ABO blood group system

Dr R AmitaAssistant Professor

Dept of Transfusion Medicine

Discovery

• Karl Landsteiner(1900) discovered human A,B,O groups.

• Von Decastello and Sturli (1902) discovered AB blood group.

• Von Dungern and Hirszfeld(1911) divided group A into 2 subgroups A1 and A2.

ABO system is classified into 6 groups: A1, A2, A1B, A2B, B, AB and O

Landsteiner’s law

• 1.If an agglutinogen is present on red blood cell membrane ,the corresponding agglutinin must be absent in the plasma.

• 2.If an agglutinogen is absent on red blood cell membrane, then corresponding agglutinin must be present in the plasma.

Antigens

• Appear in the sixth week of fetal life. • Present on red cell membrane, WBC, platelets and in other

tissues like salivary glands, pancreas, kidney, body fluids • Exception CNS

ABO gene• The ABO blood type is controlled by a single gene (the ABO

gene) with three types of alleles i, IA, and IB. • The gene encodes a glycosyltransferase• The gene is located on the long arm of the ninth

chromosome (9q34).• IA allele gives type A, IB gives type B, i gives type O.• Co dominant• O group : Only ii AB group : IAIB• A group : IAIA or IAi B group : IBIB or IBi

Genetics• Inheritance of genes follows Mendelian Law• Bernstein theory: there is one locus on a chromosome at

which any of the three alleles may be located

ABO Antigen Genetics• The presence or absence of the ABH antigens on the red blood

cell membrane is controlled by the H gene (chr 19)• The presence or absence of the ABH antigens in secretions is

indirectly controlled by the Se gene (chr 19)

• H gene – H and h alleles (h is an amorph)

• Se gene – Se and se alleles (se is an amorph)

• ABO genes – A, B and O alleles

Biochemistry • Precursor: Paragloboside/Glycan• Type I precursor : terminal galactose linked to a subterminal N-acetylgluosamine in a 1-3 linkage. • Type II precursor : same sugars combine in a 1-4 linkage

• ABH antigens on RBC are derived from Type II chains • Blood group substances in secretion are made from both types I & II precursors

Precursor Substance

Glucose

Galactose

N-acetylglucosamineGalactose

Precursor Substance (stays

the same)

RBC

H substance

• H gene (FUT1 gene) leads to production of an enzyme α-2-L-Fucosyl transferase, which transfers fucose to the terminal galactose of the precursor

Glucose

Galactose

N-acetylglucosamineGalactose

RBC

Fucose

A antigen

• The “A” gene codes for N-acetylgalactosaminyltransferase that adds N-acetylgalactosamine to the terminal sugar of the H antigen

Glucose

Galactose

N-acetylglucosamineGalactose

RBC

Fucose N-acetylgalactosamine

B antigen

• The “B” gene codes for D-galactosyltransferase that adds D-galactose to the terminal sugar of the H antigen

Glucose

Galactose

N-acetylglucosamine

Galactose

RBC

Fucose Galactose

ABO Subgroups

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

• Subgroups have fewer antigens are present on the RBC

• Subgroups are the result of less effective enzymes (not as efficient in converting H antigens to A or B antigens)

• Subgroups of A are more common than subgroups of B

Subgroups of A• A1 and A2

• Both react strongly with reagent anti-A• To distinguish A1 from A2 red cells, the lectin

Dolichos biflorus is used (anti-A1)• 80% of group A or AB individuals are subgroup

A1• 20% are A2 or A2B

A2 Phenotype• The A2 gene doesn’t convert the H3 & H4 to A

very well• The result is fewer A2 antigen sites compared to

the many A1 antigen sites. • A2 and A2B individuals may produce an anti-A1• This may cause discrepancies when a

crossmatch is done.

A1 and A2 SubgroupsAnti-A

antiseraAnti-A1 antisera

Anti-H lectin

ABO antibodies in serum

# of antigen sites per

RBC

A14+ 4+ 0 Anti-B 900 x103

A24+ 0 3+ Anti-B &

anti-A1

250 x103

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 • Missense mutation of FUT1 gene• The h gene is an amorph and results in little or no

production of L fucosyltransferase • Originally found in Bombay (now Mumbai) by Bhende

in 1952• Very rare (Frequency in India 1:10000)

Bombay group• The hh causes NO H antigen to be produced• Results in RBCs with no H, A, or B antigen (Cell group: O)• Bombay RBCs are NOT agglutinated with anti-A, anti-B, or

anti-H (no antigens present)• Bombay serum has strong anti-A, anti-B and anti-H,

agglutinating ALL ABO blood groups.• Group O RBCs cannot be given because they still have the

H antigen• Transfuse the patient with blood that contains NO H antigen

Parabombay phenotype

• H antigen is weakly expressed on RBCs. Weak expression of A, B, H antigens on the red cells (Due to passive adsorption from secretion) which react weakly with antisera to A, B, H antigens• H antigen is present in the secretions, but there is

no expression on red cells. Serum contains anti-H antibodies

• Genotype:hh/Sese or hh/SeSe

ABO Antigens in Secretions• Secretions include body fluids like plasma, saliva, synovial

fluid, etc• Blood Group Substances are soluble antigens (A, B, and H) that

can be found in the secretions. • This is controlled by the H and Se genes• Se gene (FUT2gene) encodes α2 L fucosyltransferase which

modifies type 1 precursor to H substance• If the Se allele is inherited as SeSe or Sese, the person is called

a “secretor”• 80% of the population are secretors

Secretor Status• 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 antigenB 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

