LECTURE

THE ABO BLOOD GROUP SYSTEM - discovery of the first human blood group
system, ABO.
ABO System
- marked the beginning of the concept of
- Most important of all blood groups in both
individual uniqueness defined by the RBC
transfusion and transplant medicine.
antigens present on the RBC membrane.
- only blood group system in which individuals
- first individual to perform forward and
already have antibodies in their serum to
reverse grouping.
antigens that are absent from their red blood
cells (RBCs) without any prior exposure to
RBCs through transfusion or pregnancy.
Forward Grouping (front type)
- transfusion of an incompatible ABO type may
- defined as using known sources of commercial
result in immediate lysis of donor RBCs.
antisera (anti-A, anti-B) to detect antigens on
- Testing to detect ABO incompatibility an individual’s RBCs.
between a donor and potential transfusion
recipient.
Reverse Grouping (back type)
➢ the foundation on which all other
pretransfusion testing is based. - defined as detecting ABO antibodies in the
patient’s serum by using known reagent RBCs,
- transfusion-related acute lung injury
namely A1 and B cells.
(TRALI)
➢ most frequent cause of death in fiscal
year (FY) 2015 ABO Grouping
- most frequently performed test in the blood
bank.
- Both ABO forward and reverse grouping tests
must be performed on all donors and patients.

Bacteria
- widespread in the environment, which
constantly exposes individuals to A-like and B-
like antigens.
- exposure serves as a source of stimulation of
anti-A and anti-B.

Naturally Occurring Antibodies

- antibodies in the serum to antigens that are
absent from their RBC.
- Antibody production in most other blood
group systems requires the introduction of
foreign RBCs by either transfusion or
HISTORICAL PERSPECTIVE AND ROUTINE ABO pregnancy, although some individuals can
TESTING occasionally have antibodies present that are
not related to the introduction of foreign RBCs.
Karl Landsteiner

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 LECTURE

- These antibodies are usually of the IgM type

and are not consistently present or expected in
everyone’s serum.

ABO ANTIBODIES
Naturally occurring
- produced without any exposure to RBCs.

ABO antibodies
- predominantly IgM, activate complement, and
react at room temperature or colder.
- produce strong direct agglutination reactions
during ABO testing.
- production is initiated at birth, but titers are
generally too low for detection until infants
are 3 to 6 months old.
- most antibodies found in cord blood serum are
of maternal origin.
- it is logical to perform only forward grouping
on cord blood from newborn infants.
- production peaks between 5 and 10 years of
age and declines later in life.
- antibodies may be undetectable in the reverse
grouping.
- can cause rapid intravascular hemolysis if the
wrong ABO group is transfused, potentially
resulting in patient death.
- Serum from group O individuals contains anti-
A, anti-B, and anti-A,B. Anti-A,B reacts with
both A and B cells.
- Anti-A,B antibody activity, originally thought
to be just a mixture of anti-A and anti-B, cannot
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 LECTURE

be separated into a pure specificity when - considered an amorph, as no detectable

adsorbed with either A or B cells.
- Anti-A,B antibody is not a combination of anti-
A and anti-B but is a separate “cross-reacting”
antibody that is usually IgG in nature.
- Both immunoglobulin classes of ABO
antibodies react preferentially at room
temperature (20°C to 24°C) or below and
efficiently activate complement at 37°C.
antigen is produced in response to the
inheritance of this gene.
- group O phenotype is an autosomal recessive
trait with the inheritance of two nonfunctional
O genes.
• designations group A and B = phenotypes
• AA, BO, and OO = genotypes

Anti-A,B Reagent • O individual, both phenotype and genotype are the

- routinely used for performing ABO
confirmation of group O donor units simply
because it is more economical to use one
reagent instead of both anti-A and anti-B.
same because that individual would have to be
homozygous for the O gene.
• An individual who has the phenotype A (or B) can
have the genotype AA or AO (or BB or BO).

- Use of anti-A,B reagent is not required for

routine patient ABO testing.
- Some believe anti-A,B is more effective at
detecting weakly expressed A and B antigens
than reagent anti-A or anti-B.
- prepared using blended monoclonal anti-A
and anti-B; polyclonal human anti-A,B; or a
blend of monoclonal anti-A, anti-B, and anti-
A,B.

INHERITANCE OF THE ABO BLOOD GROUPS

- theory for the inheritance of the ABO blood
groups was first described in 1924.
➢ indicating that an individual inherits one
ABO gene from each parent and that
these two genes determine which ABO
antigens are present on the RBC
membrane.
- inheritance of ABO genes follows simple
Mendelian genetics. ABO
➢ codominant in expression
- One position, or locus, on each chromosome 9
is occupied by an A, B, or O gene.

O Gene

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 LECTURE

Type 1 Precursor
- substance refers to a beta 1 → 3 linkage
between galactose and N-acetylglucosamine.

H Antigen
- the precursor structure on which A and B
antigens are made.
- Inheritance of the H gene results in formation
of the H antigen.

• FUT 1 (H) and FUT 2 (Se) genes are closely linked

and located on chromosome 19.
• ABO genes located on chromosome 9.

H and Se genes
FORMATION OF A, B, AND H RED BLOOD CELL - not part of the ABO system
ANTIGENS
- their inheritance influences A and B antigen
• results from the interaction of genes at three expression.
separate loci (ABO, Hh, and Se).
- H gene must be inherited to form ABO antigens
• genes do not actually code for the production of on the RBCs.
antigens but rather produce specific - Se gene must be inherited to form ABO
glycosyltransferases that add sugars to a basic antigens in secretions.
precursor substance.

