ABO Blood Group system

ABO BLOOD GROUP
• History
• The ABO blood group system (BGS) is the most important
human BGS in transfusion practice and was the first to be
discovered.
• The ABO system was first described by Karl Landsteiner in
1900 and was reported in 1901.
• Landsteiner drew blood from coworkers in his laboratory,
separated cells and plasma, and mixed the cells and
plasma from the various people on glass tiles.
• He was able to identify three different patterns of
reactivity, which he termed A, B, and C.
• These were later reclassified as groups A, B, and O,
respectively.
1Alick Mwambungu - BTS

ABO BLOOD GROUP
• History
• Von Decastello and Sturli discovered group AB (the
rarest of the common ABO types) and reported this
blood type in 1902.
• Landsteiners 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
Importance of ABO
• Almost all normal healthy individuals above 3-6 months
of age have “ naturally occurring Abs” to the ABO Ags
that they lack
• These Abs termed naturally occurring because they
were thought to arise without antigenic stimulation
• This theory was soon discarded after it was shown that
the naturally occurring antibodies were actually caused
by stimulation with antigenically similar substances to A
and B present in our environment.

ABO BLOOD GROUP
• Importance of ABO
• These “naturally occurring” Abs are mostly IgM class.
• This means that, they are Abs capable of agglutinating
saline/ low protein suspended red cell without
enhancement and may activate complement cascade.

Inheritance
• The ABO blood group genes code not for the
antigens directly, which are carbohydrate in
nature, but for the production of
glycosyltransferases.
• The ABO genes follow simple Mendelian
genetic laws and are inherited in a
codominant fashion because both A and B
alleles are expressed when present.

Inheritance
• The O gene is an amorphic or silent gene in
that it appears to have no gene product or to
produce a nondetectable product.
• No speciﬁc transferase has been associated
with the O gene.
• ABO gene is located on the long arm of
chromosome 9, comprises seven exons and
encodes the A and B glycosyltransferases

Inheritance
• Products of the A and B alleles differ by four amino
acids encoded by exon 7,
• Two of which determine whether the enzyme has N-
acetylgalactosaminyl-transferase (A) or D-
galactosyl transferase (B) activity.

Inheritance
9
The majority of O alleles (called O1 ) resemble
A, but have a single-base deletion in exon 6,
which creates a shift in the reading frame
generating a premature stop codon O gene
Alick Mwambungu - BTS

The ABO genes
• About 3% of O alleles(called O2) have a single-
nucleotide polymorphism (SNP) that changes
one of the vital amino acids in the catalytic
site, inactivating the enzyme.

The ABO genes
• The A2 allele has a single-base deletion immediately
before the usual termination codon, creating a
reading frameshift and abolition of this stop codon.
• This produces an A-transferase with 21 extraneous
amino acids on its C-terminus, reducing its efficiency
as a GalNAc-transferase

The ABO genes
H genes
• At least two genes, FUT1 and FUT2, on
chromosome 19, are responsible for
production of H antigen.
• Both encode α1,2 fucosyltransferases that
catalyse the transfer of fucose to the terminal
galactose of the H precursor chain

Biochemistry and biosynthesis of ABH antigens
• The inheritance of at least one H gene (HH or Hh)
elicits the production of an enzyme called, α-2-L-
Fucosyl transferase.
• Transfers the sugar from the Guanosine diphosphate
L-fucose (GDP-Fuc) donor nucleotide to the terminal
galactose of the precursor chain type 1 or 2.
• The H substance must be formed for the other sugars
to be attached in response to an inherited A and /or
B genes

Biochemistry of ABO antigens
• There are two potential precursors substance (PS)
both are comprised of identical sugar (N- acetyl
glucosamine) found in between Galactose but
different in linkage.
• Type I PS has a terminal galactose (Gal) linked to a
subterminal N-acetyl-glucosamine (GlcNAc) in 1-3
linkage.
• Type II PS, has the same sugar combined in 1-4
linkage
• ABH Ags on RBCs are derived from Type II chains.

Biochemistry of ABO antigens
• Type 1: #1 carbon of Gal is
attached to the #3 carbon
of GlcNAc.
• Type 2: #1 carbon of Gal is
attached to the #4 carbon
of GlcNAc.

H gene acts on
a Precursor
substance(PS)*
by adding
Fucose
H Antigen
*PS = oligosaccharide chain
attached to either glycosphingo-
lipid, Type 2 chain (on RBC) or
glycoprotein, Type 1 chain (in
secretions)
The H gene (FUT 1) codes for an enzyme (fucosylytranferase)
that adds a Fucose to the terminal sugar of a Precursor
Substance (PS*). The biochemical structure below constitutes
the H Antigen.

