ABO BLOOD GROUP SYSTEM

&
DISCREPANCY IN ABO BLOOD
GROUPING

PRESENTER – DR LEKSHMI G Y
MODERATOR – DR GIRIJA NANDINI
KANUNGO
 HUMAN BLOOD GROUPS
• Antigens present on individual’s RBC membrane
determine that person's blood group
• Blood group antigens are either proteins or
carbohydrates/glycolipids
• These antigens are
o unique to the individual
o recognized as foreign if transfused into another
individual
o promote agglutination of red cells if combined with
antibody
o more than 30 such antigen systems discovered

• Clinically important blood groups – ABO & Rh system

• Presently more than 700 red cell antigens have been
 ABO BLOOD GROUP SYSTEM
• Discovered by Karl Landsteiner in 1900

• First blood group system to be identified

• Only blood group system in which individuals have antibodies

in serum to antigens that are absent from their RBCs without
any prior exposure to RBCs through transfusions or pregnancy

• ABO blood group consist of

o two antigens (A & B) on the surface of the RBCs
o two antibodies in the plasma (anti-A & anti-B)
 ANTI-A & ANTI B
Naturally occurring
Predominantly IgM antibodies; small quantities of IgG may
be present
Antibody production initiated at birth, reach adult level at5
to 10 years and declines in elderly
Some food & environmental antigens (bacterial, viral or
plant antigens) are similar enough to A and B glycoprotein
antigens and may stimulate antibody development
 ABH ANTIGENS
 ABH antigens develop in early fetal life but do not increase
very much in strength during gestational period

Red cell of newborn carry 25-50 % of number of antigenic

sites found on adult RBC

A or B antigen expression fully develops at 2-4 years of age

and remain constant throughout life

These antigens form the integral part of

-membrane of RBC
-endothelial and epithelial cells
-platelets
-lymphocytes
ABO antigens & corresponding
antibodies
Landsteiner's law :
the plasma contains natural antibodies to A or
B, if these antigens are absent from the red
cells of that person

Karl
Landsteiner
 INHERITANCE OF ABO BLOOD
GROUPS
Follows Mendelian genetics
codominant expression
One position or locus of each chromosome 9 is occupied by
A,B or O gene
H antigen is the precursor structure on which A and B
antigens are made
FUT1(H) & FUT2(Se) genes are located on chromosome 19
 H and Se genes are not part of ABO system ;yet their
inheritance influences A and B antigen expression
Paragloboside or glycan is the basic precursor
substance from which A B H antigens originate
Specific enzyme transferases elicited by inherited gene
attach sugars to paragloboside or glycan
 Inheritance of H gene

α-2-L -fucosyltransferase enzyme production

Transfers the sugar L-fucose (immunodominant

sugar) to an oligosaccharide chain on the terminal
galactose of type 2 chains.

Formation of H antigen

precursor on which A and B antigens are made

 RBC precursor substance

RBC

Glucose

Precursor Galactose
Substance
(stays the
same) N-acetylglucosamine

Galactose
 Formation of the H
antigen

RBC

Glucose

H antigen Galactose

N-acetylglucosamine

Fucose Galactose
 A and B Antigen
• “A” gene codes for α-3-N-acetyl galactosaminyltransferase
 transfers N-acetyl galactosamine sugar to H substance

• “B” gene codes for α-3-D-galactosyltransferase  adds D-

galactose sugar to H substance

• “O” gene at ABO locus is referred to as amorph and does

not elicit the production of catalytically active polypeptide
transferase

As a result H substance remains unmodified in O gene

O group has highest concentration of H antigen

 Formation of the A antigen

RBC

Glucose

Galactose

N-acetylglucosamine

Fucose Galactose

N-
acetylgalactosamine
 Formation of the B antigen

RBC

Glucose

Galactose

N-acetylglucosamine

Fucose Galactose

D-Galactose
A, B, and H Soluble Antigens (secretor status)