• ABH antigens develop early in fetal life and do not

Paragloboside or Glycan
increase much in strength during the gestational
- the same basic precursor material from which period.
A, B, and H antigens all originate.
• The RBCs of the newborn have been estimated to
- Specific enzyme transferases elicited by an carry anywhere from 25% to 50% of the number
inherited gene attach sugars to the of antigenic sites found on the adult RBC.
paragloboside/glycan.
• reactions of newborn RBCs with ABO reagent
antisera are frequently weaker than reactions
with adult cells.
Type 2 Precursor
• expression of A and B antigens on the RBCs is fully
- terminal galactose on the precursor substance
developed by 2 to 4 years of age and remains
is attached to the N-acetylglucosamine in a
constant throughout life.
beta 1 → 4 linkage.
• In addition to age, the phenotypic expression of
- ABH antigens on the RBC are constructed on
ABH antigens may vary with race, genetic
oligosaccharide chains of a type 2 precursor
interaction, and disease states.
substance.

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 LECTURE

- O blood group has the highest concentration of

H antigen.
- H substance (L-fucose) must be formed for the
other sugars to be attached in response to an
inherited A and/or B gene.

H Gene
- present in more than 99.99% of the random
population.
- Its allele, h, is quite rare, and the genotype hh
is extremely rare.

Bombay
- refer to the phenotype that lacks normal
expression of the ABH antigens because of the
inheritance of the hh genotype.
- hh genotype does not elicit production of α-2-
INTERACTION OF Hh AND ABO Genes L-fucosyltransferase.
• Individuals who are blood group O inherit at least - L-fucose is not added to the type 2 chain and H
one FUT 1(H) gene (genotype HH or Hh) and two substance is not expressed on the RBC.
O genes.

A Gene
H Gene
- elicits the production of an enzyme called α-2-
L-fucosyltransferase that transfers the sugar
L-fucose to an oligosaccharide chain on the
terminal galactose of type 2 chains.
➢ L-fucose is the sugar responsible for H
specificity.
Immunodominant Sugars
- (AA or AO) codes for production of α-3-N-
acetylgalactosaminyltransferase, which
transfers an N-acetyl-D-galactosamine
(GalNAc) sugar to the H substance.
- sugar confers A specificity.
- A-specific immunodominant sugar is linked to
a type 2 precursor substance that now
contains H substance through the action of the
H gene.

- Sugars occupying the terminal positions of this - A gene tends to elicit higher concentrations of
precursor chain and conferring blood group transferase than the B gene, leading to
specificity. conversion of nearly all of the H antigen on the
RBCs to A antigen sites.

O Gene at the ABO locus

B Gene
- Referred to as an amorph.
- (BB or BO) that codes for the production of α-
- does not elicit the production of a catalytically 3-D-galactosyltransferase and attaches D-
active polypeptide transferase. galactose (Gal) sugar to the H substance
previously placed on the type 2 precursor
substance through the action of the H gene.
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 LECTURE

- sugar is responsible for B specificity.

• When both A and B genes are inherited, the B

enzyme (α-3-D-galactosyltransferase) seems to
compete more efficiently for the H substance than
the A enzyme (α-3- N-
acetylgalactosaminyltransferase).

MOLECULAR GENETICS OF ABO

• ABO gene, located on chromosome 9, consists of
seven exons.
• last two exons (6 and 7) encode for the catalytic
domain of the ABO glycosyltransferases.
• Exons 6 and 7 constitute 77% of the gene, with
most of the coding sequence lying in exon 7.
• Amino acid substitutions, resulting primarily from
deletions, mutations, or gene recombinations
within these two exons of the coding DNA of
variant ABO glycosyltransferases, are responsible
for the less efficient transfer of the
immunodominant sugar to H substance, resulting
in weak serologic reactions observed in ABO
subgroups.
• The seven common ABO alleles include A1,
A1variant (A1v), A2, B1, O1, O1variant(O1v), and O2.
• All ABO antigens arise from mutations in the single
ABO gene.

