ABO blood group system

Discovery

Karl Landsteiner(1901) discovered human A,B,O

groups.

Von Decastello and Sturli (1902) discovered AB

blood group.

Von Dungern and Hirszfeld(1911) divided group A

into 2 subgroups A1 and A2. ABO system is
classified into 6 groups: A1, A2, A1B, A2B, B, AB and
O
In 1924, the inheritance of the ABO blood group
was described.
Landsteiner’s
law
1.If an agglutinogen is
present on red blood cell
membrane ,the
corresponding agglutinin
must be absent in the
plasma.

2.If an agglutinogen is absent

on red blood cell
membrane, then
corresponding agglutinin
must be present in the
plasma.
 Inheritance of the ABO Blood
Group
• The ABO blood type is controlled by the ABO gene and
follows the simple MENDELIAN GENETICS.

• The gene encodes a glycosyltransferase

• The gene is located on the long arm of the ninth

chromosome

• The O gene is considered as an amorph, as no

detectable antigen is produced in response to the
inheritance of this gene.
Genetics
ABO Antigens

Appear in the sixth week of fetal life.

Expressions of A and B antigens on the RBCs is fully
developed by 4 to 4 years of age and remains
constant throughout life.
Present on red cell membrane, WBC, platelets and
in other tissues like salivary glands, pancreas,
kidney, body fluids
Exception CNS
ABO Antigen Genetics
• The presence or absence of the ABH antigens on the red blood cell
membrane is controlled by the H gene (chr 19)
• The presence or absence of the ABH antigens in secretions is
indirectly controlled by the Se gene (chr 19)

• H gene – H and h alleles (h is an amorph)

• Se gene – Se and se alleles (se is an amorph)

• ABO genes – A, B and O alleles

• These genes (ABO, Hh, and Se) do not actually code for the
production of antigen but rather produce specific
glycosyltransferase that add sugars to a basic precursor substance.
Biochemistry
 Precursor:
Paragloboside/Glycan
 Type I precursor :
terminal galactose
linked to a
subterminal N-
acetylgluosamine
in a 1-3 linkage.
 Type II precursor : same
sugars combine in a 1-4
linkage

 ABH antigens on RBC are

derived from Type II chains
 Blood group substances in
secretion are made from
both types I & II precursors
H Antigen A Antigen B Antigen
 The H gene is present in almost 99.99% of the
population. While there is called the genotype “hh” that
is extremely rare and its phenotype lacks the normal
expression of the ABH antigens.

hh genotype do not produce the “a-2-

Lfucosyltransferase” therefore, there is no L-fucose
and H substance is not expressed on the RBC.

This is the Bombay Type (hh genotype)

H Antigen
ABO Antigens in
Secretions
Blood Group Substances are soluble antigens (A,
B, and H) that can be found in the secretions.
This is controlled by the H and Se genes
Se gene (FUT2 gene) encodes α2 L
fucosyltransferase which modifies type 1 precursor
to form H substance.
If the Se allele is inherited as SeSe or Sese, the
person is called a “secretor”
80% of the population are secretors
sese genotypes are nonsecretors.
Secretor Status

• The Se gene codes for the presence of the H antigen in secretions,

therefore the presence of A and/or B antigens in the secretions is
contingent on the inheritance of the Se gene and the H gene

A antigen
Se gene (SeSe H antigen in and/or
or Sese) secretions B antigen

se gene (sese) No antigens secreted

in saliva or other body
fluids
ABH Soluble Antigens
ABH Soluble Antigens
ABH Soluble Antigens
ABO antibodies

Natural antibodies: does NOT require the presence

of a foreign red blood cell for the production of
ABO antibodies.
Titer of ABO Abs is often reduced in elderly and in
patients with hypogammaglobulinemia and infants
(until 3 -6 months of age)
Antibody production peaks between 5 and 10
years of age and declines later in life.
ABO antibodies

• The ABO antibodies are predominantly

IgM, activate complement, and react at
room temperature or colder.
• ABO antibodies produce strong direct
agglutination reactions during ABO
testing.
• ABO antibodies can cause rapid
intravascular hemolysis if the wrong
ABO group is transfused, potentially
resulting in patient death.
ABO Forward Typing
ABO Reverse Typing
Forward & Reverse
Reagents for ABO Testing
ABO Subgroups

• ABO subgroups differ in the amount of

antigen present on the red blood cell
membrane
• Subgroups have fewer antigens are present
on the RBC
• Subgroups are the result of less effective
enzymes (not as efficient in converting H
antigens to A or B antigens)
• Subgroups of A are more common than
subgroups of B
Subgroups of A
A1 and A2
• Both react strongly with reagent anti-A
• To distinguish A1 from A2 red cells, the
lectin Dolichos biflorus is used (anti-A1)
• 80% of group A or AB individuals are
subgroup A1
• 20% are A2 or A2B
Subgroups of A

Group A RBCs that react with both anti-A and

anti-A1 are classified as A1,

whereas those that react with anti-A and not

anti-A1 are classified as A2
 Subgroups of A
• A1 gene elicits HIGH concentration of enzyme a-3-N-
acetylgalactosaminyltransferase.
• A1 and A2 both have N-acetyl-D-galactosamine as their
immunodominant sugar.
Subgroups of A

Unexpected antibodies (Anti-A1) can be formed from A2

individuals (1%-8%) in their serum.

