The ABO Blood Group System

PREPARED BY: MA ENRICA T SANDICO, RMT MLS

MODIFIED BY: LEA ANNE D. BUCUD, RMT, MPH
COMPILED BY: DENISE M. HARMENING ,RMT, PHD, ASCP
 The ABO Blood Group System

 Most important blood group system in

transfusion practice
 Individuals have antibodies in their
serum to antigens that are absent
from their RBCs without any exposure
to RBCs through
transfusion/pregnancy
 Incompatible ABO type = immediate
lysis of donor RBCs
 Severe, fatal transfusion reaction
Transfusion-Related Fatalities by
Complication, FY 2009
COMPLICATION NUMBER FY09

TRALI 13* 30%

HTR (non-ABO) 8 18%

HTR (ABO) 4 9%

Microbial infection 5 11%

TACO 12 27%

Anaphylaxis 1 2%

Other 1** 2%
 Causes of Fatal Hemolytic Transfusion Reactions due
to ABO Incompatible Blood Transfusions, FY 2009

 Case 1: Recipient Identification error at the time of transfusion (nursing

error)

 Case 2: Patient sample labels switched (phlebotomist error)

 Case 3: Sample collected from incorrect patient (phlebotomist error)

 Case 4: Patient sample mistyped (lab error)

ABO Forward Grouping: Detection of Antigens on
Patient’s RBCs with known commercial antisera
Patient RBCs Patient RBCs Interpretation
with Anti-A with Anti-B of Blood
Group
- - O
4+ - A
- 4+ B
4+ 4+ AB
ABO Reverse Grouping: Detection of Antigens on
Patient’s RBCs with known commercial antisera

Patient Serum Patient Serum Interpretation of

with reagent A1 with Reagent B Blood Group
cells cells
4+ 4+ O
0 3+ A
3+ - B
- - AB
RECIPIENT DONOR
ABO WHOLE RED PLASMA
PHENOTY BLOOD BLOOD
PE CELLS
A A A,O A,AB
B B B,O B,AB
AB AB AB,A,B,O AB
O O O O,A,B,A
B
 ABO Phenotype frequencies of Ethnic Groups

Phenotyp Whites Blacks Hispanic Asian

es
O 45 50 56 40
A 40 26 31 28
B 11 20 10 25
AB 4 4 3 7
ABO Antibodies

 Naturally
occurring= produced without
any exposure to red blood cells.
 Predominantly IgM
 Activate complement
 Reacts at room/cold temperature
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ABO Antibodies

 Time of appearance:
 Generally present within first 4-6 months of life
 Reach adult level at 5-10 years of age
 Level off through adult life
 Begin to decrease in later years: >65 years of age
 Inheritance of the ABO Blood Groups

 Described by Bernstein in 1924

 An individual inherits one ABO gene
from each parent and that these two
genes determine which ABO antigens
are present on the RBC membrane.
 Follows simple Mendelian genetics
 O gene = amorph ; 2 O genes that
are nonfunctional, autosomal
recessive inheritance
 Groups A, B = Phenotypes
 AA, BB, OO = genotypes
 Formation of the A, B and H Antigens
 Results from interaction of genes at
three specific loci (ABO, Hh, Se) which
produce glycosyltransferases
 Enzymes that adds sugar to basic
precursor substance
 Paragloboside, type 2 chain
 Beta 1-4 linkage
 H Antigen – precursor structure
 H and Se genes = Chromosome 19
 ABO genes = chromosome 9
Type 2: Beta 1-4 linkage

 Terminalgalactose on the precursor

substance is attached to N-acetyl
glucosamine in beta 1-4 linkage
 Page 124, figure 6-3
RBC Precursor Structure

RBC

Glucose

Galactose
Precursor
Substance
(stays the N-acetylglucosamine
same)
Galactose
H antigen

 Precursor structure on which A and B antigens are made.

 Inheritance of the H gene results in the formation of H antigen.
 H and Se genes are closely linked located on chromosome 19,
these are not part of the ABO system but inheritance influences
A and B antigen expressions
 H gene must be inherited to form ABO antigens on the RBCs
 Se gene must be inherited to form the ABO antigens in
secretions
 Page 124, table 6-9
 Interaction of the A, B and H Antigens

 Group O = one H gene and 2 O  H gene = “h” allele – rare; “hh”,

genes
 H gene = a-2-L-fucosyltransferase,
present in 99.99% of the population
 Transfer a sugar responsible for H
specificity, L- fucose
 O (amorph) – does not elicit
production of a catalytically active
polypeptide transferase, no
modification
extremely rare
 Bombay – phenotype that lacks
normal expression of ABH antigens
because of “hh” inheritance
 No a-2-l fucosyltransfersase, no L-
fucose, no H substance on RBCs.
Formation of the H antigen

