ABO Blood System

Nur Najmi Mohamad Anuar

IMMUNOHEMATOLOGY

• Hematology + immunology + genetics

• serologic, genetic, biochemical and
molecular study of antigens
• associated with membrane structures
• immunologic reactions involving all
blood components and constituents
IMMUNOLOGIC
PRINCIPLES
• primary immunological components: antigens &
antibodies
• basis for blood bank testing and reactions

RULE IN BLOOD BANK:

The antigens are found on the surface of
red blood cells and the antibodies are found
in serum or plasma
 Blood Group System

• ABO
• Rh
• Minor/Other Blood Group
Immunogenicity of Blood Group Antigens

A, B and D (Rho) – most immunogenic

Minor Blood group
Kell (K)
Duffy: Fya
Fyb
Kidd: Jka
Jkb
 • mixing each others red
cells and serum
Discovery •
together
accidentally performed
- 1901 first forward and
reverse ABO groupings

• If an antigen (Ag) is on
RBC the corresponding
antibody (Ab) will NOT
be present in the
patients plasma, under
‘normal conditions’.
 Hh system
Genetics
Gene locus : chr 19q13
Gene : FUT1, FUT 2 (Fucosyltransferase)
Features: H gene is dominant
h gene is recessive
Frequency: H gene 100% in all populations
hh approx. 1 in 106
Features: abnormal phenotypes due to point
mutations in H gene eg. Bombay and
para-Bombay groups
H antigen is the precursor for the
production of A and B antigens
 H Phenotypes

Secretor (Se) – FUT 2

• H antigen is expressed on RBCs, saliva.
• No anti-H is produced.
Non secretor – FUT1
• H antigen is present on RBCs, absent from saliva.
• No anti-H is produced.
 H Phenotypes

Bombay phenotype
• H antigen is not expressed on RBCs/saliva.
• Serum contains anti-H.

Para-Bombay phenotype
• H antigen is weakly expressed on RBCs/saliva.
• Serum contains anti-H.
 Bombay Phenotype

• Discovered in Bombay - Very rare (130 worldwide) -Oh

• The hh causes NO H antigen to be produced
• Results in RBCs with no H, A, or B antigen (patient types as O)
• Bombay RBCs DO NOT agglutinate with anti-A, anti-B, or anti-H (no
antigens present)
• Bombay serum has strong anti-A, anti-B and anti-H, agglutinate
with ALL ABO blood groups
 Para-Bombay
Phenotype

• Para-Bombay groups
– Single point mutations in H gene lead to defective
fucosyl transferase
– Results in reduced H antigen production leading to
reduced A and/or B antigen expression
– Ah, Bh or ABh
– OH
 ABO system

• Gene locus chr 9q34.1-q34.2

• A and B genes are co-dominant
• O gene is an amorph - genetic null, loss of gene f(x)
• Point mutations
– enzymes with slightly different substrate specificities
→glycosylation efficiencies → variant antigens
 Antigen development

hh gene H gene

H antigen

A gene O gene
B gene

A antigen B antigen H antigen

 ABH Antigen Genetics

• The presence or absence of the ABH antigens on the red blood cell
membrane is controlled by the H gene

• The presence or absence of the ABH antigens in secretions is controlled

by the Se gene (body fluid) -- found on virtually all cells

• Genes at three separate loci control the occurrence ABO antigens

• The presence or absence of the A, B, and H antigens is controlled by the

H and ABO genes

• H antigens - required to produce either A or B antigens

 ABO Genetics

• O gene
– H antigen is found on the RBC
when you have the Hh or HH
genotype, but NOT the hh
genotype

• A gene
– A antigen is found on the RBC
when you have the Hh, HH, and
A/A, A/O, or A/B genotypes

• B gene
– B antigen is found on the RBC
when you have the Hh, HH, and
B/B, B/O, or A/B genotypes
 ABO Basics

• Blood group antigens → sugars attached to the RBC.

