ABO BLOOD GROUP SYSTEM

P SUNIL KUMAR
Dept.of Haematology
St.John’s Medical College Hospital
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Bangalore
HISTORY ………???

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 HISTORY
• ABO blood group system was discovered by Karl
Landsteiner in 1900-1901.
• It is the most important blood group system for transfusion
practice.
• A fourth blood group AB was discovered by Landsteiner’s
associates,Von Decastello and Sturli in 1902.
• The four groups are determined by the presence or
absence of blood group antigens on the RBCs and
accordingly an individual’s group is A,B,AB or O
• In 1911, Von Dungern and Hirszfeld showed that group A
could be divided into 2 main sub-groups A1 and A2

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Landsteiner’s Law……..

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 Landsteiner concluded from his
observation that……..
• 1. Individuals who lack the antigens in of ABO
system, have the corresponding Ab in their
serum.
• 2. Ab’s of ABO system are usually “naturally
occuring “ IgM type.
• 3.These are capable of causing intravascular
haemolysis in patients, if transfused with
incompatible blood.
• 4.Thus , an error in ABO grouping o donor or
recipient can be fatal.
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• Naturally occuring ………. There is a enough
evidence to suggest that the Anti-A and Anti-B
are stimulated by substances that are
ubiquitous in nature.
• Eg; bacteria, pollen or other substances
present in nature.

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 THE ABO ANTIGENS AND
CORRESPONDING ANTIBODIES
Antigen on RBC Antibody in plasma/serum Blood group

A Anti-B A

B Anti-A B

AB None AB

None Anti-A and Anti-B O

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GENETICS OF ABO SYSTEM

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 GENETICS
• In order to understand the ABO blood group
system one must have a basic knowledge of
genetic inheritance.
• The ABO system follows the mendelian laws of
inheritance .

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 GENETICS
• The locus of ABO gene is on chromosome 9 occupied by one of the
three major allelic genes i.e. A,B or O , thus there is gene
responsible for the specificity of our ABO blood groups.

• Other gene which plays an important role in the determination of

ABO blood groups is H gene with locus on chromosome 19.

• Each individual inherits two ABO genes , one from each parent and
these genes determine the ABO antigen present on their red cells

• The A and B genes are dominant while O is recessive thus is not

directly detected. Absence of A and B antigens on the cells
indicates O blood group.
• The serological typing reveals the phenotype and and the family
studies reveal the genotype

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• The ABO antigens are not fully developed at
birth and hence the cells react weakly against
the antisera till the age of 6-18 month.
• By 2-4 years of age A and B antigens
expression is fully developed and remain fairly
consistent throughout life.

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SUNIL KUMAR.P 14
 Phenotype and genotype in ABO
blood group system

Phenotype Genotype
A AA,AO
B BB,BO
O OO
AB AB

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BIOCHEMISTRY OF ABO SYSTEM..

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 BIOCHEMISTRY
• The A and B antigens are basically glycoproteins formed from a basic precursor by
addition of terminal sugar.
• The basic precursor substance is converted to H substance by enzyme L-fucosyl
transferase (a product of H gene) by addition of a terminal sugar L-fucose.
• H substance is converted to A and B antigen by N-acetyl galactosaminyl and D-
galactosyl transferase by addition of terminal sugars N-acetyl galactosamine and D-
galactose resp.
• Thus it can be seen that genes do not synthesize antigens directly. The products of
A,B and H genes are transferase enzymes.
• The O gene is an amorph (no gene product) and hence group O cells contains only
H substance.
• Some H substance remains unconverted ,thus , all A and/or B cells normally
contain some H substance along with A and B antigen.
• The amount of H substance in order of decreasing quantity in the red cells is.
• O>A2>B>A2B>A1>A1B cells