Formation of the
A Antigen
The A gene codes
for an enzyme that
adds GalNAc
(N-Acetyl-D
galactosamine)
to the terminal
sugar of the
H Antigen. The biochemical structure
constitutes the A antigen.

Formation of the
B Antigen
B gene codes for an
enzyme that adds
D-Galactose
to the terminal sugar
of the H Antigen.
The biochemical structure
constitutes the B Antigen.

The H antigen is found on
the RBCs when there is an Hh
or HH genotypes but NOT
with the hh genotype.
The A antigen is found on
the RBCs when there is Hh,
HH, and A/A, A/O or A/B
genotypes.
The B antigen is found on
the RBCs when there is Hh,
HH, and B/B, B/O or A/B
genotypes.

Amount of H Antigen According to Blood
Group
 Blood Group O people
have red blood cells
rich in H antigen.
Why?
Greatest
Amount of H
Least
Amount of H
O > A2 > B > A2B > A1 > A1B

Donor Nucleotides & Immundominant Sugars responsible for H, A,
and B Ags specificity
AntigenImmunodominant sugarGlcosyltransferaseGene
HL-fucoseL- fucosyl
transferase
H
AN-acetyl-D-
galactosamine
N
acetylgalactosaminyl
transferase
A
BD-galactoseD- galactosyl
transferase
B

ABO: ANTIGENS
• ABO Antigens can be detected on RBCs very early in
embryonic development.
• However not yet maximally developed at birth
• No. of A and B antigens on the RBCSs of newborns is
smaller than that on the RBCs of adults.
• Subgroups of A antigen include A1 and A2.
• The A1 red cells react with both anti-A and anti-A1
• The A1 has more antigenic sites for A antigen and less
for H.
• The antibody present in A1 is only anti-B.

ABO: ANTIGENS
• A2 red cells react with only anti-A and not with
anti-A1
• This is a weak A subgroup and carries more H
substance.
• The cells of approximately 80% of A
individuals are A1,
• The remaining 20% are A2

Quantitative differences between A1
and A2
• A1-enzyme is a very strong enzyme that
changes all type II H-chains into A1-antigens.
• A2-enzyme is less strong and does not change
all type II chains into A2 antigens.
• Therefore in blood group A1 there are more A1
antigens on the RBCs (850,000) compared to
the number of A2 antigens (240,000) on the
red cell membrane in blood group A2.

Qualitative differences
• Differences arise due to a mutation in the A2 glycosyl
transferase peptide chain.
• Coding region of the A2 allele differs from A1 in two ways:
• First by a single nucleotide (467 C>T) substitution.
• Creates a single amino acid change
• Proline no.156 is changed to a leucine
• And second by a 1061 del C mutation
• Which causes a frame shift
• These two mutations together create an enzyme whose
activity is significantly weakened.

Antibodies of the ABO system
• Naturally occurring ABO antibodies arise in
response to A- and B-like antigens present on
bacterial, viral or animal molecules.
• Titres of ABO antibodies vary considerably
with age, reaching a peak in young adults and
then declining in old age.
• Most group O persons, produces IgG as readily
as IgM anti-A,B.

• Consequently, mothers of children with ABO HDFN
are almost always group O.
• Immune anti-A,B are mainly IgG2, which does not
cause HDFN because there are no Fc receptors for
IgG2 on the cells of the mononuclear phagocyte
system.
• When the maternal serum contains potent IgG1
and/or IgG3 ABO antibodies, HDFN may occur,
although this is comparatively milder than Rh HDFN

• Individuals with blood group A1 have exclusively anti-B
in the serum.
• Approx 8% of all individuals with A2 blood group have
anti A1 in the serum apart from anti B.
• 35% of subjects with blood group A2B produce anti A1
and subjects with neither A1 nor A2 always have anti A
as well as anti A1 in their serum
• Serologically ,the subgroups A1 and A2 can be
distinguished because anti-A1 only reacts with A1 RBCs
and not with A2 RBCs.
• Anti-A(total) does react with both subgroups.
• Anti A2 has never been found.

Lectins
• These are extracts from certain plants.
• Dolichos biflorus (Indian cattle beans)
• React specifically with A1 antigen and not A2
antigen.
• Can also be used to differentiate A1 and A1B
from A2 and A2B.