• A, B and H antigens can be found in all body secretions

• FUT 2 (Se) gene --> production of α-2-L-fucosyltransferase
that modifies the type 1 precursor substance in secretions
to form H substance..
• The se gene is an amorph.
• Not linked to ABO locus, inherited independently
• If the corresponding gene is present, this H substance can
then be further modified to express A and B substance in
secretions such as saliva.
Se Se ( homozygous) SECRETORS
Se se (heterozygous)
se se (homozygous)  NON-SECRETORS
 ABO Subgroups
 ABO subgroups differ in the amount of antigen present on the
red blood cell membrane
o Subgroups have less antigen
o Represents phenotype showing weaker and variable
serologic reactivity with polyclonal antisera

 Subgroups are the result of less effective enzymes. They are

not as efficient in converting H antigens to A or B antigens
(fewer antigens are present on the RBC)

 Subgroups of A are more common than subgroups of B

 A SUBGROUPS
In 1911, Von Dungern described two different A antigens based on
reactions between group A RBCs and anti-A and anti-A1.
A1
SUBGROUP A2 SUBGROUP
• Reaction with anti A1 occurs • Doesn’t react with anti A1
• 80% of A/AB individuals are • 20% are A2/A2B
A1/A1B • A2 glycosyltransferase is
• A1 gene elicits production less efficient in adding
of high concentration of α-3 immunodominant sugar to
N- acetyl H antigen precursor
galactosaminyltransferase

A2 gene results only in

Most of H precursor structure 2,40,000 to 2,90,000
changes to A1 antigen antigenic sites
( 8,10,000 to 11,70,000
Anti-A1

• Naturally occurring IgM cold-reacting antibody

• Unlikely to cause a transfusion reaction because it

usually reacts only at temperatures well below 37°C.

• It is considered clinically significant if it is reactive at

37°C

• This antibody can cause discrepancies in both forward

and reverse ABO testing & incompatabilities in
crossmatching
Anti-A2

• Differentiation of A1 nd A2 phenotypes can be

serologically determined using Anti A1 lectin

• A2 glycosyl transferase are less efficient in adding

immunodominant sugars to H precursor

• A2 RBCs show increased reactivity with anti H lectin ulex

europaeus compared to A1 RBCs

• Subgroups weaker than A2 occurs infrequently and is

often recognized through ABO discrepancy
QUANTITATIVE AND QUALITATIVE DIFFERENCES OF SUBGROUPS A1
AND A2
QUANTITATIVE QUALITATIVE

• LESS NUMBER OF ANTIGEN SITES • DIFFERENCE IN PRECURSOR

OLIGOSACCHARIDE CHAINS

• LESS AMOUNT OF TRANSFERASE • SUBTLE DIFFERENCES IN TRANSFERASE

ENZYME ENZYME

• LESS AMOUNT OF BRANCHING • FORMATION OF ANTI-A1, IN PERCENTAGE

OF SOME SUB GROUPS
Weaker Subgroups of A
Reaction with antisera Antibodies Substanc
e in
AntiA Anti Anti Anti Anti Commo Unexpect Saliva
Pheno n (secretor
type
B AB A1 H ed s)

A1 +4 0 +4 +4 0 Anti-B - A and H
Sometimes
A2 +4 0 +4 0 +2-3 Anti-B A and H
Anti-A1
Sometimes
A3 +2 mf 0 +2 mf 0 +3 Anti-B A and H
Anti-A1
Weak Always
Ax 0 +2 0 +4 Anti-B H
Or 0 Anti-A1
No
Am 0 0 0 0 +4 Anti-B A and H
Anti-A1
Sometimes
Ay 0 0 0 0 +4 Anti-B H
Anti-A1
Weak Weak Sometimes
A
 B Subgroups

• B subgroups are rare

• Much less frequent than A subgroups

• B subgroups are differentiated by variations in

reaction strength with anti-B, anti-A,B

• Inheritance is similar to A subgroups

• Includes B3, Bx, Bm, and Bel phenotypes

 Bombay Phenotype
• First reported by Bhende et al in Bombay in 1952
• Genotype : hh (autosomal recessive trait)
• Frequency estimated to be about 1 in 7600 in Bombay.
• Most often due to mutation of FUT1 gene
• Absence of H, A & B antigens. No agglutination with anti-A,
anti-B or anti-H
• Presence of anti-H, anti-A and anti-B in the serum
• No A, B or H substances present in saliva