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 LECTURE
• Only three specific mutations showing a high
frequency in the population indirectly lead to
changes in the epitope structures resulting in A, B,
or O specificities.
• Two of the mutations (substitutions) change the
specificity of the enzyme from an α-N-
acetylgalactosaminyltransferase (the A enzyme)
to an α-3-D-galactosyltransferase (the B enzyme).
• glycosyltransferases introduce an α 1,3 N-
acetylgalactosamine (A) or an α 1,3 galactose (B)
carbohydrate at the ends of type H oligosaccharide
(glycan) chains.
• ends of type H oligosaccharide (glycan) chains.
The third mutation, a deletion within the 5' region
of the catalytic domain, creates a premature stop
codon that inactivates the enzyme altogether,
leaving the H glycan unmodified.
• These different glycans define the A, B, or O
antigens or epitopes.
FORMATION OF A, B, AND H SOLUBLE ANTIGENS
• ABH antigens are integral parts of the membranes
of RBCs, endothelial cells, platelets, lymphocytes,
and epithelial cells.
• ABH-soluble antigens can also be found in all body
secretions.
• presence is dependent on the ABO genes inherited
and on the inheritance of another set of genes
called Sese (secretor genes) that regulate their
formation.
• inheritance of a FUT 2 (Se) gene codes for the
production of the transferase α-2-L-
fucosyltransferase that modifies the type 1
precursor substance in secretions to form H
substance.
• 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 gene does not affect the formation of A, B, or H
antigens on the RBC.
• the presence of the Se gene– specified α-2-L-
fucosyltransferase that determines whether ABH-
soluble substances will be secreted.
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Nonsecretors
- People who inherit the sese genotype.
COMPARISON OF A, B, AND H ANTIGENS ON RBCs
WITH A, B, AND H SOLUBLE SUBSTANCES
• formation of soluble A, B, and H substances is the
same as that described for the formation of A, B,
and H antigens on the RBCs, except for a few minor
distinctions.
• tests for ABH secretion were used to establish the
true ABO group of an individual with poorly
developed RBC antigens.
Secretor
- refers only to secretion of A, B, and H soluble
antigens in body fluids.
• Both ABH red blood cell antigens and ABH-soluble
substances are formed due to the attachment of an
immunodominant sugar to an oligosaccharide
chain.
• Although several types of oligosaccharide chains
exist, types 1 and 3 are primarily associated with
body secretions, while types 2 and 4 are
associated with the red blood cell membrane.
• Type 1 and 2 chains are more abundant, and they
differ only in the linkage position of galactose (Gal)
to N-acetylglucosamine (GlcNAc).
- type 1 has a beta 1→3 linkage.
- type 2 has a beta 1→4 linkage.
• Addition of specific immunodominant sugars to
the type 2 and 4 chains leads to formation of A, B,
and H antigens on the RBC membrane, with the
majority being present in the form of type 2 chains.
 LECTURE
• Adding the same immunodominant sugars to type
1 and 3 chains in the body secretions allow for A,
B, and H soluble substances to be made in body
secretions.
ABO SUBGROUPS
• represent phenotypes showing weaker and
variable serologic reactivity with the commonly
used human polyclonal anti-A, anti-B, and anti-A,B
reagents.
A. A Subgroups
• 1911 - von Dungern described two different A
antigens based on reactions between group A
RBCs and anti-A and anti-A1.
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• A1 - Group A RBCs that react with both anti-A and
anti-A1.
• A2 - those that react with anti-A and not anti-A1.
• RBCs from A1 and A2 individuals react equally
strong with current reagent monoclonal anti-A in
ABO forward typing tests.
• A Subgroups are more common than B subgroups.
• Classification into A1 and A2 phenotypes accounts
for 99% of all group A individuals.
• The cells of approximately 80% of all group A (or
AB) individuals are A1 (or A1B), and the remaining
20% are A2 (or A2B) or weaker subgroups.
• The production of both types of antigens is a result
of an inherited gene at the ABO locus.
• Inheritance of an A1 gene elicits production of
high concentrations of the enzyme α-3-N-
acetylgalactosaminyltransferase, which converts
almost all of the H precursor structure to A1
antigens on the RBCs.
A2 Allele
- characterized by a single base substitution at
nucleotide 467 and a single base deletion at
nucleotide 1060 (1060delC) in exon 7.
- substitutions alter the active site of the coding
region and subsequently change the specificity
of the A glycosyltransferase.
N-acetyl-D-galactosamine
- immunodominant sugar on both A1 and A2
RBCs.
• Qualitative differences also exist, since 1% to 8%
of A2 individuals produce anti-A1 in their serum
and 22% to 35% of A2B individuals produce anti-
A1.
- antibody can cause discrepancies between
forward and reverse ABO testing and
incompatibilities in crossmatches with A1 or
A1B cells.
Anti-A1
 LECTURE
- a naturally occurring IgM cold-reacting
antibody and is unlikely to cause a transfusion
reaction because it usually reacts only at
temperatures well below 37°C.
- clinically significant if it is reactive at 37°C.
• A1 RBCs can also be conceptualized as having only
A1 antigen sites and A2 as only having A antigen
sites.
• routine forward grouping, reagent anti-A strongly
agglutinates both A1 and A2 phenotypes.
Anti-A1 lectin
- a reagent made from the seeds of the plant
Dolichos biflorus.
- agglutinates A1 (or A1B) cells but does not
agglutinate A2 (or A2B cells).
Lectins
- seed extracts that agglutinate human cells
with some degree of specificity.
• 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
RBCs 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.
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• Weak subgroups of the A antigen will often have
an inverse reciprocal relationship between the
amount of H antigen on the RBC and the amount of
A antigens formed.
- more A antigen formed, the less H antigen
expressed on the RBC.
• The H antigen on the RBCs of A1 and A1B
individuals is so well hidden by N-acetyl-D-
galactosamine that anti-H is occasionally found in
the serum.
Anti-H
- naturally occurring IgM cold agglutinin that
reacts best below room temperature.
- formed in response to a natural substance and
reacts most strongly with cells of group O
individuals (which have the greatest amount
of H substance on their RBCs) and weakly with
the RBCs of A1B individuals (which contain
small amounts of H substance).
- an insignificant antibody in terms of
transfusion purposes because it has no
reactivity at body temperature (37°C).
- high-titered anti-H may react at room
temperature and present a problem in
antibody screening procedures because
reagent screening cells are group O.
- Anti-H lectin, Ulex europaeus, closely
parallels the reactions of human anti-H. Both
antisera agglutinate RBCs of group O and A2
and react very weakly or not at all with groups
A1 and A1B.
• unbranched straight chains (H1, H2)
• complex branched chains (H3, H4)
• antigens H1 through H4 correspond to the
precursor structures on which the A enzyme can
act to convert H antigen to blood group A active
glycolipids.
- chains differ in length and complexity of
branching, the terminal sugars giving rise to
their antigenic specificity are identical.
• Straight chain H1 and H2 glycolipids can be
converted to Aa and Ab antigens, respectively, by
 LECTURE

both A1 and A2 enzymes, with the A2 enzyme being

less efficient.
• The more complex branched H3 and H4 structures
can be converted to Ac and Ad antigens by A1
enzyme and only very poorly by A2 enzyme.
• On the RBCs of some A2 individuals, Ac is
extremely low and Ad is completely lacking.
- individuals in whom one would likely find
anti-A1 in the serum.
- anti-A1 antibody could really be an antibody to
Ac and Ad determinants, which the A2
individuals lack.
• B enzyme transferase is usually more efficient
than the A enzyme in converting H structures to
the appropriate antigen, A2 enzymes would
probably fail completely when paired with a B
enzyme.
- A2B individuals would be far more likely to
lack Ac and Ad components with subsequent
production of anti-Ac and anti-Ad (anti-A1).
• Newborns have a deficiency of the branched H3
and H4 antigens and therefore the Ac and Ad
antigens as well, possibly accounting for the A2
phenotype.
• Adult cells contain a higher concentration of
branched H3 and H4 structures and therefore Ac
and Ad determinants of the A antigen in A1
individuals.