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 BOMBAY BLOOD GROUP
• In 1952, Bhende , Bhatia and Deshpande discovered a new
blood group called Bombay group (Oh).
• This results from the homozygous hh condition, h being
amorph, has no effect on precursor substance and hence is
not converted to H substance.
• The A and B gene specified transferase enzymes cannot act
directly on the precursor substance.
• As a result red cells of Bombay group (Oh) have no A, B and
H antigens but the plasma has Anti-A and Anti-B.
• The cells react with all other cells except those of the same
group (Oh).
• Bombay group is easily detected by the following tests:
• Negative reaction with anti H lectin, anti A and anti B
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 ABH ANTIGENS
• A,B and H antigens are present not only on the red
cells but widely distributed throughout the tissues
except the CNS.
• A,B and H antigenicity is determined by specific
sugars linked to the terminal portion of
oligosaccharides. These are present on glycoproteins
or glycolipids.
• In the red cell membrane, both glycoproteins or
glycolipids with ABH activity are present.
• In the plasma only glycolipid in soluble form are
found. SUNIL KUMAR.P 22
 Subgroups of A and AB , B
• A and AB have been divided into subgroups
• A1
• A2,
• A1B and
• A2B depending upon the reaction with anti-A1
Lectin or human anti-A1 serum.
• Weak subgroups of A include A3,Ax,Am and A
intermediate.
• Weak subgroups of B- B3 ,Bx ,Bm but they are rare.

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 Antibodies of ABO system
• In the ABO system, once the antigen is absent from the cells, the
corresponding antibody is present in the serum.
• These antibodies begin to appear during the first few 3-4 months of life,
probably from exposure to ABH antigen-like substances in the environment.
Because of this, these antibodies are called “naturally occuring”.
• Anti-A or Anti-B antibodies are naturally occuring and are mostly IgM.
However some IgG antibodies are also present.
• IgG anti-A and anti-B are found more commonly in group O individuals than
A or B individuals.
• O group individuals may have high titer of anti-A and anti-B as they are both
IgM and IgG and often referred to as high titer O group individuals.

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ABO TYPING TECHNIQUES
DIFFERENT METHODS

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 ABO TYPING TECHNIQUES
• The Method / techniques are :
• 1.Slide Method
• 2.Tube Method or Technique
• 3.DiaMed ID Microtyping System (Gel System)
• 4.Microplate Technique
• 5.Glass Microbeads Method

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 SLIDE TESTING
• This maybe used for emergency ABO typing
tests or for preliminary typing tests
particularly in outdoor camps where a
centrifuge is not available.

• REAGENTS REQUIRED :
• Monoclonal anti-A , anti-B sera.

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 METHOD :

• The slide test maybe performed on clean microscopic

slide
• Put one drop of anti-A and anti-B sera separately on
the labeled slide
• Add 1 drop of whole blood of test sample to each drop
of typing serum.
• Mix the cells and reagent using a clean stick. Spread
each mixture evenly on the slide.
• Rock/rotate the slide and leave the test for 2 mins at
room temp. Then rock again and look for agglutination.
• Record the results.

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 Disadvantages
• It is not recommended as a routine tests
because it is not reliable for weakly reactive
antigens on cells and for serum typing tests
with low titer anti-A/anti-B.
• It is less sensitive than the tube test.
• Drying of the reaction mixture can cause
aggregation of cells that may be
misinterpreted as agglutination.

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INTERPRETATION OF ABO BLOOD
GROUPS

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 TUBE TESTING
It is of two types :
1. Sedimentation method
2. Spin tube method

1.SEDIMENTATION METHOD
CELL GROUPING OR FORWARD GROUPING

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 PRINCIPLE: Cell grouping is done to detect the antigen present on
the red cells using commercially available antisera.

 SPECIMEN: Blood collected with or without anticoagulant may be

used. EDTA anticoagulated blood is preferred. Random sample can
be used.

 REAGENTS AND EQUIPMENTS:

 Refrigerator to store blood samples and reagents at 2-4 degree
 Centrifuge
 Testtubes, microscope
 Anti-A and Anti-B sera, 0.9% saline

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 METHOD
• Prepare an appropriate 2-5% cell suspension of test sample
in normal saline.
• Set up 3 rows of test tubes and label them .
• Add two drops of anti-A in the tube labeled A,
• Add two drops of anti-B in the tube labeled B
• Add two drops of anti-AB in tube labeled AB.
• Add 1 drop of 2-5% of test sample in each tube.
• Mix the contents of the tube gently and centrifuge for 1min
at 1000rpm.
• Gently resuspend the cell buttons and examine for
agglutination.
• Read, interpret and record the results.