Secretor phenomenon
• Secretor and non secretor refer to the presence of
water-soluble ABH antigen substances in body fluid.
• Influenced by the independently inherited regulator
Se gene (FUT 2) on chromosome 19.
• The presence of Se, in single (Sese) or double (SeSe)
dose, results in the presence of the H antigen in
secretions.
• If there is homozygozity for the silent se-allele
(genotype sese)-H,A or B- substances are not
secreted into the body fluids.(non-secretors).

Secretor phenomenon
• The secretor gene controls only the presence or
absence of the H substance in body secretions.
• It does not affect the presence of the H substance on
erythrocytes.
• If a person is a secretor and independently inherits
the A or B transferase,
• these transferases result in an altering of the H
substance in secretions and on the surface of the
erythrocytes, resulting in ABH antigens in the
secretions as well.

Secretor phenomenon
• 78% of population possess at least one Se
gene and secrete A,B or H water-soluble
antigens.
• The secretions have the property of reacting
with their corresponding Abs.
• Hence neutralize or inhibit the ability of the
Ab to react with the RBCs having the
corresponding Ag.

Detection Tests (Hemmaglutination inhibition)

Rare abnormalities in ABO Groups
Bombay blood group
• Individuals have no H-allele (genotype hh)
• Not able to produce H- substance.
• Antigens of the ABO-system cannot come to
expression.
• First donor with this phenotype was found in
Bombay (India)
• These cells are similar to the cells of blood group O
except that there is no H-substance on the cells.
• In the plasma there is anti H in addition to anti A and
anti B.

• Anti H causes an acute haemolytic transfusion
reaction.
• Therefore individuals can only receive blood
from donors who also have the Bombay blood
group.
• Units of these are very rare and are kept at the
European Bank of Frozen blood of the council
of Europe,located at Sanquin in Netherland.

Acquired B-antigen
• Some bacterial infections lead to the presence
of what seems to be B-antigen in people with
blood group A.
• These individuals show a weak reaction with
polyclonal anti-B sera.
• While they have anti-B antibodies.
• Gram-negative bacteria can produce enzymes
capable of changing A1-antigens.

37
• De-acetylase formed by the bacteria is responsible
for the transformation of N-acetyl-D-galactosamine
into alpha-galactosamine.
• alpha-galactosamine bears a strong resemblance to
galactose-the major determinant of B-antigen.
• When the bacteria that form the acquired B-antigen
disappears from the intestine-the acquired B-
antigen also disappears.
• Patients with an acquired B-antigen should never
receive a transfusion with blood of group B.
• The anti B in the patients serum can strongly react
with the donor B cells.

T-activation
• All normal RBCs contain antigens that are not usually
detectable by routine blood banking methods.
• T activation is the most common acquired
polyagglutination, especially in children and infants.
• It is caused by removal of portions of glycophorin A
and B chains by microbial neuraminidase on the RBC
membrane resulting in exposure of “crypt antigens.”
• The crypt antigens render the cells more susceptible
to agglutination by normal human plasma.

T-activation
• which contains naturally occurring antibodies against
the crypt antigens.
• T-activation or the unmasking of these hidden
antigens is a temporal condition.

T-activation
• The following can produce Neuraminidase:
• Bacteroides and Clostridium
• Yeast
• Viruses particularly Influenza virus.
• Protozoa
• When the infection subsides the RBCs are usually no
longer T-activated or polyagglutinable.
• All normal adult sera contain anti T
• therefore T-activated RBCs are polyagglutinable.

T-activation
• Cord serum lack anti T therefore can not
agglutinate T-activated erythrocytes.
• Anti-T is not an autoantibody hence can not
agglutinate person’s own RBCs.

Tn activation
• Tn polyagglutination is an acquired red blood cell
polyagglutination disorder caused by a mutation rather
than an infection.
• This situation results from a somatic mutation of a gene
carried on the X chromosome.
• The mutation results in incomplete synthesis of normal
residues on glycophorins A and B, with resultant
exposure of a normally hidden antigen (crypt antigen).
• And all normal sera contain the corresponding
antibody.

Tn activation
• Conditions associated with Tn activation include mainly
haematological conditions such as:
– Haemolytic anaemia and thrombocytopenia.
• Tn activation is a permanent alteration and can not be
stimulated in vitro.

Tn activation
Tn activated cells and T-activated cells share the
following characteristics:
• Reduced erythrocytic membrane glycophorins A and B
• A mixed-field agglutination with all normal adult sera
• No agglutination with cord blood
The correct ABO status of a person can be determined
by treating the Tn-activated cells with 1% ficin or
papain to destroy the Tn receptor sites.