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 LECTURE

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 LECTURE
WEAK A SUBGROUPS
- Subgroups weaker than A2 occur infrequently
and are most often recognized through an ABO
discrepancy.
- make up 1% of those encountered in the
laboratory and therefore are mainly of
academic interest.
- Characteristics of weak A subgroups include
the following:
➢ Decreased number of A antigen sites per
RBC (resulting in weak or no
agglutination with human polyclonal
anti-A)
➢ Varying degrees of agglutination by
human anti-A,B
➢ Increased variability in the detectability
of H antigen, resulting in strong
reactions with anti-H
➢ Presence or absence of anti-A1 in the
serum
- Secretor studies, adsorption-elution tests, and
molecular testing can be utilized to subdivide
A individuals into A3, Ax, Aend, etc.
- may present practical problems.
➢ if an Ax donor was mistyped as group O
and was transfused to a group O patient.
➢ potentially dangerous because the group
O patient possesses anti-A,B, which
agglutinates and lyses Ax RBCs, causing
rapid intravascular hemolysis.
- can be serologically differentiated using the
following techniques:
➢ Forward grouping of A and H antigens
with anti-A, anti-A,B, and anti-H
➢ Reverse grouping of ABO isoagglutinins
and the presence of anti-A1
➢ Adsorption-elution tests with anti-A
➢ Saliva studies to detect the presence of A
and H substances
- molecular testing for mutations or serum
glycosyltransferase studies for detecting the A
enzyme can be performed for differentiation
of weak subgroups.
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- Absence of a disease process should be
confirmed before subgroup investigation
because ABH antigens are altered in various
malignancies and other hematologic
disorders.
- can be distinguished as A3, Ax, Aend, Am, Ay, and
Ael using the serologic techniques.
Mixed field
- defined as small agglutinates within
predominantly unagglutinated RBCs.
- A3 enzyme is a product of an allele at the ABO
locus inherited in a dominant manner;
however, the A3 blood group has been
reported to be very heterogeneous at the
molecular level.
- Anti-A1 may be present in serum of A3
individuals, and A substance is detected in the
saliva of A3 secretors.
• Ax RBCs characteristically are not agglutinated by
anti-A reagent but do agglutinate with most
examples of anti-A,B.
• Anti-A can be adsorbed and then eluted from Ax
cells without difficulty.
• A transferase is not usually detectable in the
serum or in the RBC membranes of Ax individuals.
• The molecular genetics of Ax reflect the
considerable heterogeneity of the serologic
phenotypes. Ax individuals almost always produce
anti-A1 in their serum.
• Routine secretor studies detect the presence of
only H substance in Ax secretors.
- Ax secretors contain A substance detectable
only by agglutination/ inhibition studies using
Ax RBCs as indicators.
• Aend RBCs characteristically demonstrate very
weak mixed-field agglutination with some anti-A
and anti-A,B reagents.
- No A glycosyltransferase is detectable in the
serum or in the RBC membranes of Aend
individuals.
- Aend is inherited as an allele at the ABO locus.
 LECTURE

- Secretor studies detect the presence of only H - Ael individuals usually produce an anti-A1 that
substance in the saliva of Aend secretors. Anti- is reactive with A1 cells and sometimes
A1 is found in some Aend sera. produce anti-A, which agglutinates A2 RBCs.
• Am RBCs are characteristically not agglutinated, or • It is assumed that the patient’s medical history,
are agglutinated only weakly, by anti-A or anti-A,B such as recent transfusion, pregnancy history,
reagents. disease states, and medications, has been
investigated and excluded as a source of the
- A strongly positive adsorption or elution of
discrepancy.
anti-A confirms the presence of A antigen sites.
- An A enzyme of either the A1 or A2 type
previously described is detectable in the
serum of Am subgroups.
- usually do not produce anti-A1 in their sera.
- Normal quantities of A and H substance are
found in the saliva of Am secretors.
• Ay RBCs are not agglutinated by anti-A or anti-A,B
reagents.
- Adsorption and elution of anti-A is the method
used to confirm the presence of A antigens.
- Activity of eluates from Ay RBCs is
characteristically weaker than that of eluates
from Am RBCs.
- Trace amounts of A glycosyltransferase is
detectable in the serum of Ay individuals, and
saliva secretor studies demonstrate H and A
substance, with A substance present in below-
normal quantities.
- usually do not produce anti-A1.
WEAK B SUBGROUPS
- Ay phenotype can be observed in siblings,
implicating a recessive mode of inheritance. - very rare and much less frequent than A
subgroups.
➢ phenotype does not represent
expression of an alternate allele at the - usually recognized by variations in reaction
ABO locus but rather as a germline strength using anti-B and anti-A,B.
mutation of an A gene within a family.
- Inheritance of B subgroups is considered to be
• Ael RBCs typically are unagglutinated by anti-A or a result of alternate alleles at the B locus.
anti-A,B reagents.
- Criteria used for differentiation of weak B
- adsorption and elution can be used to phenotypes include the following techniques:
demonstrate the presence of the A antigen.
➢ Strength and type of agglutination with
- No detectable A enzyme activity can be anti-B, anti-A,B, and anti-H
demonstrated in the serum or in the RBC
➢ Presence or absence of ABO
membranes of Ael individuals by
isoagglutinins in the serum
glycosyltransferase studies.
➢ Adsorption-elution studies with anti-B
- Ael phenotype is inherited as a rare gene at the
ABO locus. ➢ Presence of B substance in saliva