• compatible with Bombay phenotype only

• A recessive mode of inheritance (identical phenotypes in

children but not in parents)
 Para-Bombay phenotype

 Rare phenotypes

 RBCs either completely lack H antigen or have small

amounts of H antigen

 The genetic basis for the para-Bombay phenotype is

either,
 - a mutated FUT1 (H gene) with or without an active
FUT2 gene (Se gene), or
 - a silenced FUT1 gene with an active FUT2 gene.
 Practical aspects of ABO grouping

• Routine ABO grouping must include both cell & serum

testing as each test serves as a check on the other

• Test should be done at room temperature or lower;

testing at 37oC weakens the reactions

• Tubes, slides should be dry and labeled properly

• Serum should always be added before adding cells

• Results should be recorded immediately after

observation

• Hemolysis is interpreted as positive result

 Blood Grouping

 There are 2 components to blood typing:

o Test unknown cells with known antibodies

o Test unknown serum/plasma with known red cells

 The patterns are compared and the blood group is

determined.
Blood Sample for Blood Grouping

Blood sample
• Clearly labeled blood samples in sterile tubes (plain &
EDTA)
• Test should be performed on the fresh sample for best
results. In case the test can not be performed
immediately, sample can be stored at 4oC & should be
tested with in 48 hours
• No signs of hemolysis should be there
• If serum is not completely separated, centrifuge tube at
1000-3000 rpm for 3 min
 Red Cell Suspensions for Blood Grouping

 30-50%: Slide Method

 2-5%: Test Tube

Method

 0.8-1%: Gel
technology
5 % cell suspension 0.8 % cell suspension
for for
 1%: Microplate Tube grouping Gel card grouping
 Slide Method for ABO Grouping

 Not recommended as a routine method

 Very rudimentary method for determining blood

groups.

 CANNOT be used for transfusion purposes as false

positives and negatives do occur. Drying of reaction
mixture can cause aggregation - false positive

 Less sensitive, not reliable for weakly reactive

antigens and antibodies

 Can only be used for emergency ABO grouping or for

selection of platelet apheresis donors
 Slide Method for ABO Grouping
 Put 1 drop anti-A & anti- B
separately on slide

 Add 1 drop of 40-50% suspension

of test red cells to each drop of
typing antisera

 Mix & spread each mixture

evenly on the slide over an area
of about 15 mm diameter

 Leave the test for 2 min at room

temp (20-24oC)

 Record the results immediately

 Test Tube Method of ABO Grouping
 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)

Lay Out of Tubes for ABO & Rh grouping

Forward grouping Reverse grouping

Rh grouping
Cell grouping Sera grouping
 2 vol of anti- 1 vol of 2-5%
A / anti-B/ red cell
Anti-AB 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
 1 vol of 5%
2 vol of
suspension of
test
reagent red cells
serum/pla
in respective
sma
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
cell grouping
Tube Agglutination Grading
 Microplate Method
 It is ideal for testing large number of blood samples.

 More sensitive to detect weaker antigen-antibody reactions

 Results can be photographed for archival storage

 Microplate can be incubated & centrifuged

 There is significant saving in time and in the cost of

disposables and reagents.

 Microplates are intended to be disposable however they can

be reused after cleaning them properly making sure that all
foreign protein are removed.

 Microplates can be adapted for automation

 Microplate Method

Reaction in the microplate after 1 hour incubation at room

temperature
 Column Agglutination Technology

 One card is basically a set of 6

microtubes

 Microtubes contain either

Sephadex Gel or glass
microbeads impregnated with
antisera

 Antigen-antibody reaction takes

place in the reaction chamber of
microtube

 Gel matrix or glass beads act as

sieve and allow free cells (un-
agglutinated) to pass through
and settle at the bottom of
microtube while agglutinated
Grading Result
 MOLECULAR TYPING
• Molecular typing of blood group genes in the transfusion
setting is recommended for transfusion-dependent patients,
as part of antibody identification process, since the
identification of the patient’s predicted phenotype allows the
laboratory to determine to which antigens the patient can
and cannot respond to make alloantibodies.