RBC

Glucose

H antigen Galactose

N-acetylglucosamine

Galactose

Fucose
Formation of the A antigen

RBC

Glucose

Galactose

N-acetylglucosamine

Galactose

N-acetylgalactosamine
Fucose
Formation of the B antigen

RBC

Glucose

Galactose

N-acetylglucosamine

Galactose

Galactose
Fucose
 Glycosyltransferases and Immunodominant
Sugars
Gene Glycosyltransferase Immunodominant Antigen
Sugar
H a-2-L fucosyltransferase L-fucose H
A a-3-N- N- A
acetylgalactosaminyltra acetylgalactosami
nsferase ne
B a-3-D- D-galactose B
galactosyltransferase
 A, B, A,B genes

 A genes = elicit higher concentrations

of transferase than B gene
 810,000 -1,170,000 antigen sites on A1
adult
 B gene = 610,000 – 830,00 antigen
sites on B adult RBC
 AB genes – B competes for more H
substance than A
 A- 600,000; B -700,000
 A,B and H Soluble Antigens

 Can be found in body secretions

 Presence dependent on ABO genes ABO Group A B H
inherited and inheritance of Sese O None None ++
genes
A ++ None +
 “Secretors” – SeSe or Sese B None + +
 Codes for production of a-2-L AB ++ ++ +
fucosyltransferase, modifies type 1
precursor substance to form H
substance.
 Comparison of A,B and H Antigens on
RBCS with A,B and H Soluble Substances
ABH Antigens on RBCS
RBC antigens can be glycolipids,
glycoproteins, or glycosphingolipids
Synthesized only on type 2 precursor chains
Type 2 chain refers to a beta 1-4 linkage in
which the carbon 1 of galactose is attached
to the 4th carbon of N acetylglucosamine
sugar of the precursor substance
Enzyme produced by the H gene acts on
type 2 chains which are prevalent in RBC
membrane.
A,B, and H Soluble Substances
Secreted substances are glycoproteins
Type 1 precursor chains
Type 1 chain refers to beta 1-3 linkage on
which carbon 1 of galactose is attached to
the 3rd carbon on N-acetylglucosamine sugar
of the precursor substance
Enzyme produced by Se gene acts on type 1
chains in secretory tissues
Fluids in which A, B and H Substances can
be Detected in Secretions
Body Fluids
Saliva
Tears
Urine
Digestive juices
Bile
Milk, Amniotic fluid, Pathological fluids
 A Subgroups
 Classification: 99% of all group A1 and
 Von Dungern described two A2
different A antigens based on  A1 = 80%
reactions between group A  A2 = 20 %
RBCs with both Anti-A and Anti-  A1 gene inheritance: high
A1 concentrations of A enzyme
 Generally more common than  810,000 – 1,170,000 antigen sites
B subgroups  A2 gene inheritance: 240,000 – 290,000
antigen sites; a2 allele –single base
 Attributed to decreased substitution at nucleotide 467 and a
number of red cells single base substitution at nucleotide
1059
A1versus A2 PHENOTYPES

 REACTIONS OF PATIENT’S RED BLOOD CELLS WITH

BLOOD GROUP ANTI-A1 REAGENT ANTI-A1 LECTIN

(ANTI-A PLUS ANTI-A1) REAGENT

A1 (+) (+)

A2 (+) 0
 Qualitative and Quantitative
Differences of Subgroups A1 and A2
Quantitative Qualitative

• Decreased number of antigen • Differences in the precursor

sites oligosaccharide chains

• Decreased amount of • Subtle differences in transferase

transferase enzyme enzymes

• Decreased amount of branching • Formation of anti-A1, in a

percentage of some subgroups
REACTIVITY OF ANTI-H ANTISERA AND ANTI-H
LECTIN WITH ABO BLOOD GROUPS

 O-A2-B-A2B-A1-A1B
 GREATEST-LEAST
 PAGE 129, TABLE 6-14
 Lectins used in Blood Banking

Lectins
Dolichos biflorus – agglutinates A1 or A1B

Bandereia simplicifolia – agglutinates B cells

Ulex europaeus – agglutinates O cells and other ABO blood

groups depending on the amount of H antigen available.
 Weak A Subgroup Characteristics
 Decreased number of A antigen sites  Tests to subdivide A individuals
per RBC include:
 Varying degrees of agglutination by  Secretor studies
anti A,B
 Adsorption-elution studies
 Increased variability in the  Molecular testing
detectability of H antigen, resulting in
strong reactions with anti-H  Forward Grouping of A and H
antigens with anti-A, anti-A,B and
 Presence or absence of Anti-A1 in anti-H
serum
 Reverse grouping of ABO
isoagglutinins and the presence of
anti-A1 and saliva studies
CHARACTERISTICS OF WEAK ABO-PHENOTYPES

