ABO BLOOD GROUP SYSTEM

ABO System indv have Abs in their

serum to Ags that are absent from
their RBCs; discovered by Landsteiner
Forward grouping (FG) using
known sources of commercial antisera
(anti-A, anti-B) to detect Ags on an
indvs RBCs
Steps:
1. Label test tubes
2. Make a 2-5% px red cell suspension
3. Add 1 gtt rgt antisera
1 gtt rgt Anti-A antisera
1 gtt rgt Anti-B antisera
4. Add 1 gtt 2-5% suspension of px
red cells to each tube
5. Mix and Centrifuge (20 sec)
6. Read and Record aggn rxns
Group B agg w/ Anti-B
Group A agg w/ Anti-A
Group AB agg w/ Anti-A and Anti-B
Group O doesnt agg w/ Anti-A or
Anti-B

Reverse grouping (RG) detecting

ABO Abs in the pxs serum by using
known rgt RBCs (A1 and B cells)
Steps:
1. Label test tubes
2. Add 2 gtts of px serum to each
tube
3. Add 1 gtt of rgt cells to each test
tube
1 gtt rgt A1 cells
1 gtt rgt B cells
4. Mix and Centrifuge (20 sec)
5. Resuspend cells button; Interpret
and Record results
Group A agg w/ B cells
Group B agg w/ A1 cells
Group O agg w/ A1 and B cells
Group AB doesnt agg w/ both

Group O and A are the most

common blood types
Group AB is the rarest
ABO Abs are initially produced at birth
but test results may not be valid
because some may be IgG maternal
Abs (best to use Forward Grouping).
Titers are low until the 3-6 mos of age.
Peak is at 5-10 yrs then declines.
Elderly people have lower levels of
antisera, therefore Abs are
undetectable in Reverse Grouping.
Wrong ABO group to ABO Abs Rapid
Intravascular Hemolysis Death
Anti-A and Anti-B, from Group B and
Group A respectively, contains
predominantly IgM (w/ small quantities
of IgG).
Group O has Anti-A, Anti-B and AntiA,B.
Anti-A,B not a combination of Anti-A
and Anti-B; cross-reacting Ab usually
in IgG in nature
Rgt can be prepared using:
Blended monoclonal anti-A and
anti-B
Polyclonal human anti-A,B
Blend of monoclonal anti-A,
anti-B, and anti-A,B
Igs of ABO Abs prefer to react at RT or
below and activate complement at
37C.
Bernstein described the theory for
the inheritance of the ABO Blood
Group; Indv inherits one ABO gene
from each parent that these genes
determine which ABO Ags are present
on the RBC membrane.

A, B, and O are codominants.

Chromosome 9 locus occupied by

the ABO genes
O gene amorph (no detectable Ag is
produced in response to the
inheritance of this gene); therefore,
inheritance of the OO gene is an
autosomal recessive trait.
Group A, Group B etc Phenotypes
AA, BO etc Genotypes
ABH Ags results from the interaction
of genes at three separate loci (ABO,
Hh and Se)
These genes produce specific
glycosyltranferases (those that
add sugars to substances).
Paragloboside or Glycan basic
precursor material from which ABH
Ags are formed
H Ag precursor structure on w/c A
and B Ags are made

Indv w/ blood group O inherit at least

one H gene (HH or Hh) and two O
genes.
H gene elicits production of -2-Lfucosyltransferase, which transfers Lfucose (responsible for H specificity)
to an oligosaccharide chain on the
terminal Galactose of Type 2 chains
Immunodominant sugars sugars
that occupy the terminal positions of
the precursor chain and confer blood
group specificity
In O gene, H substance remains
unmodified.
Bombay phenotype refers to the
lack of normal expression of the ABH
Ags because of the hh genotype;
extremely rare
The A gene codes for the production of
-3-N-acetylgalactosaminyltransferase
(transfer an N-acetylgalactosamine
sugar to H substance)

Chromosome 19 inheritance of H
and Se genes

N-acetylgalactosamine
responsible for A specificity; linked to
type 2 precursor substance

Se gene forms the ABO Ags in

secretions

The A gene has higher conc of

transferases than the B gene.