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 SERUM GROUPING (Reverse
grouping)
• PRINCIPLE: It is done to detect the presence
of ABO blood group antibody in the serum
using pooled A,B and O cells.

• SPECIMEN REQUIRED: Serum from clotted

blood less than 48 hr old.

• TYPE OF SPECIMEN: Randon non – fasting

sample

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• REAGENTS AND EQUIPMENTS:
• Centrifuge
• Microscope
• Testtubes
• Group A,B and O pooled cells
• 0.9% saline

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 PREPARATION OF POOLED CELLS (A,B and O)

• Take 3 clean testtubes and label as ‘A’ cell, ‘B’ cell and ‘O’ cell
• To the tube labeled as ‘A’ cell, add 3-4 drops of 3-4 different
individual’s samples of ‘A’ Rh positive red cells.
• To the tube labeled as ‘B’ cell, add 3-4 drops of 3-4 different
individual’s samples of ‘B’ Rh positive red cells.
• To the tube labeled as ‘AB’ ,add 3-4 drops of 3-4 different
individual’s samples of ‘AB’ Rh positive red cells.
• To all the tubes add isotonic saline (3/4th of the tube ), mix well and
centrifuge for 5 mins at 3000-4000 rpm.
• Discard the clear supernatant from top with the help of a pasteur
pipette, resuspend and repeat step 5, 3 times.
• After the third wash a suspension of A cell, B cell and O cell is made.

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 PREPARATION OF 5% RED CELL SUSPENSION:

• To 20 ml normal saline add 1 ml of

sedimented RBCs. Pooled cells are prepared
freshly everyday

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 METHOD:
• Label 3 tubes as A, B and O.

• Add 2 drops of serum to each tube.

• Add 1 drop of 5% suspension of ‘A’ reagent cells to the tube labeled as A.

• Add 1 drop of 5% suspension of ‘B’ reagent cells to the tube labeled as B.

• Add 1 drop of 5% suspension of ‘O’ reagent cells to the tube labeled as O.

• Mix the contents of the tubes gently and centrifuge for 1 min at 1000 rpm.

• Gently resuspend the cell button and examine for agglutination(positive).

• Read, interpret and record the results. Compare the test results with those
obtained in the forward grouping.

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Reactions of test red cells Reaction of serum with Interpretation (Blood
with pooled cells group of test cells)

Anti-A Anti-B Anti- A B O

AB
+ + + - - - AB
+ - + - + - A
- + + + - - B
- - - + + - O
- - - + + + Oh

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 GRADING OF AGGLUTINATION
REACTION
++++ : Single clump of agglutination with no free cells
+++ : 3 or 4 individual clumps with few free cells
++ : Many fairly large clumps with many free cells
+ : Fine granular appearance visually, but definite
small clumps (10-15 cells) per low power field.
W : 2-3 cells sticking together per low power field,
uneven distribution.
O : All cells are free
+H haemolysis (partial or total) must be
interpreted as positive.

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• ADVANTAGES OF TUBE METHOD

• It allows longer incubation of antigen and antibody mixture without

drying.
• Tubes can be centrifuged which enhances antigen and antibody reaction
• Weaker antigens and antibodies can be detected
• Less reagents are required
• Results can be read comfortably as there is no drying.

• SPIN TUBE METHOD

• All steps are similar to sedimentation technique except in step 4 of the
sedimentation technique, the serum and cell mixture is centrifuged after
5-10 mins incubation at room temp and see the results.
• It is very useful in urgent cases.

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ANTI-A1 LECTIN TEST:

*RBCs possessing the A Ag can be subdivided into A1 and A2. Group A Ags which are
agglutinated with Anti-A1 lectin are of the subgroup A1 , those which are not
agglutinated fall into subgroups weaker than A1, the majority being classified as A2 .
*Group AB RBCs can be similarly classified as A,B and A2B.

SAMPLE: EDTA anticoagulated blood

METHODS:
1.SLIDE METHOD:
*Prepare a 10% suspension of the RBCs to be tested in isotonic saline.
*On a glass slide at room temp, place 1 drop of Anti-A1 lectin
*Add 2 drops of the red cell suspension, mix well.
*Gently tilt the slide back and forth and observe for agglutination.
*Tests that show agglutination within 2 mins are confirmed as A2/A 2B.