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 LECTURE

➢ Molecular testing B plasma incubated with Bm

RBCs and uracil diphosphate
- RBCs demonstrating serologic activity that is
(UDP)-galactose transforms this
weaker than normal are designated weak B
subgroup into a normal group B
phenotypes or B subgroups and include B3, Bx,
phenotype.
Bm, and Bel phenotypes.
✓ Bm phenotype is usually the
- Serologic techniques can be used to
result of inheritance of a rare
characterize B subgroups in the following
allele at the ABO locus, although
categories: B3, Bx, Bm, and Bel.
the subgroup Bm may be the
➢ B3 phenotype generally results from the product of an interacting
inheritance of a rare gene at the ABO modifying gene linked closely to
locus and is characterized by a mixed- the ABO locus.
field pattern of agglutination with anti-B
✓ Bm subgroup is reported to be
and anti-A,B antisera.
more frequent in Japan.
✓ B glycosyltransferase is present
✓ Normal quantities of H and B
in the serum but not in the RBC
substance are found in the saliva
membranes of these individuals.
of Bm secretors.
Anti-B is absent in the serum of
B3 phenotypes, yet B substance ➢ Bel RBCs are unagglutinated by anti-B or
is present in normal amounts in anti-A,B antisera.
the saliva of secretors.
✓ extremely rare phenotype must
✓ B3 subgroup is the most be determined by adsorption
frequent weak B phenotype. and elution of anti-B.
➢ Bx RBCs typically demonstrate weak ✓ Bel is inherited as a unique
agglutination with anti-B and anti-A,B mutation in exon 7 of the B
antisera. gene at the ABO locus.
✓ B glycosyltransferase has not ✓ Only H substance is
been detected in the serum or in demonstrated in saliva of Bel
the RBC membranes of Bx secretors.
phenotypes, but a weakly
reactive anti-B usually is
produced.
✓ Family studies suggest that Bx is
a rare allele at the ABO locus.
➢ Bm RBCs are characteristically
unagglutinated by anti-B or anti-A,B
antisera.
✓ Bm RBCs easily adsorb and elute
anti-B. B glycosyltransferase is THE BOMBAY PHENOTYPES (Oh)
present in the serum of Bm - first reported by Bhende in 1952 in Bombay,
phenotypes but is usually lower India.
in activity and varies from
individual to individual. - phenotype results from the inheritance of a
double dose of the h gene, producing the very
✓ Reduced activity of B enzyme in rare genotype hh.
hematopoietic tissue is clearly
the defect causing the formation
of the Bm subgroup, since normal

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 LECTURE

- the ABO genes cannot be expressed and ABH

antigens cannot be formed, since there is no H
antigen made in the Bombay phenotype.
- The (Oh) Bombay phenotype is inherited as
an autosomal recessive trait.
- underlying molecular defect is most often a
mutation in the gene FUT1 (H gene),
producing a silenced gene incapable of coding
for the enzyme, α-2-L-fucosyltransferase (H
transferase).
➢ enzyme normally catalyzes the transfer
of L-fucose in an α-1,2 linkage to the
terminal galactose of the precursor
molecule on RBCs forming the H antigen.
➢ mutation underlying the Bombay
phenotype is also associated with a
silenced FUT2 gene (Se gene), which
usually encodes a very similar α-2-L-
fucosyltransferase that when active,
transfers a fucose to form H antigens
in secretions.
- Since these RBCs lack normal ABH antigens,
they fail to react with anti-A, anti-B, and anti-H
antisera. In RBC testing using anti-A and anti-
B, the Bombay would phenotype as an O blood
group.
THE PARA-BOMBAY PHENOTYPES
- RBCs of the Bombay phenotype (Oh) do not
react with the anti-H lectin (Ulex europaeus), - those rare phenotypes in which the RBCs
unlike those of the normal group O individual, either completely lack H antigens or have
which react strongly with anti-H lectin. small amounts of H antigen present.
- Bombay serum contains anti-A, anti-B, anti- - The genetic basis for the para-Bombay
A,B, and anti-H phenotype is either a mutated FUT1 (H gene)
with or without an active FUT2 gene (Se
- Bombay anti-H can often be potent and
gene) or a silenced FUT1 gene with an
reacts strongly at 37°C.
active FUT2 gene.
➢ It is an IgM antibody capable of binding
- With the mutated FUT1 gene para-Bombay,
complement and causing RBC lysis.
the encoded α-2-L-fucosyltransferase enzyme
- Transfusing normal group O blood (with the activity is greatly reduced, and very small
highest concentration of H antigen) to a amounts of H, A, and B antigens are produced.
Bombay recipient (anti-H in the serum) would
- RBCs from these individuals expressing weak
cause immediate cell lysis.
forms of A and B antigens are detected only
- Only blood from another Bombay individual using adsorption and elution techniques.
will be compatible and can be transfused to a
- If a person is genetically A or B, the respective
Bombay recipient.
enzymes can be detected, but no H enzyme
- ABH substance is also absent in the saliva of is detectable, even though it has been shown
individuals with the Bombay phenotype. that there is limited production of H antigen on
the RBC.
15 | P a g e
 LECTURE
➢ The notations Ah and Bh, respectively,
have been used to describe these
individuals.
- ABh individuals have also been reported. Ah,
Bh, and ABh have been reported mainly in
individuals of European origin.
➢ No H, A, or B antigen is present in the
saliva, yet anti-H is present in the serum.
- The serum of Ah individuals contains anti-B
and no anti-A, although anti-A1 is usually
present.
- In Bh serum, anti-A is always present and anti-
B may be detected.
➢ postulated that homozygous inheritance
of a mutant H (FUT1) gene codes for the
production of low levels of H transferase
activity.
➢ The small amount of H substance on the
RBC is completely consumed by the A or
B transferase present, resulting in small
quantities of A or B antigen being
present on the RBC with no detectable H
antigen.
➢ The anti-H present in the serum is
weaker in reactivity than the anti-H
found in the Bombay phenotype,
although it may be reactive at 37°C.
- the silenced FUT1 gene with the active FUT2
gene para-Bombays, the α-2-L-
fucosyltransferase enzyme asso ciated with
the FUT2 gene (α2FucT2) produces H, A, and B
type 1 antigens in secretions, including
plasma.
➢ type 1 antigens in the plasma may
adsorb onto the RBC membrane.
- RBCs have little or no A, B, and H antigens.
RBCs of Oh secretors are not agglutinated by
most examples of anti-H but may be
agglutinated by strong anti-H reagents.
➢ Adsorption and elution of anti-H may
reveal the presence of some H anti gen
on the RBC.
- Cells are not usually agglutinated by anti-A
and anti-B; however, some Oh A RBCs can
mimic the behavior of Ax cells and can be
16 | P a g e
agglutinated by anti-A,B and potent examples
of anti-A.
Anti-IH
- weak H-like antibody that is reactive at low
temperature is almost always present in the
serum.
- antibody is nonreactive with cord cells and is
not inhibited by secretor saliva.
- normal levels of H substances are present in
the saliva.
- A and B substances are present in the
secretions when A and B genes are present.
ABH ANTIGENS AND ANTIBODIES IN DISEASE
- “hangover” in A blood group
- “criminality” in group B blood groups
- “good teeth” in group O individuals
- Various disease states seem to alter RBC
antigens, resulting in either progressively
weaker reactions or additional acquired
pseudoantigens, which can be seen during
forward grouping.
- induces stress hematopoiesis (e.g.,
thalassemia) have been shown to depress
antigen strength.
➢ Leukemia
➢ chromosome 9 translocations
➢ any hemolytic disease
Mixed-field Agglutination
- tiny agglutinates in a sea of unagglutinated
cells.
Hodgkin’s disease
- reported to weaken or depress ABH RBC
antigens, resulting in variable reactions during
forward grouping similar to those found in
leukemia.
 LECTURE

