ABO blood group system

1. ABO blood group system
Dr R Amita
Assistant Professor
Dept of Transfusion Medicine

2. 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

3. 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.

5. 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

6. 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

7. 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

8. 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

9. 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

10. Precursor Substance
Glucose
Galactose
N-acetylglucosamine
Galactose
Precursor
Substance
(stays the
same)
RBC

11. 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-acetylglucosamine
Galactose
RBC
Fucose

12. A antigen
• The “A” gene codes for
N-acetylgalactosaminyltransferase
that adds N-acetylgalactosamine to
the terminal sugar of the H antigen
Glucose
Galactose
N-acetylglucosamine
Galactose
RBC
Fucose N-acetylgalactosamine

13. 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

14. 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

15. 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

16. 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.

17. 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

18. 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

21. 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

22. 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)

23. 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

24. 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

26. 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

27. 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 antigen
B antigen
se gene (sese) No antigens secreted
in saliva or other body
fluids
and/or

28. 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

29. 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>A1B
Greatest
amount of H
Least
amount of H

30. 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

31. 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)

32. 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)

33. 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.

34. 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

35. 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

36. Anti-H
Auto-Anti-H
Clinically
Significant
No
Abs class
IgM
Thermal range
4 - 15
HDNB
No
Transfusion Reactions
Extravascular Intravascular
No yes
Allo-Anti-H (Bombay group)
Clinically
Significant
Yes
Abs class
IgM, IgG
Thermal range
4 - 37
HDNB
Yes
Transfusion Reactions
Extravascular Intravascular
Yes Yes

37. 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

38. Transfusion

39. 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

40. • 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

41. • 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

42. • 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

43. • 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

44. • 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.

45. Resolving ABO discrepancy