ABO BLOOD GROUP

History: Karl Landsteiner

• He and his five co-workers began mixing each others red cells and serum together and inadvertently performed the

http://www.nobelpreis.org/castellano/medizin/images/landsteiner.jpg

Why is it important?

ABO ANTIGENS

Biochemical & Genetic Considerations

ABO INHERITANCE

• In 1924, Bernstein described the theory for the

inheritance of ABO blood groups

• Codominant in expression

• Genotypes:

• Phenotypes:

ABO and H Antigen Genetics

• Genes at three separate loci (ABO, Hh, and Se)

control the occurrence and location of ABO

antigens

• There are three common alleles at the ABO

• The H and Se (secretor) loci,

• The presence or absence of the A, B, and H

antigens is controlled

Location

• The presence or absence of

the ABH antigens on the red

blood cell membrane is

controlled by the

• The presence or absence of

the ABH antigens in secretions

is indirectly controlled by the

ABO Antigen Genetics

• H gene –

• Se gene –

• ABO genes –

H Antigen

• The H gene codes for an enzyme that adds the

sugar fucose to the terminal sugar of a

• The precursor substance (proteins and lipids) is

formed on an

RBC Precursor Structure

Glucose

Galactose

N-acetylglucosamine

Galactose

Precursor

Substance

(stays the

same)

RBC

Formation of the H antigen

Glucose

Galactose

N-acetylglucosamine

Galactose

RBC

H antigen

• The H antigen is the foundation upon which A and B

antigens are built

• A and B genes code for enzymes that add an

immunodominant sugar to the H antigen

– ________________________are present at the

terminal ends of the chains and confer the ABO

antigen specificity

A and B Antigen

• The “A” gene codes for an enzyme (transferase)

that adds _______________________to the

terminal sugar of the H antigen

– N-acetylgalactosaminyltransferase

• The “B” gene codes for an enzyme that adds

________________to the terminal sugar of the

H antigen

– D-galactosyltransferase

Formation of the A antigen

Glucose

Galactose

N-acetylglucosamine

Galactose

RBC

Fucose

Formation of the B antigen

Glucose

Galactose

N-acetylglucosamine

Galactose

RBC

Fucose

Genetics

• The _____________ is found on the RBC when you

have the Hh or HH genotype, but NOT from the hh

genotype

• The _____________is found on the RBC when you have

the Hh, HH, and A/A, A/O, or A/B genotypes

• The _____________is found on the RBC when you have

the Hh, HH, and B/B, B/O, or A/B genotypes

H antigen

• Certain blood types possess more H

antigen than others:

Greatest

amount of H

Least

amount of H

ABO Antigens in Secretions

• Secretions include

• Blood Group Substances are soluble

antigens (A, B, and H) that can be found in

the secretions. This is controlled by the

Secretor Status

• The secretor gene consists of 2 alleles (Se and se)

• The Se gene is responsible for the

• If the Se allele is inherited as SeSe or Sese, the person

is called a

– 80% of the population are secretors

Secretors

• Secretors express soluble forms of the H antigen in secretions that can then be

• Individuals who inherit the sese gene are called

– The se allele is an amorph (nothing expressed)

– sese individuals do not convert antigen precursors to H antigen and has neither soluble H antigen nor soluble A or B antigens in body fluids

Secretor Status Summary

• The Se gene codes for the presence of the H antigen in

secretions, therefore the presence of A and/or B antigens

in the secretions is contingent on the inheritance of the Se

gene and the H gene

Se gene

(SeSe or Sese)

H antigen in

secretions

A antigen

B antigen

se gene

(sese)

No antigens secreted

in saliva or other

body fluids

and/or

ABO Group ABH

Substances

Secretors (SeSe or Sese): A B H

A +++ 0 +

B 0 +++ +

O 0 0 +++

AB +++ +++ +

Non-secretors (sese):

A, B, O, and AB 0 0 0

Sese + h/h (no H

antigen) no antigens

in secretions

COMPARISON OF ABH ANTIGENS

ON RBCs AND IN SECRETIONS

ABH Antigens on Red Cells A, B, and H Soluble substances

RBC antigens can be glycolipids,

glycoproteins, or glycosphingolipids

Secreted substances are glycoproteins

RBC antigens are only synthesized on type

2 precursor chains

Secreted substances are primarily

synthesized on type 1 precursor chains

Type 2 chain refers to a beta1 4 linkage

in which the number one carbon of the

galactose is attached to the number three

carbon of the N-acetylglucosamine sugar

of the precursor substance

Type 1 chain refers to a beta 1 3 linkage

in which the number one carbon of the

galactose is attached to the number three

carbon of the N-acetylglucosamine sugar

of the precursor substance

Enzyme produced by the H gene (α-2-L-fucosyltransferase)acts primarily on type 2 chains, which are prevalent on the RBC membrane