• The isoagglutinins (anti-A, anti-B, or anti-A,B) also

may be weak or absent in leukemias
TECHNICAL ERRORS
demonstrating hypogammaglobulinemia.
- can also cause ABO discrepancies
➢ chronic lymphocytic leukemia (CLL)
➢ blood sample and test tube labeling
- Various lymphomas, such as the malignant
errors
(non-Hodgkin’s) variety, may yield weak
isoagglutinins, owing to moderate ➢ failure to add reagents
decreases in the gamma globulin fraction.
➢ addition of incorrect reagents or sample
- Immunodeficiency diseases, such as
congenital agammaglobulinemia, will also - Serum and antiserum should always be added
yield weak or absent isoagglutinins. first, followed by the patient or reagent RBCs
to avoid both reagent contamination and
➢ simple serum protein electrophoresis potential omission of either patient sample or
will confirm or rule out this condition. reagent.
- Individuals with intestinal obstruction, - Results must be recorded immediately after
carcinoma of the colon or rectum, or other obtaining them to avoid transcription errors.
disorders of the lower intestinal tract may
have increased permeability of the intestinal - Always examine reagent vials concurrently
wall, which allows passage of the bacterial while performing ABO testing and quality
polysaccharides from Escherichia coli control testing for possible contamination.
serotype O86 into the patient’s circulation and
results in the acquired B phenomenon in
group A1 individuals.
- The patient’s group A RBCs absorb the B-like
polysaccharide, which reacts with human
source anti-B.
- A lack of detectable ABO antigens can occur in
patients with carcinoma of the stomach or
pancreas.
➢ The patient’s red blood cell antigens
have not been changed, but the serum
contains excessive amounts of blood
group–specific soluble substances
(BGSS) that may neutralize the antisera
utilized in the forward grouping.
RESOLUTION
- If initial testing was performed using RBCs
ABO DISCREPANCIES suspended in serum or plasma
- occur when unexpected reactions are obtained ➢ repeat testing of the same sample using
in the forward and/or reverse grouping. a saline suspension of RBCs can usually
- can be due to problems with the patient’s resolve the ABO discrepancy
serum (reverse grouping), problems with the - It is important to make sure any and all
patient’s RBCs (forward grouping), or technical factors that may have given rise to
problems with both the serum and cells. the ABO discrepancy are reviewed and
- The unexpected reaction(s) may be due to an corrected.
extra positive reaction or a weak or missing - It is also essential to obtain adequate
reaction in the forward and reverse grouping. information regarding the patient’s age,
17 | P a g e
 LECTURE
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
18 | P a g e
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.
 LECTURE
CATEGORIES OF ABO DISCREPANCIES
- ABO discrepancies may be arbitrarily divided
into four major categories.
A. GROUP I DISCREPANCIES
- associated with unexpected reactions in the
reverse grouping due to weakly reacting or
missing antibodies.
- more common than those in the other groups
- When a reaction in the serum grouping is weak
or missing, a group I discrepancy should be
suspected because, normally, RBC and serum
grouping reactions are very strong (4+).
- One of the reasons for the missing or weak
isoagglutinins is that the patient has
depressed antibody production or cannot
produce the ABO antibodies.
- Common populations with discrepancies in
this group are:
➢ Newborns (ABO antibody production is
not detectable until 3 to 6 months of age)
➢ Elderly patients (production of ABO
antibodies is depressed)
➢ Patients with a leukemia (e.g., chronic
lymphocytic leukemia) or lymphoma
(e.g., malignant lymphoma)
demonstrating
hypogammaglobulinemia
➢ Patients using immunosuppressive
drugs that yield
hypogammaglobulinemia
➢ Patients with congenital or acquired
agammaglobulinemia or
immunodeficiency diseases
➢ Patients with bone marrow or
hematopoietic progenitor stem cell
(HPC) transplants (patients develop
hypogammaglobulinemia from therapy
and start producing a different RBC
population from that of the transplanted
bone marrow)
➢ Patients whose existing ABO
antibodies may have been diluted by
19 | P a g e
plasma transfusion or exchange
transfusion
➢ ABO subgroups
• Resolution of Common Group I Discrepancies
- Obtaining patient clinical history may
immediately resolve this type of discrepancy.
- If the history indicates the patient is an elderly
individual or has a diagnosis indicative of
hypogammaglobulinemia
➢ enhance the weak or missing reaction
➢ incubating the patient serum with
reagent A1 and B cells at room
temperature for approximately 15 to
30 minutes.
- If there is still no reaction after centrifugation
➢ serum-cell mixtures can be incubated
at 4°C for 15 to 30 minutes.
- An auto control and O cell control must
always be tested concurrently with the
reverse typing when trying to solve the
discrepancy since the lower temperature of
testing will most likely enhance the reactivity
of other commonly occurring cold agglutinins
- The RBC results present a group O individual
and the serum results indicate an AB
individual.
➢ Since serum problems are more
common, it is more likely that the serum
immunoglobulins are decreased.