Type 2 RBC precursor substance;

ABH Ags are constructed on
oligosaccharide chains of this
substance

The B gene codes for the production of

-3-D-galactosyltransferase (attaches
D-Galactose to precursor substance

Terminal Galactose (from the

precursor substance) is
attached to the Nacetylglucosamine in a 14
linkage between Galactose and
N-acetylglucosamine

D-Galactose responsible for B

specificity
When A and B genes are inherited, B
enzymes competes more than the A
enzyme.
H

A gene

B gene

Se gene secretor (SeSe or Sese);

codes for the production of -2-Lfucosyltransferase (determines if ABHsoluble substances will be secreted)
that modifies Type 1 precursor
substance to form H substance

gene
Enzyme
produce
d
Specifici
ty

o
Molecular Genetics of ABO:
ABO gene is located at chrm 9 and
consists of 7 exons
7 common ABO Alleles:
A1
O1
A1variant (A1v)
(O1v)
A2
O2
B1

O1variant

Mutations in the ABO gene may result

to:
-1,3-N-acetylgalactosamine (A
specificity)
-1,3-galactose (B specificity)
Deletion within the 5 region of
the catalytic domain that results
in a frameshift and inactivates
enzymes leaving H unmodified.
Recombination gene conversion;
may lead to mutations [weak
subgroups, non-deletional null (O)
alleles, hybrids] that result in
unexpected ABO phenotypes
Crossing over between exons
6 and 7 of chrm 9
Less efficient transfer of the
immunodominant sugar to H
substance
Weak serologic reactions

sese genotype are nonsecretors

ABH Ags vs ABH Soluble

Substances
Secretor term that refers to the
secretion of A, B, and H soluble Ags in
body fluids
Glycoprotein-soluble substances
(or Ags) normally found found in the
saliva of secretors
ABH Ags and Soluble Substances are
formed due to the attachment of an
immunodominant sugar to an
oligosaccharide chain
Types 1 and 3 assoc with body
secretions
Types 2 and 4 assoc with red cell
membrane
Type 1 and 2 are the most abundant.
Linkage position
of Galactose (Gal)
to N-acetylglucosamine
(GlcNAc)
Type 1 (ABH
soluble
substances)
Type 2 (ABH
Ags)

13
14

Immunodominant sugars + Type 2 and

4 ABH Ag formation on red cell
membrane

Immunodominant sugars + Type 1 and

3 ABH soluble substances in the
body secretions
ABH soluble substances are found in:
Saliva
Bile
Tears
Milk
Urine
Digestive Juices
Pathological fluids* Amniotic fluid
* Pleural, Peritoneal, Pericardial,
Ovarian cyst
ABO Subgroups
-

von Dungern described 2 diff A

Ags
show weaker variable serologic
reactivity compared to
polyclonal antisera

A Subgroups

Bandeiraea simplifolia agg B

cells
Ulex europaeus aggs blood
groups depending on the amt of
H Ag
Results of high titered anti-H:
Reaction in RT and may be a
problem in Ab screening
because rgt screening cells are
grp O
May also be a problem in
compatibility testing
O > A2 > B > A2B > A1 > A1B
Four diff H Ags:
Unbranched straight chains: H1 and H2
Complex branched chains: H3 and H4
H1 Aa by A1 enzyme

A1 Group A RBCs that react w/ anti-A

and anti-A1

H2 Ab by A2 enzyme

A2 reacts with anti-A and not anti-A1

H3 Ac by A1 enzyme

A1 gene elicits the production of -3-Nacetylgalactosaminyltransferase that

converts H to A Ag.

H4 Ad by A2 enzyme

N-acetyl-D-galactosamine
immuno-dominant sugar on both A1
and A2
To differentiate A1 and A2, use Dolichus
biflorus seeds (src of anti-A1). It is also
known as anti-A1 lectin.
Lectins seed extracts that agg
human cells w/ some degree of
specificity; aggs A1 (or A1B) but not A2
(or A2B); Lectins used are:
Dolichus biflorus aggs A1 or
A1B

* A2 enzyme is less efficient.

More unconverted H3 and H4 are
available on grp A2 RBCs. Only Aa and
Ab determinants are formed.
A2B indv would be far more likely to
lack Ac and Ad cmpts w/ subsequent
production of anti-Ac and anti-Ad (antiA1)
A1 red cells - Aa Ab Ac Ad; converted H3
and H4 Ag sites
In infants, most are A2, then develops
to A1. Def of H3 and H4 may account to
A2 phenotype.

Adults have higher conc of H3 and H4

and therefore may lead to A1
phenotype.
Weak A Subgroups
-

Weaker than A2 and may cause

unexpected rxns in the forward
and reverse grouping

Characteristics:
Decreased number of A Ag
sites per RBC (weak or no
aggn w/ polyclonal anti-A)
Varying degrees of aggn by
human anti-A,B
Increased variability in the
detectability of H Ag (strong
rxns w/ anti-H)
Presence or absence of antiA1 in the serum
Differentiation of Weak A phenotypes:
FG of A and H Ags w/ anti-A,
anti-A,B and anti-H
RG of ABO isoagglutinins and
the presence of anti-A1
Adsorption-elution tests w/ antiA
Saliva studies to detect
presence of A and H substances
Special procedures such as
Molecular Testing (mutations) or
Serum Glycosyltransferase
studies (A enzyme)
Subgroups:
A3 mixed-field* pattern of
aggn w/ anti-A and most antiA,B rgts
* Small agglutinates w/in
predominantly unagglutinated
RBCs