Enzyme produced by the Se gene (α-2-L-fucosyltransferase) preferentially acts on

type 1 chains in secretory tissues

Lewis (Le)

• The Lewis Blood Group System is mentioned here

because it is related to secretor status

• Lewis antigens are plasma antigens formed by tissues

and are released into plasma where they adsorb onto

the RBCs (they are not an integral part of the RBC

membrane)

• Consists of 2 antigens

– Lea

– Leb

Lewis

• Lea and Leb are a single gene (Le) and its amorph (le)

– Lea is a precursor to Leb

• The Le gene codes for a transferase, which attaches L-fucose to the precursor chain to form the Lea antigen (designated Le(a+b-)

• If the H and Se genes are inherited, the Lea is converted to Leb and is designated Le(a-b+)

• In childhood, both may be on the RBC, Le(a+b+)

• If a person is lele, they will have no Lewis antigens in plasma or on red blood cells

ABO Subgroups

• ABO subgroups differ in the amount of antigen present on the red blood cell membrane

– Subgroups have less antigen

• Subgroups are the result of less effective enzymes. They are not as efficient in converting H antigens to A or B antigens (fewer antigens are present on the RBC)

• Subgroups of A are more common than subgroups of B

Subgroups of A

• The 2 principle subgroups of A are: A1 and A2

A2 Phenotype

• Why is the A2 phenotype important?

– A2 and A2B individuals may produce an anti-A1

– This may cause discrepancies when a crossmatch is done (incompatibility)

• What’s the difference between the A1 and A2 antigen?

A1 and A2 Subgroups*

Anti-A

antisera

Anti-A1

antisera

Anti-H

lectin

ABO

antibodies

in serum

# of

antigen

sites per

RBC

A1 4+ 4+ 0 Anti-B 900 x103

A2 4+ 0 3+ Anti-B &

anti-A1

250 x103

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

Other A subgroups

• There are other additional subgroups of A

– Aint (intermediate), A3, Ax, Am, Aend, Ael, Abantu

• A3 red cells cause mixed field agglutination when

polyclonal anti-A or anti-A,B is used

• Mixed field agglutination appears as small agglutinates

with a background of unagglutinated RBCs

• They may contain anti-A1

B Subgroups

• B subgroups occur less than A subgroups

• B subgroups are differentiated by the type

of reaction with anti-B, anti-A,B, and anti-H

• B3, Bx, Bm, and Bel

Other ABO conditions

• Bombay Phenotype (Oh)

• Inheritance of hh

• The h gene is an amorph and results in little or no production of L-fucosyltransferase

• Originally found in Bombay (now Mumbai)

• Very rare (130 worldwide)

Bombay (Oh) Phenotype

• Total Lack of H, A, and B antigens

• Develop strong anti-H, anti-A, and anti-B

• “O” forward, “O” reverse; with positive antibody

screen

GENERAL CHARACTERISTICS OF

BOMBAY Oh+ (Hnull) Phenotypes

• Absence of H, A, and, B antigens; no agglutination with anti-A,-B, -H

lectin

• Presence of anti-A, anti-B, and anti-A,B and a potent wide thermal

range of anti-H lectin

• A,B,H nonsecretor (no A, B, or H substances present in saliva)

• Absence of α-L-fucosyltransferase (H enzyme) in serum and H antigen on red cells

• Presence of A or B enzymes in serum (depending on ABO genotype)

• A recessive mode of inheritance (identical phenotypes in children but not in parents)

• RBCs of the Bombay phenotype will not react with the anti-H lectin

• RBCs of the Bombay phenotype are compatible only with the serum from another individual

H deficient phenotypes

• Basic Concepts

– Rare phenotypes in which the RBCs are completely

devoid of H antigens or that have small amounts of H

antigen present

– Three categories:

1) Category 1:

2) Category 2:

3) Category 3:

CLASSIFI-

CATION

Proposed

Genes

inherited

Glycosyl-

transferase

Red Cell

antigens: A,

B, and H

detected

A, B, and H

soluble

substances

in secretions

Antibodies in

serum

Category 1

Oh, OhB, Oh

A,

OhAB

hh sese None or A

and/or B in

serum or

RBC stroma

None

detectable

None

detectable

Anti-A, anti-

B, anti-H

Category 2

Oh, Ah, Bh,

ABh

A and/or B

hh sese

A and/or B in

serum and

RBC stroma

Weak A/B

Residual H

when A or B

immunodomi

nant sugar is

removed

with

appropriate

enzyme

None

detectable

Anti-H, Anti-

A/Anti-B

Category 3

OhO, Oh

A, OhB,

OhAB

Se A and/or B in

serum/

RBCs, H in

serum

(weak)

Weak A/B

and H

H substance

(normal

amounts)

A/B (all

normal

amounts)

Weak IH

Anti-A/anti-B

ABO DISCREPANCIES

• Group I

• Group II

• Group III

• Group IV

***Assignment: Resolution of the different group

discrepancies (1/2 crosswise)

GROUP I DISCREPANCIES

• Unexpected reactions in reverse grouping due to weakly reacting or

missing antibodies

• More common than most of the other groups

• Suspected when reaction in the serum grouping is weak or missing

• Reason: Patient has depressed antibody production or cannot

produce ABO antibodies

• Rare group I discrepancies: Chimerism: presence of two cell

populations in a single individual

GROUP I DISCREPANCIES

• Some of the more common populations with discrepancies in

this group are:

– Newborns

– Elderly patients

– Patients with leukemias demonstrating hypogammaglobulinemia or

lymphomas

– Patients using immunosuppressive drugs that yield

hypogammaglobulinemia

– Patients with congenital agammaglobulinemia or immunodeficiency

diseases

– Patients with bone marrow transplantations

– Patients whose existing ABO antibodies may have been diluted by

plasma transfusion or exchange

– ABO subgroups

GROUP II

DISCREPANCIES • Unexpected reactions in the forward grouping due to

weakly reacting or missing antigens

• Least frequently encountered

• Some of the causes are: – Subgroup of A (or B) may be present

– Leukemias may yield weakened A or B antigens

– Hodgkin’s disease has been reported in some cases to mimic the

depression of antigens found in leukemia

– Acquired B phenomenon is most often associated with diseases of the

digestive tract ( cancer of the colon)

GROUP II

DISCREPANCIES

• Rare Group II discrepancies

– Excess amounts of Blood group-specific soluble

(BGSS) substances present in the plasma in

association with certain diseases such as carcinoma

of the stomach and pancreas

– Antibodies to low-incidence antigens in reagent anti-A

or anti-B

– Chimerism

GROUP III

DISCREPANCIES • Between forward and reverse grouping caused by protein or

plasma abnormalities and result in roleaux formation or

pseudoagglutination attributable to:

– Elevated levels of globulin from certain disease states (MM,

Waldenstrom’s macroglobulinemia, plasma cell dyscrasias,

moderately advanced cases of Hodgkin’s lymphomas)

– Elevated levels of fibrinogen

– Plasma expanders (dextran and polyvinylpyrrolidone)

– Wharton’s jelly in cord blood samples

GROUP IV

DISCREPANCIES • Between forward and reverse groupings due to

miscellaneous problems and have the following :

– Cold reactive autoantibodies in which RBCs are so heavily

coated with antibody that they spontaneously agglutinate,

independent of the specificity of the reagent antibody

– Patient has circulating RBCs of more than one ABO group due

to RBC transfusion or marrow transplant

– Unexpected ABO isoagglutinins

– Unexpected non-ABO alloantibodies

GROUP IV

DISCREPANCIES

• Rare Group IV discrepancies

– Antibodies other than anti-A or anti-B may react to

form ag-ab complexes that may then adsorb onto

patient’s RBCs

– Some individuals have antibodies against acriflavin in

their serum

• Px’s ab combines with the dye and attaches to the px’s rbcs,

resulting in agglutination in the forward grouping

ABO Blood Group:

ABO Antibodies

Landsteiner’s Rule:

• Normal, Healthy

individuals possess

ABO antibodies to

the ABO antigen

absent from their

RBCs

ABO Blood Group System

• The ABO Blood Group System was the first to be

identified and is the most significant for transfusion

practice

• It is the ONLY system that the reciprocal antibodies are

consistently and predictably present in the sera of people

who have had no exposure to human red cells

Blood Group Systems

• Most blood group systems (ABO and others) are made up of:

– An antigen on a red cell and the absence of it’s corresponding antibody in the serum (if you’re A, you don’t have anti-A)

• If you do NOT have a particular antigen on your red cells then it is possible (when exposed to foreign RBCs) to illicit an immune response that results in the production of the antibody specific for the missing antigen

ABO

• Remember:

– The ABO Blood Group System does NOT

require the presence of a foreign red blood

cell for the production of ABO antibodies

– ABO antibodies are “non-red blood cell

stimulated” probably from environmental

exposure and are referred to as “expected

antibodies”

ABO antibodies

• group A serum contains anti-B

• group B serum contains anti-A

• group AB serum contains no antibodies

• group O serum contains anti-A, anti-B, and

anti-A,B

Anti-A1

• Group O and B individuals contain anti-A in their

serum

• However, the anti-A can be separated into

different components: anti-A and anti-A1

Anti-A,B

• Found in the serum of group O individuals

• Reacts with A, B, and AB cells

• Predominately IgG, with small portions

being IgM

• Anti-A,B is one antibody, it is not a mixture

of anti-A and anti-B antibodies

ABO antibodies

• Activate complement

• React at room temperature or colder

• IgM is the predominant antibody in Group A and Group B

individuals

• IgG (with some IgM) is the predominant antibody in

Group O individuals

ABO antibody facts

• Reactions phase:

• Complement can be activated with ABO

antibodies (mostly IgM, some IgG)

• High titer:

ABO Antibodies

• Usually present within the first 3-6 months of life

• Stable by ages 5-6 years

• Decline in older age

• Newborns may passively acquire maternal antibodies

(IgG crosses placenta)

– Reverse grouping (with serum) should not be

performed on newborns or cord blood

Nature of antibodies

• Non-red blood cell stimulated (previously discussed)

– ABO antibodies

• Red blood cell stimulated

– Antibodies formed as a result of transfusion, etc

– Usually IgG

– Active at 37°C

– Can occur in group O (may occur in group A or B)

– These antibodies also occur in the other Blood Group Systems

Laboratory Testing:

ABO typing

The Use of Lectins for Antigen

Confirmation

• Dolichos biflorus = anti-A1

• Ulex europaeus = anti-H

58

ABO Blood Groups

ABO

Group

Antigen

Present

Antigen

Missing

Antibody

Present

A A B anti-B

B B A anti-A

O None A and B anti-A, anti-B, anti-A,B

AB A and B None None

Forward & Reverse Typing

anti-A anti-B A cells B cells ABO

group

1 0 0 + + O

2 + 0 0 + A

3 0 + + 0 B

4 + + 0 0 AB

Reaction of cells

tested with:

Reaction of serum

tested with:

ABO Ag LOCATION IN THE

BODY

• Body fluids

– Saliva

– Tears

– Urine

– Digestive juices

– Bile

– Milk

– Amniotic fluid

• Pathologic fluids

– Pleural

– Peritoneal

– Pericardial

– Ovarian cyst

ABO Antigens and Antibodies

• ABO antigens based on combinations of three genes: A,

B, and O

• Antibodies are clinically significant and “naturally

occurring”

– causing most fatal acute HTRs

– some causing HDFN

• ABO antibodies neutralized with secretor saliva.

Group O

• Generally the most common blood group

• Genotype: OO

• Antigen: H

• Antibodies: anti-A, anti-B, and anti-A,B

– Antibodies are naturally occurring and very strong

– Anti-A,B (mostly IgG) may cross placenta to cause

HDFN

Group A

• Genotype:

• Antigen:

• Antibodies:

• A subgroups

Group B

• Genotype:

• Antigen:

• Antibodies:

• B subgroups: Not important

Group AB

• Genotype:

• Antigen:

• Antibodies:

• B subgroups: Not important

• A2B:

ABO Testing

• Cell typing (forward grouping) to determine antigen

types on RBCs

• Serum/plasma typing (reverse grouping or

backtyping) to determine type of antibody in serum:

• Note the opposite reactions

– If the forward reactions are opposite of reverse, an

ABO discrepancy is not present.