Mixed-field Agglutination
- may look like small to large agglutinates with
unagglutinated cells.
- appear as a “halo” or “puff of smoke” of
unagglutinated RBCs as the RBC button is
dislodged from the test tube bottom.
- Common causes:
➢ receiving non-ABO-type specific RBCs
➢ ABO subgroups (A3)
 LECTURE
➢ bone marrow or hematopoietic stem cell
transplantation.
- it is imperative to investigate the patient’s
transfusion records and clinical history to
verify the cause of the mixed-field
agglutination.
Chimerism
- defined as the presence of two cell populations
in a single individual.
- was discovered in twins born to a group O
mother and group B father with a mixture of
both B and O cells instead of the expected
group of either B or O.
True Chimerism
- occurs only in twins and is rarely found.
- two cell populations will exist throughout the
lives of the individuals.
Utero exchange of blood = vascular anastomosis
- two cell populations emerge, both of which are
recognized as self, and the individuals do not
make anti-A or anti-B.
Dispermy
- two sperm fertilizing one egg
- cause if the patient or donor has no history of
a twin and indicates mosaicism.
• More commonly, artificial chimeras occur, which
yield mixed cell populations as a result of the
following:
➢ Blood transfusions (e.g., group O cells
given to an A or B patient)
➢ Transplanted bone marrow or
hematopoietic progenitor stem cells
(HPC) of a different ABO type
20 | P a g e
➢ Exchange transfusions
➢ Fetal-maternal bleeding
HPC Engraftment
- unrestrained in the company of circulating
ABO antibodies since pluripotent and early
committed HPCs lack ABO antigens.
Major ABO Incompatibility
- occurs when the donor’s red RBCs are
incompatible with the recipient’s plasma
- the donor is group B and the recipient is group
O with naturally occurring anti-B
- complications:
➢ hemolysis of RBCs at time of infusion
and continued antibody production
directed against erythroid progenitors
and donor RBCs by the engrafted HPCs.
Minor ABO Incompatibility
- occurs when the donor’s plasma is
incompatible with the recipient’s RBCs
- the donor is group O with naturally occurring
anti-B and the recipient is group B
Bidirectional ABO Incompatibility
- occurs when both a major and minor
incompatibility are present
- the donor is group A and the recipient is group
B
• Following an ABO-incompatible HPC transplant,
the pretransplant ABO type will remain in these
tissues for the rest of the patient’s life.
• the patient will never make antibody against the
ABO type the body still sees as self (e.g., a group B
recipient converted to group A will never make
anti-B).
 LECTURE
• RBCs, platelets, and plasma products must be
compatible with both the donor and recipient
blood types.
GROUP II DISCREPANCIES
- associated with unexpected reactions in the
forward grouping due to weakly reacting or
missing antigens.
- the least frequently encountered
- some of the causes of discrepancies in this
group:
➢ Subgroups of A or B may be present
(see the “ABO Subgroups” section).
➢ Leukemias may yield weakened A or B
antigens, and Hodgkin’s disease has
been reported in some cases to mimic
the depression of antigens found in
leukemia.
➢ The “acquired B” phenomenon will
show weak reactions with anti-B
antisera and is most often associated
with diseases of the digestive tract (e.g.,
cancer of the colon).
• Resolution of Common Group II Discrepancies
- The agglutination of weakly reactive antigens
with the reagent antisera
➢ enhanced by incubating the test mixture
at room temperature for up to 30
minutes to increase the association of
the antibody with the RBC antigen.
- If the reaction is still negative
21 | P a g e
➢ incubate the text mixture at 4°C for 15
to 30 minutes
- Include group O and autologous cells as
controls.
- RBCs can also be pretreated with enzymes and
retested with reagent antisera.
- The acquired B antigen arises when bacterial
enzymes modify the immunodominant blood
group A sugar (N-acetyl-D-galactosamine)
into D-galactosamine, which is sufficiently
similar to the group B sugar (D-galactose) to
cross-react with anti-B antisera.
Pseudo-B antigen
- formed at the expense of the A1 antigen and
disappears following the patient’s recovery.
- reaction of the appropriate antiserum with
these acquired antigens demonstrates a weak
reaction, often yielding a mixed-field
appearance.
• Blood group reagents of a monoclonal anti-B
clone (ES4) strongly agglutinate cells with the
acquired B antigen.
- pH of reagents containing ES4 has been
lowered, and consequently, only those cells
with the strongest examples of acquired B
antigen react with the antisera.
- Testing the patient’s serum or plasma against
autologous RBCs gives a negative re action,
because the anti-B in the serum does not
agglutinate the patient’s RBCs with the
acquired B antigen.
- The acquired B antigen is also not agglutinated
when reacted with anti-B that has a pH greater
than 8.5 or less than 6.
• Secretor studies can be performed when trying
to characterize the acquired B phenomenon.
- If the patient is in fact a secretor, only the A
substance is secreted in the acquired B
phenomenon.
• Treating RBCs with acetic anhydride reacetylates
the surface molecules, then markedly decreases
the reactivity of the cells tested with anti-B.
 LECTURE