45
ABO FORWARD AN REVERSE GROUPING

Examples of Discrepancies in ABO Forward and
Reverse grouping
• Technical errors and various clinical conditions
can lead to a discrepancy between erythrocytes
and serum results in ABO grouping.
• Possibility of technical errors must be excluded
when the reverse and forward grouping don’t
produce the complementary matching results.

Technical and Clerical problems leading to false
results
False Positive results
1. Contaminated reagents or dirty glassware
2. Over centrifugation
3. Incorrect interpretation or recording of test results
False Negative results
1. Lack of specimen or reagents in the test system
2. An incorrect serum: cell ratio
3. Under centrifugation
4. Old or inactive reagents
5. Failure to recognize haemolysis as a positive reaction
or an error in the recording of results. 47Alick Mwambungu - BTS

Unexpected Antigen Reaction
Acquired B-like antigen
• RBCs react as AB, while the serum contain only
anti B.
• A substance is present in the saliva of secretors
but B substance is not.

49
Reaction of Cells Tested With
Reaction of Serum
Tested Against Auto
Control
Anti-A Anti-B Anti-AB Cells1A B Cells
4+ 1+ 4+ O 4+ O
Antigen reaction example of a Typical Unexpected Antigen Reaction
.

50
Observation of the 4+ forward and reverse grouping
reactions is typical in a Group A
However the 1+ reaction of RBCs with Anti B should be
investigated.
Additional Laboratory testing
A substance but not B substance was present in the
saliva of the patient.
In addition, this patient had been admitted to the
Hospital for severe gastrointestinal infection.
The conclusion was that the patient was group A with
an acquired B-like antigen

Unexpected Antigen Reaction
Mixtures of Blood
• Mixture of cell types in recently transfused
patients
• or recipients of bone marrow transplants can
produce unexpected results in forward typing.

Weak and missing Antibody reactions
• Missing antibody reactions on reverse
grouping can be caused by the following:
1. Age
2. Hypogammaglobulineamia
3. Agammaglobulineamia

Age
• Newborn and young infants and the elderly
may exhibit weak or missing isoantibodies.
• Reverse grouping is not routinely done on
newborn infants.
• In most cases detectable antibodies in
newborn infants are usually acquired in utero
from the mother

Hypogammaglobulineamia
• Decreases in the gamma globulin fraction of
plasma can lead to weak or missing antibodies.
• Conditions that may cause
hypogammaglobulineamia include the use of
immunosuppressive drugs,
• lymphomas,
• Leukaemias and immunodeficiency disorders

 Agammaglobulineamia
• Absence of gamma globulins can either be
congenital or acquired.
• Congenital: Burton’s Agammaglobulineamia
• Acquired: exposure to radiation
• And cytotoxic drugs

Unexpected Antibody reactions
• Conditions producing unexpected reactions on
reverse grouping include:
1. Rouleaux formation
2. Cold auto antibodies
3. Unexpected antibodies

 Rouleaux formation
• Abnormal concentration of serum proteins
• Elevated globulin levels in diseases such as
multiple myeloma,Waldenstron’s
macroglobulineamia and Hodgkin's lymphoma.
• can cause RBCs to stick together in a manner that
may resemble agglutination.
• Washing a patient’s red cells three times in saline
usually remove proteins that cause rouleaux.

 Cold auto antibodies
• There may be auto antibodies in the serum that
reacts with antigens other than the A1 or B cells
used for reverse grouping.
• Anti I is the commonly encountered autoantibody
in reverse grouping.
• Ant I usually agglutinates all RBCs from adult
donors including the patient’s own and those used
in reverse grouping.

59
Reaction of Serum
Tested Against Auto
Control
4+ O 4+ 1+ 4+ O
The strong (4+)forward agglutination with Anti A and Anti AB and reverse
agglutination with B cells represents a typical group A reaction.
The reaction with A1 cells suggests the presence of an additional weakly
reacting antibody
Additional laboratory testing
Agglutination wascells.2and A1The serum was tested against A
In addition thecells.2RBCs but none of the A1observed in all of the A
patient’s RBCs were tested with Dolichos biflorus . The test came out
negative
phenotype with an2The conclusion was that this patient was a group A
antibody.1A-anti

• The presence of a positive auto control invalidates the initial
testing results.
 Additional lab testing
• A cold autoabsorption method was performed and absorbed
serum was tested against A1 ,and B cells.
• No agglutination was observed with the absorbed serum and
reagent cells.
• The conclusion was that the patient was a group AB phenotype
with an auto anti-I.
60
Reaction of Serum
Tested Against Auto
Control
4+ 4+ 4+ 2+ 2+ 2+
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