• Molecular typing provides improved accuracy and more

information on the antigenic profile of the patients, especially
those where no serological reagents are available.
MOLECULAR TYPING
 ABO DESCREPANCIES

• When unexpected reactions occur in forward and or reverse grouping

• Can be due to problems with patients serum or RBC or with both

• May be due to extra positive reaction or a weak or missing reaction

• All ABO discrepancies should be resolved prior to reporting

TECHNICAL ERRORS
 RESOLUTION

1 2 3 4
Repeat testing of the Make sure technical Gather adequate If discrepancy
sample using saline factors are reviewed information about persists, new sample
suspension of RBCs and corrected patients age, must be drawn and
diagnosis, repeat testing
transfusion history,
medications and
history of pregnancy
CATEGORIES OF ABO DISCREPANCIES
Group- I Discrepancy

• Unexpected reactions in reverse grouping

• Weakly reacting or missing antibodies
Newborns
Elderly patients
Patients with leukemia or lymphoma demonstrating
hypogammaglobulinemia
Congenital or acquired agammaglobulinemia or
immunodeficiency diseases
Bone marrow or HPC transplants
Post plasma transfusion or exchange transfusion dilution
ABO subgroups
 Group- I Discrepancy Resolution
• Obtain patient clinical history
• Enhance weak/missing reaction in serum by incubating
patient’s serum with reagent A1 and B cells at room
temperature for 15 to 30 minutes
• Incubating serum cell mixture at 4°C for 15 to 30
minutes
• Concurrent testing of auto control and O cell control
 Group II Discrepancies

• Unexpected reactions in forward grouping due to weakly

reacting or missing antigens
Subgroups of A or B may be present
Leukemias yield weakened A or B antigens
Acquired B phenomenon(most commonly associated with
digestive tract disorders)
• Rare group II discrepancies : due to excess BGSS (blood
group specific soluble substances) in plasma
 Group II Discrepancies Resolution
•Agglutination is enhanced by incubating test mixture in room
temperature up to 30 minutes

Incubate at 4 °C for 15 to 30 minutes

• In acquired B phenomenon :
o Incubating patients own serum against autologous RBC
gives negative reaction
o Acquired B doesn’t agglutinate at pH less than 6 or more
than 8.5
o Secretor studies can be done
oTreating RBC s with acetic anhydride
 Group III Discrepancies
• Discrepancies between forward and reverse groupings
Caused by protein or plasma abnormalities resulting in
rouleaux formation or pseudo agglutination
Due to elevated levels of globulin (multiple myeloma ,
Waldenstroms macroglobulinemia)
Elevated levels of fibrinogen
Plasma expanders
Whartons jelly in cord blood samples
 Group III Discrepancies Resolution
• In rouleaux formation : cell grouping done after
washing RBC several times
• Saline replacement technique in reverse grouping
• In Whartons' jelly : thoroughly washing the red cells 6
to 8 times
 Group IV discrepancies
• Discrepancies between forward and reverse grouping
due to miscellaneous problems like:
Cold reactive auto antibodies : RBC heavily coated
with antibodies and spontaneously agglutinate
RBC transfusion or marrow/ stem cell transplant
Unexpected ABO isoagglutinin
Unexpected non-ABO alloantibodies
Group IV Discrepancies Resolution
• If Cold autoantibodies present : positive DCT
Incubate patient's RBC at 37 °C then wash with saline
and retype
Patient's RBC treated with 0.01 M DTT to disperse IgM
related agglutination
Reagent RBC and serum can be warmed to 37 °C ,
mixed, tested and read at 37 °C
If reverse grouping is still negative : cold auto
absorption (patient's cell with patient's serum)
 REFERENCES
• Modern blood banking and transfusion practices by
Denise M Harmening
• DGHS technical manual
• AABB Technical manual
• NACO guidelines
Thank you

Dr. Lekshmi G Y