- The reactivity of normal B cells is not free of contaminating antibodies to low-

affected by treatment with acetic prevalence antigens.
anhydride.

• Rare Group II Discrepancies

- Weakly reactive or missing reactions in RBC
grouping
GROUP III DISCREPANCIES
➢ excess amounts of blood group–
specific soluble (BGSS) substances - discrepancies between forward and reverse
present in the plasma, which sometimes groupings are caused by protein or plasma
occurs with certain diseases. abnormalities and result in rouleaux
formation or pseudoagglutination,
- Excess amounts of BGSS substances will attributable to the following:
neutralize the reagent anti-A or anti-B,
leaving no unbound antibody to react with ➢ Elevated levels of globulin from certain
the patient cells and yields a false-negative or disease states, such as multiple
weak reaction in the forward grouping. myeloma, Waldenström’s
macroglobulinemia, other plasma cell
- Washing the patient cells free of the BGSS dyscrasias, and certain moderately
substances with saline should alleviate the advanced cases of Hodgkin’s lymphomas
problem, resulting in correlating forward and
reverse groupings. ➢ Elevated levels of fibrinogen
- Antibodies to low-prevalence antigens in ➢ Plasma expanders, such as dextran
reagent anti-A or anti-B can also cause weakly and polyvinylpyrrolidone
reactive or missing reactions in RBC grouping.
➢ Wharton’s jelly in cord blood samples
- additional antibody in the reagent antisera has
➢ ABO discrepancy caused by rouleaux
reacted with the corresponding low-
formation
prevalence antigen present on the patient’s
RBCs.
➢ produces an unexpected reaction of the
patient’s cells with anti-A or anti-B or
both, mimicking the presence of a weak
antigen.
• Resolution of Common III Discrepncies
➢ repeating the forward type, using
antisera with a different lot number. - Rouleaux
- If the cause of the discrepancy is indeed a low- ➢ a stacking of erythrocytes that adhere in
prevalence antibody in the reagent antisera a coin-like fashion, giving the
reacting with a low-prevalence antigen on the appearance of agglutination.
patient’s cells
- Cell grouping can usually be accomplished by
➢ the antibody will most likely not be washing the patient’s RBCs several times with
present in a different lot number of saline.
reagent.
- Performing a saline replacement technique
- phenomenon is only seen when human source will free the cells in the case of rouleaux
antiserum is used. formation in the reverse type.
- Most ABO reagents in use today are ➢ serum is removed and replaced by an
monoclonal antibodies and these reagents are equal volume of saline.

22 | P a g e
 LECTURE

- In true agglutination, RBC clumping will still

remain after the addition of saline.
- Cord blood samples can pose a problem in
ABO testing, since cord cells may be
contaminated with a substance called
Wharton’s jelly.
➢ a viscous mucopolysaccharide material
present on cord blood cells that may
cause the red blood cells’ aggregation.
- 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.

• Resolution of Common Group IV Discrepancies

GROUP IV DISCREPANCIES
- discrepancies between forward and reverse
groupings are due to miscellaneous problems
that have the following causes:
➢ 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
- Potent cold autoantibodies can cause
spontaneous agglutination of the patient’s
cells.
- If the antibody in the serum reacts with all
adult cells
➢ the reagent A1 and B cells used in the
reverse grouping also agglutinate.
- the patient’s RBCs could be incubated at 37°C
for a short period, then washed with saline at
37°C three times and retyped.
- If this is not successful in resolving the
forward type
➢ the patient’s RBCs can be treated with
0.01 M dithiothreitol (DTT) to
disperse IgM-related agglutination.
➢ reagent RBCs and serum can be warmed
to 37°C for 10 to 15 minutes, mixed,
tested, and read at 37°C.
➢ test can be converted to the antihuman
globulin phase if necessary.
- Weakly reactive anti-A or anti-B may not react
at 37°C, which is outside their optimum
thermal range.
- If the reverse typing is still negative (and a
positive result was expected)

23 | P a g e
 LECTURE
➢ a cold autoabsorption (patient cells
with patient serum) could be performed
to remove the cold autoantibody from
the serum.
➢ The absorbed serum can then be used to
repeat the serum typing at room
temperature.
- Unexpected ABO isoagglutinins in the patient’s
serum react at room temperature with the
corresponding antigen present on the reagent
cells.
- Examples:
➢ A2 and A2B individuals
➢ who can produce naturally occurring
anti-A1, or A1 and A1B
➢ individuals who may produce naturally
occurring anti-H.
- Serum grouping can be repeated using at least
three examples of A1, A2, B cells; O cells; and
an autologous control (patient’s serum mixed
with patient’s RBCs).
- The specificity of the antibody can be
determined by examining the pattern of
reactivity.
- The patient’s RBCs can be tested with
Dolichos biflorus to confirm the presence of
the ABO subgroup.
➢ Dolichos biflorus will agglutinate cells
of the A1 but not the A2 phenotype.
- Unexpected alloantibodies in the patient’s
serum other than ABO isoagglutinins (e.g.,
anti-M) may cause a discrepancy in the reverse
grouping.
- Reverse grouping cells possess other antigens
in addition to A1 and B, and it is possible that
other unexpected antibodies present in the
patient’s serum will react with these cells.
➢ an antibody identification panel should
be performed with the patient’s serum.
➢ Once the unexpected alloantibodies are
identified, reagent A1 and B cells
negative for the corresponding antigen
should be used in the reverse grouping.
24 | P a g e
• Rare Group IV Discrepancies
- Antibodies other than anti-A and anti-B may
react to form antigen-antibody complexes that
may then adsorb onto patient’s RBCs.
➢ an individual with an antibody against
acriflavine, the yellow dye used in some
commercial anti-B reagents.
➢ The acriflavine antiacriflavine
complex attaches to the patient’s RBCs,
causing agglutination in the forward
type.
➢ Washing the patient’s cells three times
with saline and then retyping them
should resolve this discrepancy.
Cis-AB
- inheritance of both AB genes from one parent
carried on one chromosome and an O gene
inherited from the other parent.
- results in the offspring inheriting three ABO
genes instead of two.
- used to distinguish this mode of in heritance
from the more usual AB phenotype in which
the alleles are located on different
chromosomes.
- first discovered in 1964, when a Polish family
was described in which the father was group O
and the mother was group AB and gave birth
to children who were all group AB.
- was resolved by the fact that the A and B genes
were inherited together and were both on the
same, or cis, chromosome.
- RBCs with the cis-AB phenotype (a rare
occurrence) express a weakly reactive A
 LECTURE
antigen (analogous to A2 cells) and a weak B
antigen
- The B antigen usually yields a weaker reaction
with the anti-B from random donors, with
mixed-field agglutination typical of subgroup
B3 reported in several cases.
25 | P a g e
- Weak anti-B (present in the serum of most cis-
AB individuals) leads to an ABO discrepancy in
the reverse grouping.
- The serum of most cis-AB individuals contains
a weak anti-B, which reacts with all ordinary B
RBCs, yet not with cis-AB RBCs.
- A and B transferase levels are lower than those
found in ordinary group AB sera.
 LECTURE

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 LECTURE

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