugar of a precursor substance (PS).
-Fucosyltransferase
The precursor substance (proteins and lipids) is formed on an

oligosaccharide chain (the basic structure).
FO R M AT IO N OF THE H A N T IG E N
RBC
Glucose
H antigen Galactose
N-acetylglucosamine
Fucose Galactose
The H antigen is the foundation upon which A and B antigens
are built.
A and B genes code for enzymes that add an

immunodominant sugar to the H antigen.
Immunodominant sugars are present at the terminal ends of

the chains and confer the ABO antigen specificity.
The “A” gene codes for an enzyme (transferase) that adds

N-acetylgalactosamine to the terminal sugar of the H
antigen
N-acetylgalactosaminyltransferase
The “B” gene codes for an enzyme that adds D-galactose

to the terminal sugar of the H antigen
D-galactosyltransferase
FO R M AT IO N OF THE A A N T IG E N
RBC
H Antigen
Glucose
Galactose
N-acetylglucosamine
Fucose Galactose
N-acetylgalactosamine
FO R M AT IO N OF THE B A N T IG E N
RBC
H Antigen
Glucose
Galactose
N-acetylglucosamine
Fucose Galactose
D-Galactose
The O gene does not encode a functional enzyme;
group O individuals commonly have a deletion at nucleotide

261 (the O1 allele), which results in a frame-shift and
premature termination of the translated polypeptide.
Hence, a specific sugar is not attached to the red cell

membrane .
BO M B AY PH E N O T Y P E (OH)
The individual is homozygous for the inactive h allele of FUT1 and

hence cannot form the H precursor of the A and B antigen.
Their red cells type as group O, but their plasma contains anti-H in
addition to anti-A, anti-B and anti-A,B, which are all active at 37 °C.
As a consequence, individuals with an Oh Bombay phenotype can

only be safely transfused with other Oh red cells.
Serologically, the red cells group as O; unlike the true O phenotype,

red cells from Bombay phenotype lack H antigen
Blood Group Antigen H Substance Antibody
A A antigen + Anti-B
B B antigen + Anti-A
AB AB antigen + No Antibody
O H antigen ++ Anti-A anti-B
Bombay blood None No H substance Anti-H anti-A

group Anti- B
ABO SU B G R O U P S
ABO subgroups differ in the amount of antigen present on the

red blood cell membrane
Subgroups have less antigen
Subgroups of A are more common than subgroups of B
Results from from mutant forms of the glycosyltransferases

produced by the A gene that are less efficient at transferring
N-acetyl-D- galactosamine onto H substance.
ABO SU B G R O U P S
Two common subgroups of the A antigen.
Approximately 20% of group A and group AB individuals belong to

group A2 and group A2B, respectively.
The remainder belonging to group A1 and group A1B.
These subgroups arise as a result of inheritance of the A1 or A2

alleles.
The A2 transferase is less efficient in transferring N-acetyl-D-

galactosamine to available H antigen sites.
A2 red cells have fewer A antigen sites than A1 cells.

ABO A N T IG E N S A N D D IS E A S E .
The inheritance of ABH antigens is also known to be weakly

associated with predisposition to certain diseases.
Group A individuals have 1.2 times the risk of developing

carcinoma of the stomach than group O or B
Group O individuals have 1.4 times more risk of developing

peptic ulcer than non-group O individuals
The ABO group also affects plasma von Willebrand factor

(VWF) and factor VIII levels, group O healthy individuals have
levels around 25% lower than those of other ABO groups
ABO A N T IG E N S A N D D IS E A S E .
ABO A N T IB O D IE S
IgM is the predominant antibody in Group A and Group B

individuals
Anti-A
Anti-B
IgG (with some IgM) is the predominant antibody in Group O

individuals.
Hyperimmune IgG anti-A and/or anti-B cross the placenta and
cause ABO haemolytic disease of the newborn (HDN). These
antibodies react over a wide thermal range are more effective
haemolysins than the naturally occurring antibodies.
RH SYSTEM
The Rh system, formerly known as the Rhesus system, was so

named because the original antibody that was raised by
injecting red cells of rhesus monkeys into rabbits and guinea
pigs reacted with most human red cells.
Discovered by, Landsteiner and Wiener In 1940.

RH GROUP
Rh is a blood group system with many antigens, one of

which is D (RH1) .
Presence or absence of blood antigen D
Unlike the ABO system, individuals who lack the D antigen do

not naturally produce anti-D.
Production of antibody to D requires exposure to the antigen.