ABO Grouping Reagents

• Forward Grouping Reagent

• Reverse or Back Tying Cells

Forward Grouping Reagent

Forward Grouping

• Reagent: Monoclonal antibody

– Highly specific

– IgM

– Expected 3+- to 4+ reaction

– 1 drop

– Anti-A=Blue; anti-B=Yellow (Acroflavin dye)

• A and B antigens on patient red cells are agglutinated by known sera (anti-A, anti-B)

Reverse or Back Tying

Reagent Cells

Reverse or Back Typing

• Reagent Cells: Human Source

– Expected 2+ to 4+ reaction

– 4-5% cell suspension

– 1 drop

• Anti-A or anti-B antibodies in patient serum (or plasma)

agglutinate with A1 and B antigens on Reagent cells

Forward Typing Procedures

• To determine what antigens are present

on RBCs.

Step 1. Label test tubes.

Step 2: Make a 2-5%

patient red cell suspension.

Step 3: Add reagent

antisera (1 drop).

Step 3A: Add reagent Anti-A antisera

(1 drop).

Step 3B: Add Anti-B reagent antisera

(1 drop).

Step 4: Add one drop of 2-5% suspension

of patient RBC to each tube.

80

Step 5: Mix and centrifuge

(approximately 20 seconds).

Group A: 4+ Agglutination with Anti-A

0 Agglutination with Anti-B

Group B: 4+ Agglutination with Anti-B

0 Agglutination with Anti-A

Group AB: 4+ Agglutination with Anti-A

and Anti-B

Group O:

No Agglutination with Anti-A or Anti-B

Back Typing

• To determine what antibodies are present

in patient’s plasma.

Step 1: Label Test Tubes

Step 2: Add two drops of

patient serum to each tube

Step 3: Add one drop of

reagent cells to each test tube

Step 3A: Add one drop of

Reagent A1 cells

Step 3B: Add one drop of

Reagent B cells

Step 4: Mix and centrifuge

(approximately 20 seconds)

Group A: 4+ Agglutination with B Cells

0 Agglutination with A1 Cells

Group B: 4+ Agglutination with A1 Cells

0 Agglutination with B Cells

Group O: 4+ Agglutination with A1 Cells

3+ Agglutination with B Cells

Group AB:

No Agglutination with A1 and B Cells

What can Cause ABO

Discrepancies?

• Disagreement between the

interpretations of forward and

reverse grouping

• Antigen problems

• Antibody problems

Antigen Problems

• Lack of expected antigens

• Presence of unexpected antigens

Antibody problems

• Lack of expected antibodies

• Presence of unexpected antibodies

A Subgroups

• A1

• A2

• A3

• Ax

• Aend

• Am

• etc

A1 vs A2 Phenotypes

Blood Group Anti-A Anti-A1 lectin

A1 (80%) + +

A2 (20%) + 0

• A1 & A2 account for 99% of A group

A1vs A2 Phenotypes

• Quantitative differences:

• Qualitative differences between A1 and

A2 antigens:

B Subgroups

• Very rare and are less frequent than A

subgroups.

• B subgroups demonstrate variations in

the strength of the reaction using anti-

B and anti-A,B

• Examples are: B3, Bx, Bm, Bel

Acquired B phenotype

• Occurs in type A individuals with:

• Bacteria deacetylate group A sugar

(GalNAc); remaining galactosamine

crossreacts with reagent anti-B.

Acquired B phenotype

Acquired B phenotype

• AB forward (with weak reactions with

reagent anti-B)

• A reverse

• Reaction with anti-B is negative, if:

Acquired B typing result

Forward Reverse

Anti-A Anti-B Interp A1 cells B cells Interp

4+ 1-2+ AB 0 4+ A”B”

Blood Type:

Antigens vs Antibodies

Blood Type Antigens Antibodies

on rbcs in Plasma

A A Anti-B

B B Anti-A

AB A,B None

O None Anti-A, Anti-B

Consequences of

ABO incompatibility

• Severe acute hemolytic transfusion

reactions

– One of the most frequent causes of blood

bank fatalities

– Clerical errors

• Most frequent HDFN; usually mild.

Sources of Technical Errors

Resulting in ABO Discrepancies

• Inadequate identification of blood samples

• Cell suspension too heavy or too light

• Clerical errors

• A mix-up in samples

• Missed observation of hemolysis

• Failure to add reagents

• Failure to follow manufacturer’s instructions

• Uncalibrated centrifuge

• Contaminated reagents

• Warming during centrifugation

Resolving ABO

Discrepancies

Problems with RBCs Resolution Techniques

Rouleaux

MF agglutination

Unusual phenotype (hh)

Disease processes (Acq. B)

Resolving ABO

Discrepancies (Cont’d) Problems with serum Resolution Techniques

Rouleaux

Presence of unexpected Ab

Absence of expected Ab