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 2. TUBE METHOD
• Prepare a 5% suspension of the RBCs in isotonic saline.
• Label 2 test tubes as test and control.
• To both test tubes add 1 drop of Anti-A1 lectin.
• To the tube labelled ‘test’, add 1 drop of 5% suspension of the
patient.
• To the tube labelled ‘control’, add 1 drop of 5% of A1 cells.
• Mix well, centrifuge at 1000rpm for 1 min.
• Agglutination should be seen in the control tube
• If no agglutination in the tube labelled as ‘test’, it is confirmed that
the patient is A2.
• If there is agglutination in the tube labelled ‘test’, it is confirmed
that the patient is A1.

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• ANTI-H LECTIN TEST
*RBCs possessing the H antigen are agglutinated with anti-H lectin. Those which are
not agglutinated by anti-H lectin are classified as Bombay phenotype (Oh).
*The general pattern of strength of agglutination of red cells with anti-H lectin is as
follows:
O>A2 >A2 B> B> A1 >A1B

METHODS:
1.SLIDE METHOD
*Use whole blood or prepare a suspension of red cells of an approx equal
concentration to whole blood in their own serum or plasma.
*On a glass slide at room temp, place 1 drop of anti-H lectin.
*Add 1 drop of whole blood, mix well, gently tilt the slide back and forth and observe
for agglutination.
*Test that show no agglutination within 2 mins are considered negative.

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2. TUBE METHOD:
i) Prepare a 5% suspension of the RBCs in isotonic saline.
ii) To the testtube add 1 drop of anti-H lectin.
iii) Add 1 drop of the cell suspension , mix well and centrifuge at 1000 rpm
for 1 min.
iv) Gently dislodge the cell button and observe for agglutination.

GROUPING OF CORD OR INFANT BLOOD

i) Special precautions should be taken while testing cord or new-born
infant blood since ABO antigens are not fully developed and allo-
agglutinins are usually absent.
ii) Cord RBCs should be washed 3 times to avoid error due to Wharton’s
jelly. Serum grouping is not recommended in new born upto 3 months.

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 GEL TECHNOLOGY

• Gel technology can be used for any immunohematological test that has
hemagglutination as its endpoint such as
1. ABO and Rh typing
2. Typing for other blood group system
3. Antibody sreening and identification
4. Compatibilty testing including crossmatching

PRINCIPLE:
The basic principle is that instead of testtube, the serum and cell reaction takes place
in a microtube consisting of a reaction chamber that narrows to become a column
Red cells or mixture of cells & serum (as appropriate) are added into the gel
The cells are always added prior to the serum so that the serum does not come into
contact with the gel
This eliminates the need for washing as in conventional techniques
Incubation takes place followed by centrifugation under strictly controlled conditions

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 METHODS:
1.FORWARD GROUPING
ABO AND Rh DETERMINATION
i) A 5% suspension of the test red cell is prepared in LISS
ii) 50µl of this suspension is added to each of the microtubes (1-4 having Anti-A,
Anti-B, Anti-AB and Anti-D).
iii)After incubation at room temp for 10 min s the card is centrifuged.
iv) Read results

2.REVERSE GROUPING
ABO SERUM GROUPING
i)For reverse grouping 50µl of known 5% cell suspension of A1 and B cells are added
to the respective microtubes (5 & 6 resp)
ii) 25µl of each patients serum is added to microtubes 5 & 6
iii) Incubate at room temp for 10 mins
iv) The card is then centrifuged and the results are read

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• ADVANTAGES
• The gel technique is simple, reliable, rapid to use, reproducible and
sensitive
• Greater uniformity between repeat test
• Less specimen volume are needed to perform a large number
number of test
• The cards have a shelf life of 1 year and easy storage at room temp
(18-25̊ ̊c)

• DISADVANTAGES
• Special centrifuge to accommodate the microtube cards
• Special incubators to incubate the microtube cards
• Expensive

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 MICROPLATE TECHNIQUE
• Microplate technique has been successfully applied in
blood bank laboratory
• All routine test of blood grouping, antibody screening and
cross-matching can be done in microplate without loss of
sensitivity of antibodies or any problem in blood grouping

• PRINCIPLE: Microplate is a matrix of 96 short testubes.