RH G E N E T IC S
Rh antigens encoded by two genes: RHD and RHCE

Locus of genes is on short arm of chromosome 1
RHD encodes for the D antigen
RHCE encodes for the C, c E and e antigens

D antigen is only immunogenic and clinically significant.
RH G E N E T IC S
The Rh antigens are present only on red cells and are a

structural part of the cell membrane.
Complete absence of Rh antigens (Rh-null phenotype) may

be associated with a congenital haemolytic anaemia with
spherocytes and stomatocytes in the blood film, increased
osmotic fragility and increased cation transport.
AN T IB O D IE S OF THE RH SYSTEM
The vast majority of Rh antibodies are IgG and do not fix

complement.
Anti - D may occasionally be partly IgA.
IgM anti - D is very rare.

CL IN IC A L SIG N IF IC A N C E OF RH BLO O D
GROUPS
Hemolytic disease of newborn

when there is incompatibility of blood group between mother
and baby
Known as erythroblastosis foetalis.
occurs when mother is Rh –ve ( dd) and father is Rh+ve

resulting in fetus which is Rh +
HE M O LY T IC D IS E A S E O F N E W B O R N
The chance of the first pregnancy to suffer from hemolytic

disease of newborn is relatively small
In subsequent pregnancies: the risk of developing Ab (Anti –D

IgG) increases causing hemolytic disease of newborn
So anti-D Ig prophylaxis is given.
-Anti–D Ig (Rhogam) administered to Rh-ve mothers at 28

weeks and within 72 hrs of delivery.
Also administered following abortions
Maternal blood can be exposed to fetus by
1.Transplacental hemorrhage: Rh + fetal blood exposed to Rh-

mother
During delivery
Abortion
Amniocentesis
Trauma
2. Transfusion of Rh incompatible blood or blood product.

If Rh – mother has received Rh+ blood during transfusion
First baby may be affected
ABO HA E M O LY T IC DIS E A S E OF THE
NE W B O R N
Occur in babies that are group A or B born to

mothers that are group O as result of high-titre
maternal IgG anti-A and anti-B antibodies.
Can cause prolonged neo n a t a l j a u n d i c e a n d

anaemia associated with spherocytosis on the blood
film.
Treatment : Phototherapy or Exchange Transfusion.

AN T IG E N –A N T IB O D Y R E A C T IO N S
Agglutination or lysis is a visible indication (endpoint) of an

antigen–antibody reaction.
The reaction occurs in two stages:

in the first stage the antibody binds to the red cell antigen
(sensitisation) and
the second stage involves agglutination (or lysis) of the

sensitised cells.
The first stage is reversible and the strength of binding

(equilibrium constant) depends on the ‘exactness of fit’ between
antigen and antibody.
This is influenced by the following:

1. Temperature
2. PH (serum or cells are diluted to a fixed pH, usually 7.0)
3. Ionic strength of the medium (Low ionic strength increases
the rate of antibody binding).
Agglutination is brought about by antibody crosslinking

between cells.
Depends on various laboratory manipulations to promote

agglutination or lysis of sensitised cells.
1. Reducing intercellular distance by pretreatment of red
cells with protease
2. Adding polymers (e.g. albumin)
3. Bridging between sensitised cells with an antiglobulin
reagent in the antiglobulin test
ER Y T H R O C Y T E AN T IG E N S AND
AN T IB O D IE S
ABO grouping is the single most important test performed in

the blood bank because it is the fundamental basis for
determining blood compatibility.
ABO grouping is determined by reacting erythrocytes with

licensed antisera to identify the A or B antigens they carry (the
forward, or cell, grouping) and by reacting the
corresponding serum or plasma with known A and B cells to
identify the antibodies present (the reverse, or serum,
grouping).
AN T IB O D IE S
2 components :
Test unknown cells with known antibodies (forward)

Test unknown serum/plasma with known red cells (reverse)
The patterns are compared and the blood group is determined.
Positive reactions are seen as hemagglutination or hemolysis,

and the results of one test should confirm the results of the
other.
AN T IB O D IE S
If results are discrepant or reactions are weaker than expected,

the cause must be investigated before the ABO group can be
interpreted with confidence.
Discrepancies can be related to red cell anomalies, serum

anomalies, or both and may be associated with disease.
AG G L U T IN AT IO N O F R ED C ELLS BY
A N T IB O D Y
Carried out in tubes, microtitre plates or using column

agglutination (gel) technology, centrifugation or sedimentation.
Rarely slide tests are used for emergency ABO and D grouping
BL O O D SAM PLE
Clearly labeled blood samples in sterile tubes (plain & EDTA).
Test should be performed on the fresh sample for best results.
If serum is not completely separated, centrifuge tube at 1000-3000

rpm for 3 min.
No signs of hemolysis should be there
Preferably use saline washed red cells and make 2-5% red cell
suspension.
RE D CE L L SU S P E N S IO N S FOR BL O O D
GR O U P IN G
50%: Slide Method