 PAGE 131, TABLE 6-15

 Weak A Subgroups
 A3, Ax, Aend, Am, Ay, Ael
 A3 RBCs: mixed field pattern of  Ax RBCS: not agglutinated with anti-A
agglutination with anti-A and most reagent but do agglutunate with anti
anti A,B reagents A,B
 Weak transferase detected in serum  transferase not detected in
serum/RBCs
 Molecular heterogeneity
 Produces an anti-A1 in serum
 35,000 antigenic sites per RBC
 4,000 antigenic sites per RBC
 Molecular genetics reveal subgroup
heterogeneity
 Weak A Subgroups
 A3, Ax, Aend, Am, Ay, Ael
 Aend RBCs: mixed field agglutination  Am RBCs: not agglutinated/weakly
with anti-A and anti-A,B but only a agglutinated by anti-A/anti-A,B
small percentage of RBCS  200 -1,900 Antigenic sites per RBC
agglutinate.
 A enzyme detectable in serum
 3,500 antigenic sites per RBC
 Do not produce anti-A1 in their sera
 No transferase detected in
serum/RBC membranes
 Afinn and Abantu: variants of Aend
subgroup
 Weak A Subgroups
 A3, Ax, Aend, Am, Ay, Ael
 Ay individuals: do not produce anti-  Ael RBCs: unagglutinated by anti-A or
A1 anti-A,B
 Phenotype can be observed in  No detectable enzyme activity can
siblings, recessive mode of be demonstrated in serum/RBCs
inheritance  Ael inherited as a rare gene in ABO
 Expression of an alternate allele at locus
ABO locus
 Germline mutation of A gene within a
family
 Weak A Subgroups
Phenotypes Anti-A Anti-B Anti-A,B Anti-A1 Common Unexpected Substances Number of
Present in antigenic
Secretors sites 10^3

A1 4+ 0 4+ 4+ Anti- None A,H 810-

B 1,170
A2 4+ 0 4+ 0 Anti- Anti- A,H 240-
B A1 (1- 290
8% of
cases
 Weak B Subgroups
 Very rare, much less frequent
 Recognized by variations of reaction
in anti-B and anti-A,b
 Inheritance is a result of alternate
alleles at B locus
Criteria for differentiation:
 Strength and type of agglutination
with B, anti A,B, and anti-H
 Adsorption-elution studies with anti-B
 Presence of B substance in saliva
 Molecular testing
 B3, Bx, Bel and Bm phenotypes
 B3: inheritance of a rare gene at the
ABO locus
 Bx: can only be detected by
inhibiting agglutination of Bx cells by
anti-B
 Bm: reduced activity of B enzyme in
hematopoietic tissues
 Bel: unique mutation in exon 7 of B
gene in ABO locus
CHARACTERISTICS OF B PHENOTYPES

PAGE 134- TABLE 6-16

 Bombay Phenotypes
 Reported by Bhende in 1952 in  ABH substance also absent in saliva
Bombay, India  Gene FUT1 (H) gene mutation
 Double dose of h gene, hh (silenced) incapable of coding for the
enzyme a 1,2 fucosyltransferase
 No H antigen made in the Bombay
phenotypes  Silenced FUT2 gene (Se gene)
 130 Bombay phenotypes in various
parts of the world
 RBCs of the Bombay phenotype Oh
do not react with the anti-H lectin
 Bombay serum: Anti- A, Anti-B, Anti-H
(IgM antibody that causes RBC lysis)
Parabombay Phenotypes
 RBCs completely devoid of H antigens/small
amounts of H antigen present.
 Even if a person is genetically A or B, Enzymes
can be detected, but no H is detectable though
there is a limited production of H antigen on
RBCs.
 No H, A or B angtigens in the saliva, anti-H
present in serum
 Homozygous inheritance of a mutant H(FUT1)
gene that codes for production of low levels of H
transferase activity; with or without active FUT2
gene (Se gene)
ASSOCIATION OF ABH ANTIGENS AND ANTIBODIES IN
DISEASES

A= hangover
B= criminality
O= good teeth
Diseases/disorders

 Alter red cell antigens

 Caused production of antigens
 Leukemia- chromosome 9 translocation= decreased
ABH antigens
 Hemolytic disease inducing thalassemia- depressed
antigen strength
 Hodgkin’s disease- depressed ABH antigen
 CLL= weaker anti-A1, anti-B, anti-A,B
Individuals with intestinal obstruction, carcinoma of
colon/rectum, increased permeability of intestinal wall