…and 22%-35% from A2B individuals.

This anti-A1 can cause discrepancies between forward

and reverse ABO testing and incompatibilities in
crossmatches with A1 or A1B cells.
 Due to the A2 glycosyltransferase being less efficient at adding
the immunodominant sugar to the H antigen precursor, A2 RBCs
show increased reactivity with anti-H lectin, Ulex europaeus,
compared to A1 RBCs.
H antigen is found in greatest concentration on the RBC of
group O individuals. H antigen may not be detectable in group A1
individuals, because in the presence of the A1 gene, almost all of the
H antigen is converted to A1 antigen by placing the large N-acetyl-D-
galactosamine sugar on the H substance. Due to the presence of so
many A1 antigens, the H antigen on A1 and A1B RBCs may be hidden
and therefore may not be available to react with anti-H antisera. In
the presence of an A2 gene, only some of the H antigen is converted
to A antigens, and the remaining H antigen is detectable on the cell
 Other A subgroups
Weak A Subgroups
Characteristics:

1. Decreased number of A antigen sites per RBC (resulting in weak

or no agglutination with human polyclonal anti-A)
2. Varying degrees of agglutination by human anti-A,B
3. Increased variability in the detectability of H antigen, resulting
in strong reactions with anti-H
4. Presence or absence of anti-A1 in the serum
Weak A phenotypes can be serologically differentiated using the
following techniques:
1. Forward grouping of A and H antigens with anti-A, anti-A,B, and
anti-H
2. Reverse grouping of ABO isoagglutinins and the presence of
anti-A1
3. Adsorption-elution tests with anti-A
4. Saliva studies to detect the presence of A and H substances
 Mixed-field - defined as small
agglutinates within
Weak A Subgroups predominantly
unagglutinated RBCs.
B Subgroups

⚫ B subgroups occur less than A subgroups

⚫ B subgroups are differentiated by the type
of reaction with anti-B, anti-A,B, and anti-H
⚫ B3, Bx, Bm, and Bel

Criteria used for differentiation of weak B phenotypes

include the following techniques:
• Strength and type of agglutination with anti-B, anti
A,B, and anti-H
• Presence or absence of ABO isoagglutinins in the
serum
• Adsorption-elution studies with anti-B
• Presence of B substance in saliva
• Molecular testing
Weak B Subgroups
Bombay Phenotype (Oh)
Bombay Phenotype (Oh)
First reported in Bhende in 1952 in Bombay, India.
Inheritance of hh genotype
Phenotypes as blood group O.
Anti-A, anti-B, and anti-H are present in the serum. (IgG)
Missense mutation of FUT1 gene (H gene) and silenced FUT2 (Se
gene)
The h gene is an amorph and results in little or no production
of L fucosyltransferase
Very rare (Frequency in India 1:10000)
Individuals with Bombay phenotype can only be transfused with
Bombay blood group only.
Parabombay phenotype
H antigen is completely lacking or
have only small amounts.
Basis can either of the 2 types: (1)
Mutated FUT1 (H gene) with or without
an active FUT2 gene (Se gene), or (2)
Silenced FUT1 gene with an active
FUT2 gene.
 Parabombay phenotype
Mutated FUT1 gene para bombay Silenced FUT1 gene parabombay
Enzyme activity of H antigen is reduced/weakly
expressed.
Very small amounts of H, A, and B antigen are Have little or no H, A, and B antigens.
produced in RBC
If a person is genetically Type A or B, their
enzymes can be detected but they don’t have the
H antigen. Thus, they are called Ah and Bh
respectively. Same with Type AB (ABh )
No H, A, and B antigen in saliva. Has H, A, and B type 1 antigen in secretions,
including plasma.
Antibodies present:
Type A (Anti-B and Anti-A1 )
Type B (Anti-A and Anti-B )
ABO
Phenotype
Frequencies
of Ethnic
Groups in
the US
ABO Discrepancies