5%: Test Tube Method
1%: Gel technology
1%: Microplate
AN T I - S E R U M
SL ID E TESTS.
These are used rarely in a few parts of the world.
Because of evaporation, slide tests must be read within about 5

min.
Reagents that produce strong agglutination within 1–2 min are

normally used for rapid ABO and RhD grouping.
Because the results are read macroscopically, strong cell

suspensions should be used (50% cells in their own serum or
plasma).
SL ID E ME T H O D FOR ABO G R O U P IN G
• Take a slide and label A, B and D.

• Put 1/1 drop of 40-50% suspension
of test red cells on labeled slide
• Add 1/1 drop of anti-A, anti- B and
anti-D to respective drop of blood as
labeling.
• Mix the contents.
• Tilt the slide gently back and forth.
• Examine for agglutination
SL ID E TESTS.
ADVANTAGES:
– Preliminary typing tests
DISADVANTAGES:
– Not routine test
– Less sensitive
– Drying of reaction giving to false positive results
SL ID E TESTS.
Anti-A Anti-B Anti-D

TE S T TU B E ME T H O D O F ABO
GR O U P IN G
Recommended method
Allows longer incubation of antigen and antibody mixture without

drying
Tubes can be centrifuged to enhance reaction
Can detect weaker antigen / antibody
Two steps in ABO grouping
Cell grouping (Forward grouping)

Tests the patients red cells with known Anti-A & Anti-B to determine
the antigen expressed
Serum grouping (Reverse grouping)

Test the patients serum with known A & B cells to determine the
presence of antibody
LAY OU T O F TU B E S FO R ABO & RH
G R O U P IN G
2 vol of anti- 1 vol of 2-5%
A/ anti-B/ red cell
anti-D suspension
Incubate at room
temp (20-24oC) for
5 min
Forward Centrifuge at 1000

Grouping rpm for 1 min
Check for agglutination

against well lighted
background O+ve
1 vol of 5%
2 vol of test
suspension of
serum/
reagent red cells
plasma
in respective
tubes
Shake & leave at room

temp (20-24oC) for 5
min
Reverse
Grouping Centrifuge at 1000 rpm
for 1 min
Centrifuge & record the

results similarly as for
“O”
cell grouping
RE C O R D IN G R E S U LT S O F ABO
G R O U P IN G
Reaction of red Reaction of Interpretatio

cells with serum with n
pooled cells
Anti- Anti- Anti- A B O
A B D cell cell cell
+ 0 + 0 +/H 0 A +ve
+ 0 0 0 +/H 0 A-ve
0 + + +/H 0 0 B+ve
0 + 0 +/H 0 0 B-ve
+ = agglutination, 0 = no agglutination H = hemolysis

RE C O R D IN G R E S U LT S O F ABO
G R O U P IN G
Reaction of red Reaction of Interpretatio

cells with serum with n
pooled cells
Anti-A Anti- Anti-D Ac Bc Oc
B
+ + + 0 0 0 AB+ve
+ + 0 0 0 0 AB-ve
0 0 + +/H +/H 0 O+ve
0 0 - +/H +/H 0 O-ve
0 0 +/H +/H + Oh
+ = agglutination, 0 = no agglutination H = hemolysis

GR A D IN G OF AG G L U T IN AT IO N
GR A D IN G OF AG G L U T IN AT IO N
AN T IB O D Y SC R E E N
The antibody screen, or indirect antiglobulin test, detects

"atypical" or "unexpected" antibodies in the serum (i.e., other
than anti-A and anti-B) using group O reagent red cells .
Serum or plasma and screening cells are incubated at 37°C