Weak -3-N-acetylgalactosaminyltransferase activity

Presents molecular
heterogeinity (anti-A1 in serum
and A substance in saliva)
Ax not agglutinated by anti-A
rgt but does w/ most anti-A,B
Has considerable homogeinity
(Ax produce anti-A in serum; H
substance found in secretions;
Ax secretors contain A
substance detectable only by
aggn/inhn studies).
Ax as indicator cells.
Aend mixed-field aggn w/ anti-A
and anti-A,B (small amt of
RBCs)
Afinn and Abantu variants of Aend
Am weak or no aggn by anti-A
or anti-A,B.
A enzyme (A1 and A2) is present
in the serum
Rare allele in ABO locus
No anti-A1 in sera
Has normal quantities of A and
H substance in saliva
Ay not agglutinated by anti-A
or anti-A,B
Adsorption and elution is used
Eluates have weaker activity
than Am
Trace amts of A enzyme is in the
serum. H and A substances are
present in saliva
Doesnt produce anti-A1
Observed in siblings; recessive
mode of inheritance
Germline mutation of A gene
Ael unagglutinated by anti-A or
anti-A,B
Adsorption and elution is used

No A enzyme in serum; H
substance in saliva
Rare gene in ABO locus
Produces anti-A1 reactive to A1
cells, and anti-A that aggs A2
Weak B Subgroups
-

Very rare and much less

frequent
Usually classified in the
variations of strength of rxn
using anti-B and anti-A,B
Inheritance is due to alternate
alleles at the B locus

Criteria used for weak B phenotypes:

Strength and type of aggn
w/anti-B, anti-A,B, and anti-H
Presence or absence of ABO
isoagglutinins in the serum
Adsorption-elution studies w/
anti-B
Presence of B substance in
saliva
Subgroups:
B3 inheritance of rare gene;
mixed-field pattern of aggn w/
anti-B and anti-A,B
B glycosyltransferase is present
in serum but not in RBC
membranes
Anti-B is absent in serum
B substance is present in
normal amts in saliva
Most frequent weak B
phenotype
Bx weak aggn w/ anti-B and
anti-A,B
B glycoyltransferase not
detected in serum or in RBC
membranes

Weakly reactive anti-B is

produced
Readily adsorb and elute anti-B
Large amts of H substance
Some B substances can only be
detected if aggn of Bx w/ anti-B
is inhibited
Rare allele
Bm unagglutinated by anti-B or
anti-A,B
Easily adsorb and elute anti-B
B glycosyltransferase is present
in serum (low and varying
activity)
Small amt of B enzyme is in RBC
membranes
Normal plasma B plasma
incubated w/ Bm RBCs and uracil
diphosphate-galactose
transforms Bm to normal B
phenotype
Anti-B is not present in the
serum
Normal quantities of H and B
substances are found in the
saliva f Bm secretors
Rare allele
ABO Modifier gene may depress
B gene expression and
subsequently decrease B
enzyme activity
Bel unagglutinated by anti-B or
anti-A,B
Extremely rare
Determined by adsorption and
elution of anti-B
No B enzyme in serum or RBC
membrane
Unique mutation in exon 7 of B
gene
Weak anti-B may be present in
serum
H substance is in saliva
Bombay Phenotypes (Oh)

First reported by Bhende

hh gene
ABO cant be expressed and
ABH Ag cant be formed
Fail to react with anti-A, anti-B
and anti-H
Dont react w/ anti-H lectin
(Ulex europaeus)
Serum contains anti-A, anti-B,
anti-A,B and anti-H
Anti-H can be potent and react
strongly at 37C; IgM that can
bind complement and cause
RBC lysis

Group O (highest conc of H Ag) +

Bombay recipient (anti-H) = cell lysis
* Only blood from another
Bombay indv will be compatible to a
recipient with the same phenotype
-

ABH substance is absent in

saliva
Autosomal recessive trait
Molecular defect in FUT1 (H
gene) that produces a silenced
gene*

* Silenced gene incapable of

coding enzyme
(1,2)fucosyltransferase (H
transferase)
H enzyme transfers fucose in 1,2 to the terminal galactose of
the precursor molecule forming
H Ag
Mutation is assoc w/ silenced
FUT2 gene (Se gene) that
transfers fucose to form H Ag in
secretions
ABO genes inherited in the Bombay
phenotype: OhA, OhB, OhAB
Para-Bombay Phenotypes
-

RBCs contains a small amt or

are completely devoid of H Ags
Weak form of A and B Ags
(detected by Adsorption and
Elution studies
No H enzyme detectable
Notations: Ah, Bh and ABh
No ABH Ag is presnt in saliva
Anti-H is in serum