Principle of agglutination by test-tube method is also
applicable here. Micro plate maybe rigid or flexible either
with U-shaped or V-shaped wells. U-bottom plates are
more widely used because results can be read by observing
the characteristics of resuspended RBCs or the streaming
pattern of RBCs when the plate is placed at an angle.

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 METHODS
1.SALINE TECHNIQUE
2.ENZYME TECHNIQUE
3.INDIRECT ANTIGLOBULIN TEST (IAT)
4.DIRECT ANTIGLOBULIN TEST (DAT)

ADVANTAGES
 It is cost effective for testing large number of blood samples
 Enhanced sensitivity of reaction
 Saving in reagents and equipments
 Less laboratory space required
 Less time for technologists
 Results can be observed visually or can be evaluated with automatic
photometric readers which eliminates human errors

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GLASS MICROBEADS TECHNOLOGY
• This system is for irregular antibody screening
• The test is performed in a microcolumn prefilled with glass microbeads in
suspension of antihuman globulin serum, any diagnostic reagent or
neutral isotonic solution. The sensitized cells are trapped by the
microbead suspension during column centrifugation.
• The detection of sensitized cell is based on the seiving effect of glass
microbeads.

• Cassettes of different programs are available like

- ABO-Rh(D) typing with ABO reverse grouping
- AHG polyspecific (anti -IgG + anti-C3d)
- AHG-IgG monospecific
- Cross-matching
- ABO-Rh(D) tying in newborn

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• ADVANTAGES
• Minimum incubation time of 10 mins for antibody screening
or cross-matching
• More objective, consistent and reproducible interpretation of
results

• DISADVANTAGES
• Special centrifuge to accommodate glass beads cassettes
• Special incubators to incubate the glass bead cassettes
• Expensive

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 PROBLEMS IN ABO GROUPING
• Problems arise when there is discrepancy between results of cell
and serum grouping and are related to one or more of the following

• EQUIPMENTS AND GLASSWARES

• An inaccurate centrifuge may result in over or under-centrifuge to
give false-positive or negative results
• Dirty glasswares may give false positive result
• Thermostatic equipment may not have proper temperature

• REAGENTS AND SPECIMEN

• Substandard or deteriorated grouping sera due to improper storage
• Contaminated or haemolysed reagents

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 TECHNIQUES

• Any of the following can lead to incorrect result

i)Failure to add proper reagent, cell or serum
ii)Incorrect cell: serum ratio
iii)High ambient temperature in the lab
iv)Incorrect reading or interpretation of test results
v)Failure to confirm negative results under the
microscope may cause false negative result
vi)Failure to interpret haemolysis as a positive
reaction causes false negative results

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 RED CELL MEDIATED DISCREPANCIES
1.Weakening or loss of antigens due to age, diseases such as leukemias and
other malignancies and inherited weak expression of A and B antigens which
lead to false negative results
2.Acquired B antigen like activity is associated with gram negative
septicemia, intestinal obstruction and carcinoma of colon or rectum. Red
cells may give false positive reaction with anti-B serum
3.Polyagglutinable cells
4.Antibody coated on red cells as in HDN,AIHA, incompatible blood
transfusions may mask the antigen on the red cell surface give false negative
results
5.Presence of 2 separate cell population as in chimera e.g. a patient recently
transfused with non-specific blood group (massive or exchange transfusion),
bone marrow transplant or fraternal twins which lead to mixed reaction
6.Substances in plasma or serum-excess blood group substances, Wharton’s
jelly
7.Reagents maybe weak or deteriorated

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SERUM MEDIATED DISCREPANCIES
• Unexpected antibodies in serum like anti-A1 in A2 or A2B individuals,
anti –I, anti-H or anti-P1 cause problems in serum grouping
• Auto-agglutinins
• Rouleaux formation
• Absence or loss of antibodies in serum
• Haemolysis

• SOLVING PROBLEMS/DISCREPANCIES
• Once a discrepancy is detected in ABO cells and serum grouping,
repeat the test before additional investigations are carried out
• Quality assurance of reagents, correct technique, careful
observation and interpretation of results resolve many problems

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THANK YOU…

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