(98.6°F) with an additive to potentiate antibody-antigen
reactions, then an indirect antiglobulin test is performed.
Hemagglutination or hemolysis at any point is a positive

reaction, indicating the presence of naturally occurring or
immune alloantibody or autoantibody.
IN D IR E C T CO O M B ’S TE S T
DIR E C T AN T IG L O B U L IN TE S T
The direct antiglobulin test (direct Coombs test) detects

antibody or complement bound to RBCs in vivo.
Red cells are washed free of serum and then mixed with
antiglobulin reagents that agglutinate RBCs coated with IgG or
the C3 component of complement.
DIR E C T AN T IG L O B U L IN (CO O M B ’S )
TE S T
DIR E C T AN T IG L O B U L IN TE S T
Positive direct antiglobulin test results are associated with the following:
(⑴) Transfusion reactions, in which recipient alloantibody coats

transfused donor RBCs or transfused donor antibody coats recipient
RBCs
(2) HDFN, in which maternal antibody crosses the placenta and coats
fetal RBCs
(3) autoimmune hemolytic anemias, in which autoantibody coats the

patient's own RBCs;
(4) drug or drug–antibody complex interactions with RBCs that

sometimes lead to hemolysis; (adsorb onto circulating RBCs.
(5) Hypergammaglobulinemia, in which Ig nonspecifically adsorb onto

circulating RBCs.
CO M PAT IB IL IT Y TE S T IN G
Compatibility testing refers to a collection of donor and recipient

tests that are performed prior to red cell transfusion.
If the recipient has a negative antibody screening test result and

no history of clinically significant antibodies, a serologic
immediate spin cross-match between recipient serum and donor
red cells is required to confirm ABO compatibility.
Can be used in conjuction with antiglobulin crossmatch
Sensitivity can be optimised by using two volume of

plasma and 1 volume of 2-3% of cells .
Incubating at room temperature for 2-5 minutes ,

centrifuging at 1000 rpm for 1 minute and reading using
tip and roll technique.
No HEMOLYSIS or AGGLUTINATION
indicates COMPATIBILITY

Abo Blood

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Abo Blood

Uploaded by

Copyright:

Available Formats

HE M AT O L O G Y TO P IC P R E S E N TAT IO N

Dr. Ramesh Paudel

Discovered by Landsteiner in 1901

Blood group antigens are structures on the outer surface of

Presence or absence of these antigens is used to classify

The International Society of Blood Transfusion recognizes 35

Each blood group system consists of a group of antigens

Source : Williams Hematology Ninth Edition,

Major blood groups – ABO & Rh

Most blood group genes have been assigned to specific

RBC antigens are inherited carbohydrate or protein structures

Although most of the protein blood group antigens are carried

The main clinical importance of a blood group system:

depends on the capacity of alloantibodies (directed against

To cross the placenta and give rise to haemolytic disease in

On these criteria, the ABO and Rh systems are of major

Discovery of the ABO system by Landsteiner marked the

Discovered by Karl Landsteiner in 1901

When he noticed that the red cells of some individuals could be

If an antigen (Ag) is present on a patients red blood cells the

ABO blood group consist of

Expressed on a wide variety of human tissues and are

Important histocompatibility antigens.

Transplantation of ABO-incompatible solid organs increases

There are four main blood groups: A, B, AB and O

In Caucasian population, the frequency of group A is 42%,

The genes from each locus are inherited in pairs as

Presence or absence of the ABH antigens on the red cell membrane

Presence or absence of the ABH antigens in secretions is indirectly

H gene – H and h alleles

Three alleles of the ABO gene (A, B, and O)

A and B are co-dominant alleles.

Four different phenotypes: A, B, AB and O

The precursor substance (proteins and lipids) is formed on an

A and B genes code for enzymes that add an

Immunodominant sugars are present at the terminal ends of

The “A” gene codes for an enzyme (transferase) that adds

The “B” gene codes for an enzyme that adds D-galactose

The O gene does not encode a functional enzyme;

group O individuals commonly have a deletion at nucleotide

Hence, a specific sugar is not attached to the red cell

The individual is homozygous for the inactive h allele of FUT1 and

As a consequence, individuals with an Oh Bombay phenotype can

Serologically, the red cells group as O; unlike the true O phenotype,

Blood Group Antigen H Substance Antibody

O H antigen ++ Anti-A anti-B

Bombay blood None No H substance Anti-H anti-A

ABO subgroups differ in the amount of antigen present on the

Subgroups have less antigen

Subgroups of A are more common than subgroups of B

Results from from mutant forms of the glycosyltransferases

Approximately 20% of group A and group AB individuals belong to

The remainder belonging to group A1 and group A1B.

These subgroups arise as a result of inheritance of the A1 or A2

The A2 transferase is less efficient in transferring N-acetyl-D-

A2 red cells have fewer A antigen sites than A1 cells.

The inheritance of ABH antigens is also known to be weakly

Group A individuals have 1.2 times the risk of developing

Group O individuals have 1.4 times more risk of developing

The ABO group also affects plasma von Willebrand factor

IgM is the predominant antibody in Group A and Group B

IgG (with some IgM) is the predominant antibody in Group O

The Rh system, formerly known as the Rhesus system, was so