• Individuals inherit gene which encodes for specific
sugar(s) to be added to the red cell.
• The type of sugar added determines the blood group
 Formation of the H antigen

• The precursor substance (proteins and lipids) is formed on an

oligosaccharide chain (the basic structure)

RBC

Glucose

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

H gene codes for an enzyme

(fucosyl transferase) that RBC
adds the sugar fucose to the
terminal sugar of a
precursor substance (PS)

Glucose

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

• The H antigen is the foundation of A and B antigens

• A and B genes code for enzymes that add an immunodominant sugar to

the H antigen
– Immunodominant sugars are present at the terminal ends of the chains and confer the
ABO antigen specificity

• Certain blood types possess more H antigen than others:

O>A2>B>A2B>A1>A1B
 A Antigen

• The “A” gene codes for N-

acetylgalactosaminyltransfer
ase
• adds N-acetylgalactosamine RBC

to the terminal sugar of the H

antigen

Glucose

Galactose

N-acetylglucosamine

Galactose
A antigen

N-acetylgalactosamine
Fucose
 B Antigen

• The “B” gene codes for D-

galactosyltransferase
RBC
• adds D-galactose to the
terminal sugar of the H
antigen
Glucose

Galactose

N-acetylglucosamine

Galactose

D-galactose B antigen
Fucose
 • differ in the amount of antigen
ABO present on the red blood cell
Subtypes membrane
• Subgroups have less antigen
• Due to less effective enzymes.
• not as efficient in converting H
antigens to A or B antigens (fewer
antigens are present on the RBC)
 A Subtypes

• 2 principle subgroups of A
• A1 and A2
• Both react strongly with reagent anti-
A
• To distinguish A1 from A2
• the lectin Dolichos biflorus is
used (anti-A1)
• 80% of group A or AB -- subgroup A1
• 20% -- A2 and A2B
 A Subtypes

Why is the A2 • A2 and A2B individuals may produce

an anti-A1
phenotype • cause discrepancies in crossmatch
important? (incompatibility)

What’s the • The A2 gene doesn’t convert the H to

difference A very well
between the A1 • The result is fewer A2 antigen sites
and A2 antigen? compared to the many A1 antigen sites

O>A2>B>A2B>A1>A1B
 Blood Group Phenotype

Antigen Genotype Antibodies

A1 A1A1, A0, A1A2 anti-B

A2 A2A2, A2O anti-B (2-8% anti-A1)

B BB, BO anti-A, -A1

O OO anti-A, -A1, -B, -A,B

A1B A1B none

A2B A2B anti-A1 (25-30%)

Oh hh anti-A, -A1, -B, -H
 Phenotype Frequency
O 46.1%
A1 29.7%
A2 9.3%
B 11.4%
A1B 2.3%
A2B 1.2%
Phenotype
frequencies Note: these are Caucasian
frequencies, they differ in
other racial groups
ABH antigens and disease

Translocations involving
Reduced expression chromosome 9
Leukemia

Acquired B antigen
Acquired antigens (should be A group)
Grouping Reagents
• Polyclonal antisera
– Anti-A(blue), anti-B(yellow) and
anti-A,B(colorless)
• Collected from volunteers
who have been stimulated to
produce high titre antibodies
• Lectins
– Ulex europaeus H lectin
– Dolichos biflorus A1 lectin
 A1 and A2 Subgroups*

Anti-A Anti-A1 Anti-H ABO # of

antisera antisera lectin antibodies antigen
in serum sites per
RBC
A1 4+ 4+ 0 Anti-B 900 x103