Occurs when unexpected reactions are observed in the

forward and reverse grouping.
Can be due to the following:
• Patient’s serum (reverse grouping)
• Patient’s RBC (forward grouping)
• Both the Serum and Red cells.
Technical Errors
Resolution
If initial testing was performed using RBCs suspended in serum
or plasma, repeat testing of the same sample using a saline
suspension of RBCs can usually resolve the ABO discrepancy. It is
important to make sure any and all technical factors that may have
given rise to the ABO discrepancy are reviewed and corrected. It is
also essential to obtain adequate information regarding the patient’s
age, diagnosis, transfusion history, medications, and history of
pregnancy. If the discrepancy persists and appears to be due to an
error in specimen collection or identification, a new sample must be
drawn from the patient and all RBC and serum testing repeated.
When a discrepancy is encountered, all results must be
recorded, but interpretation of the ABO type must be delayed until
the discrepancy is resolved. If the blood is from a potential
transfusion recipient, it may be necessary to administer group O, Rh-
compatible RBCs before the discrepancy is resolved. In general,
when investigating ABO discrepancies, always remember that RBC
and serum grouping reactions are very strong (3+ to 4+) and the
weaker reactions typically represent the discrepancy.
ABO Group I Discrepancies
• These are associated with
discrepancies unexpected reactions in the
reverse grouping due to weakly
reacting or missing antibodies.

Includes:
• Infants less than 3-6 month of age
• Elderly patients
• Leukemia patients
• Severe hypogammaglobulinemia
• ABO incompatible HPC
transplantation
• ABO Subgroups
Resolution of Group I

Enhancing weak or missing reaction by incubating

the patient’s serum with reagent A1 and B cells at
room temperature for 15-30 minutes
Serum cell mixture is incubated at 4⁰C for 15-30
minutes
An autocontrol and O cell control must always
be tested concurrently to detect reactivity of
other commonly occurring cold agglutinins eg:
anti I
Group II discrepancies
These are associated with unexpected reactions in forward
grouping due to weakly reacting or missing antigen
Includes:
Weak subgroups of A or B
Weakening of A or B antigen in malignancies (leukemia /
Hodgkins’s dse)
Acquired B phenotypes:
results from the action of bacterial deacetylase,
which converts N-acetylgalactosamine to ẞ-
galactosamine, which is very similar to galactose, the
chief determinant of B. ‘passenger antigen’ type is
caused by adsorption of B-like bacterial products on to
O or A cells but occurs only in vitro.
Out of group transfusion or ABO mismatched HPSCT
Neutralization of anti A and anti B typing reagent by high
concentration of A or B soluble substances in serum with
serum or plasma suspended red cell
 Resolution of Group II
✓Weaker reactions with antisera can be resolved by
enhancing reaction of antigen with respective antisera
by incubating test mixture at room temperature for 15-
30 minutes. If the reaction is still negative, incubate
the text mixture at 4°C for 15 to 30 minutes.
✓Sub groups causing group discrepancies can be
resolved by adsorption elution studies
✓Acquired B phenomenon can be resolved by lowering
PH of monoclonal antisera. Anti B in the serum of
acquired B person does not agglutinate autologous
red cells (auto control negative).
✓Secretor status of person can resolve acquired B,
saliva of acquired B person contains A substance
not B substance.
✓High concentration of A or B substance causing group
discrepancies can be resolved by saline washing of
red cells
Group III discrepancies
These discrepancies between forward and reverse groupings are
are associated with protein or plasma abnormalities, rouleaux
formation and pseudoagglutination.

Includes
• Elevated level of globulin from e.g. multiple myeloma,
waldenstorm macroglobulinemia, Hodgkin lymphoma.
• Elevated level of fibrinogen.
• Small fibrin clot in plasma or incompletely clotted serum can
be mistaken for red cell agglutinates in reverse grouping.
• Sample with abnormal concentration of serum proteins,
altered serum protein ratio, or high molecular weight volume
expanders can aggregate reagent red cells and can mimic
agglutination.
• Plasma expanders (dextran and polyvinylpyrrolidone)
• Wharton’s jelly in cord blood samples
 Resolution of Group III
✓ Performing a saline replacement technique
will free the cells in the case of rouleaux
formation in the reverse type.

✓ Thoroughly washing cord cells six to eight

times with saline should alleviate spontaneous
rouleaux due to Wharton’s jelly and result in
an accurate ABO grouping.
Group IV
Discrepancies
Group IV discrepancies between forward
and reverse groupings are due to
miscellaneous problems.
✓ Cold reactive autoantibodies in which RBCs are
so heavily coated with antibody that they
spontaneously agglutinate, independent of the
specificity of the reagent antibody
✓ Circulating RBCs of more than one ABO group
due to RBC transfusion or marrow/stem cell
transplant
✓ Unexpected ABO isoagglutinins
✓ Unexpected non-ABO alloantibodies
Resolving ABO
discrepancy