 E.coli
infections= passage of bacterial
polysaccharides into patient’s circulation
 A1 individuals will absorb B-like
polysaccharide which may reacts with
anti-B= ACQUIRED B PHENOMENON
Carcinoma of stomach or pancreas

 Lack of detectable ABO antigens

 Patient’s red cells do not change but serum
may contain BLOOD GROUP SPECIFIC SOLUBLE
SUBSTANCES (BGSS)
 All these disease states may lead to
discrepancy in forward and reverse grouping
and typing
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ABO Discrepancies

 ABO discrepancies happen when there is no match in results

between forward and reverse grouping.

 ABO discrepancies are usually technical in nature and can be

simply resolved by correctly reporting the testing and carefully
checking reagents with meticulous reading and recording of
results.
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ABO Discrepancies

 There are some ABO discrepancies that can happen due to

technical errors and may lead to false positive or false
negative reactions.

 False positive reactions are due to;

 Uncalibrated centrifuges
 Contaminated reagents
 Dirty tubes or glassware
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ABO Discrepancies

 False negative reactions can be due to many causes

 Failure to add serum or reagents

 Use of incorrect reagents or samples
 Cell suspension is too heavy or too light
 Inadequate identification of samples or test
tubes
RESOLUTION

 Repeat testing
 Acquire medical history of patient
 Document
 Repeat collection
 “ when investigating ABO discrepancies, it should be
noted that red blood cells and serum grouping
reactions are very strong (+3/+4); therefore the weaker
reactions usually represent the discrepancy”
ALGORITHM FOR RESOLVING ABO
DISCREPANCIES

 PAGE 137, FIGURE 6-14

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ABO Discrepancies

 Group I discrepancies
These discrepancies are due to unexpected reactions in
reverse grouping due to weak reaction or missing
antibodies.
These kind of discrepancies are the most common.
The reason for the missing antibody or weak reaction is
that the patient has depressed antibody production or
cannot produce the ABO antibodies.
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ABO Discrepancies

 Group I discrepancies
 Newborns
 Elderly patients
 Patients with leukemia or lymphoma, hypogammaglobulinemia
 Patients using immunosuppressive drugs
 congenital/acquired agammaglobulinemia
 Bone marrow/stem cell transplantation
 ABO subgroups
ABO DISCREPANCY 1= WEAK/MISSING
ANTIBODY

 PAGE 138, TABLE 6-17

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ABO Discrepancies

 Group II discrepancies
 Due to unexpected reactions in the forward grouping due to
weakly reacting/missing antigens
 Least encountered
 Subgroups of A or B
 Leukemias
 Acquired “B” phenomenon
ABO DISCREPANCY 2

 PAGE 138- TABLE 6-18

RARE GROUP 2- DISCREPANCIES

 BGSS
 Low incidence antibodies
 Ex: page 139, table 6-20
MORE RARE ABO DISCREPANCY 2

 Chimerism- presence of two cell populations in a single

individual
 Ex: page 140, Table 6-21
 Blood exchange in utero= vascular anastomosis
 Dispermy
 Blood transfusions- o cells given to A or B
 Bone marrow transplant
 Exchange transfusion
 Fetal-maternal bleeding
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ABO Discrepancies

Group III discrepancies

These discrepancies are between forward and reverse

grouping due to protein or plasma abnormalities.

These can be caused by elevated levels of globulin from

certain diseases such as multiple myloma, hodgkin’s
lymphoma. Some are caused by (Rouleaux formation).
ABO DISCREPANCY 3 EXAMPLE

 PAGE 140- TABLE 6-22

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ABO Discrepancies
Group IV discrepancies

 These kind of discrepancies are between forward and

reverse groping due to miscellaneous problems.
 Bacterial contamination in vitro or vivo produces an
enzyme that alters and exposes the hidden Ag. on red
cell leading to T activation.
 Cold reactive autoantibodies
 UNEXPECTED ABO ISOAGGLUTININS
 UNEXPECTED NON ABO ALLOANTIBODIES
 PAGE 141, TABLE 6-23
 PAGE 142, TABLE 6-24
 PAGE 142, TABLE 6-25
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ABO Discrepancies

 Some examples of discrepancies

 Example 1
 Forward grouping: anti-A =O, anti-B =O, anti-AB= O
 Reverse grouping: A1 cells= O, B cells =O
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ABO Discrepancies
 Example 3

 Forward grouping: anti-A 4+,anti-B 2+,anti-AB 4+

 Reverse grouping: A1 cells 4+, B cells 4+