H antigen• Certain blood types possess more H antigen than others:• The O gene is a silent allele. It does not alter the structure

of the H substance….that means more H antigen sites

O>A2>B>A2B>A1>A1BGreatest

amount of HLeast

amount of H

Group O Group A

Many H antigen sites

Most of the H antigen sites in a Group A individual have been

converted to the A antigen

Fewer H antigen

sites

A

A A

AA

Group O Group A

ABO antibodies• Natural antibodies: 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.

• Titer of ABO Abs is often reduced in elderly and in patients with hypogammaglobulinemia and infants (until 3 -6 months of age)

ABO antibodies

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

• Anti-A• Anti-B

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

• Anti-A,B (with some anti-A and anti-B)

Anti-A• 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-A1 only agglutinates the A1 antigen, not the A2 antigen

• Occurs in 1-8% of A2 and 22-30% of A2B• There is no anti-A2.

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

Anti H antibody

• A1 gene very efficiently converts all of H substance to A antigen,

• Therefore some A1 and A1B individuals may have anti H

• IgM cold agglutinin• Best reacts at room temperature

Anti-HAuto-Anti-H

Clinically Significant

No

Abs classIgM

Thermal range4 - 15

HDNBNo

Transfusion Reactions

Extravascular Intravascular

No yes

Allo-Anti-H (Bombay group)

Clinically Significant

Yes

Abs classIgM, IgG

Thermal range4 - 37

HDNBYes

Transfusion Reactions

Extravascular Intravascular

Yes Yes

Frequency of blood group system

RBC Phenotype Frequency (%) Serum Ab

A 24 Anti-B

B 30 Anti-A

AB 6 --------

O 40 Anti-A,B

Transfusion

ABO discrepancies

• Group I Discrepancies - • These are associated with unexpected reactions in the

reverse grouping due to weakly reacting or missing antibodies.

• Includes:• Infants less than 4-6 month of age• Elderly patients• Severe hypogammaglobulinemia • ABO incompatible HPC transplantation

• Resolution:• Enhancing weak or missing reaction by incubating the

patient’s serum with reagent A1 and B cells at room temperature for 15-30 minutes

• Serum cell mixture is incubated at 4⁰C for 15-30 minutes • An autocontrol and O cell control must always be tested

concurrently to detect reactivity of other commonly occurring cold agglutinins eg: anti I

• Group II discrepancies• These are associated with unexpected reactions in forward grouping due to weakly

reacting or missing antigen• Includes:• Weak subgroups of A or B• Weakening of A or B antigen in malignancies• Acquired B phenotypes: • results from the action of bacterial deacetylase, which converts N-

acetylgalactosamine to ẞ-galactosamine, which is very similar to galactose, the chief determinant of B.

• ‘passenger antigen’ type is caused by adsorption of B-like bacterial products on to O or A cells but occurs only in vitro.

• Out of group transfusion or ABO mismatched HPSCT • Neutralization of anti A and anti B typing reagent by high concentration of A or B

soluble substances in serum with serum or plasma suspended red cell

• Resolution of group II discrepancies • Weaker reactions with antisera can be resolved by enhancing reaction of

antigen with respective antisera by incubating test mixture at room temperature for 15-30 minutes

• Sub groups causing group discrepancies can be resolved by adsorption elution studies

• Acquired B phenomenon can be resolved by lowering PH of monoclonal antisera. Anti B in the serum of acquired B person does not agglutinate autologous red cells (autocontrol negative).

• Secretor status of person can resolve acquired B, saliva of acquired B person contains A substance not B substance.

• High concentration of A or B substance causing group discrepancies can be resolved by saline washing of red cells

• Group III discrepancies • These are associated with protein or plasma abnormalities, rouleaux formation

and pseudoagglutination.• Includes • Elevated level of globulin from e.g. multiple myeloma, waldenstorm

macroglobulinemia, Hodgkin lymphoma. • Elevated level of fibrinogen. • Small fibrin clot in plasma or incompletely clotted serum can be mistaken for

red cell agglutinates in reverse grouping. • Sample with abnormal concentration of serum proteins, altered serum protein

ratio, or high molecular weight volume expanders can aggregate reagent red cells and can mimic agglutination.

• Rouleaux will disperse when suspended in saline. True agglutination is stable in the presence of saline

• Group IV discrepancies are due to miscellaneous problems.• Recent transfusion of out of group plasma containing

component. • Cold alloantibodies (e.g. anti M) or autoantibodies (e.g. anti I),

pH dependent autoantibodies, a reagent dependent antibody (e.g. EDTA, paraben) leading to unexpected positive eaction.

• Recent infusion of IvIg which can contain ABO isoagglutinins. • Mixed field agglutination with circulating red cell of more than

one ABO type.

Resolving ABO discrepancy