A2 4+ 0 3+ Anti-B & 250 x103

anti-A1

*Adapted from Flynn, J. (1998). Essentials of Immunohematology

Classification • Alloantibodies
• Reacts with foreign Ag not

Antibodies:
present on patient’s own RBC
of Blood

• Ab to an Ag that the person

Group
lack
• immune stimulation via
transfusion or pregnancy
(usually during delivery)
• Autoantibodies
• Reacts with an Ag on
patient’s own cells & with that
same Ag on the cells of other
individuals
 • NATURALLY occurring Ab to the ABO Ag that
they lack - without antigenic stimulation
• Reactions phase: Room temperature (4C –
20C, can react at 37C)
ABO • Complement can be activated with ABO
antibodies
antibodies • mostly IgM, some IgG
• Usually present within the first 3-6 months of
life
• Stable by ages 5-6 years
• Decline in older age
• React best at techniques: saline, enzyme and
AHG
• Newborns may passively acquire maternal
antibodies (IgG crosses placenta)
– Reverse grouping (with serum) should not be
performed on newborns or cord blood
 Anti-H
Anti-H type
Anti-H reactivity
Transfusion reaction
Hemolytic disease of the newborn
IgM is more common than IgG
Anti-H is naturally occurring in people
with H antigen deficiency.
Capable of hemolysis
Anti-H can activate the complement
cascade which lyses RBCs while they
are still in the circulation (intravascular
hemolysis).
Yes—can cause an acute hemolytic
transfusion reaction
Possible
HDN could arise in mothers with the
Bombay phenotype (Oh, h/h)
 Anti-A, B

Antibody type IgG and IgM

Naturally occurring. Anti-A is found in the serum of
people with blood groups O and B. Anti-B is found in
the serum of people with blood groups O and A.

Antibody reactivity Capable of hemolysis

Anti-A and anti-B bind to RBCs and activate the
complement cascade, which lyses the RBCs while they
are still in the circulation (intravascular hemolysis).

Transfusion reaction Yes — typically causes an acute hemolytic

transfusion reaction
Most deaths caused by blood transfusion are the result
of transfusing ABO-incompatible blood.

Hemolytic disease of the newborn No or mild disease

HDN may occur if a group O mother has more than
one pregnancy with a child with blood group A, B, or
AB. Most cases are mild and do not require treatment.
COMPLEMENT
ACTIVATION
 ABO • Forward - Ab that are specific at
routine detecting a particular ABO antigen
on RBCs.
testing
– Patient blood (Ag) + known anti
(Grouping) sera (Ab)

• Reverse – Cells/Ag (known antigen)

agglutinate with naturally occurring
antibodies in the person's serum.
– Patient Serum (Ab) + known cell
(Ag)
 Forward Grouping

• Reaction of patient red blood cells tested with Reagent anti-A and anti-B
antisera

• Slide/Tiles: 15-25% RBC suspension + anti-serum

• Tube (12x75mm): 2-5% RBC suspension + anti-serum (centrifuge before

read) Blood group Agglutination Agglutination with
with Anti-A Anti-B
A + -

B - +

AB + +

O - -
 Reverse grouping

• serum is combined with cells having known Ag content

• commercially prepared reagents containing saline-suspended A1 and B
cells

Blood Group Agglutination Agglutination

with A cells with B cells
A - +

B + -

AB - -

O + +
Grading of Agglutination

No clumps or aggregates

Tiny clumps or aggregates barely

visible macroscopically
Few small aggregates visible
macroscopically
Medium-sized aggregates
One solid aggregate
Grading of Agglutination
 • Invent by Dr Yves Lapierre
(France 1988)
• Diamed – ID card system
• Each column – Sephacryl gel
• Depend on the card – gel will
premix with anti sera, AHG/
other reagents
– contains a mixture of
human polyclonal and
monoclonal anti-A, human
polyclonal anti-B and
human polyclonal anti-D
antibodies.
– Contain cell - reverse
grouping with A1 and B
cells.
• Large agglutinate stuck at the
top
• Small agglutinate pass through
gel

GEL card system

GEL card system
GEL card system
GEL card system

• Fully-automatic walk-away for ID-

Cards
• Stand alone instrument
• Continuous sample loading
• Continuous reagent loading
• Priority samples
• Reagent stock
• High throughput
 GEL card system

• Optimized for small blood volumes

• Dispense verification
• Easy-to-use
• Full test menu
• Wi-Fi
• Touchscreen 17"
• Host connectivity
• Internal & external validation
• Capacity
180 samples
240 ID-Cards
28 reagent vials