PART III

BLOOD GROUP
SYSTEM
 Blood Group Antigens

• An inherited character of the red cell

surface.
• Blood group antigens may be:
Glycoproteins. Ex: Lewis
Glycolipids. Ex: ABO
• Incidence:
Low Incidence. Ex: Kell
High Incidence. Ex: I
Immunogenicity of Blood Group Antigens
A, B and D (Rho) – most immunogenic
Kell (K)
Duffy
Kidd
Blood Group Antibodies

1. Autoantibody

1. Alloantibody
• Naturally occurring
• Immune antibodies
ABO and
H
Systems
 ABO SYSTEM
• The first blood group system to be
discovered by Landsteiner in 1900
• Most Immunogenic.
• Naturally occurring; IgM; React at room
temp.
• ISBT 001.
• Three antigens: A, B, H
• Two major antibodies: anti-A and anti-B
• Four phenotypes: A, B, AB, O
 Landsteiner Law
1. Ag on the RBC determines the
blood group.
2. The corresponding Ab is never
found in the individual’s serum.
3. The opposite Ab is always present
in the individual’s serum.
 ABO NOMENCLATURE

Moss Jansky Landsteiner

I IV AB

II III A

III II B

IV I O
 ABO Inheritance &
Genetics
• The ABO gene is autosomal.
• The ABO gene locus is located on the
chromosome 9.
• A and B blood groups are dominant over the O
blood group
• A and B group genes are co-dominant.
• Two genes inherited, one from each parent.
• Individual who is A or B may be homozygous or
heterozygous for the antigen.
 Heterozygous: AO or BO
 Homozygous: AA or BB
 Basic Biochemistry
Type 1 and 2 Chains
 Linkages
 Type 1: Terminal galactose (Gal) linked to a sub-
terminal N-acetylglucosamine (GlcNAc) in a 1,3
linkage
 Type 2: Same sugars combine in a 1,4 linkage
 Nature:
 Type 1: Glycoproteins in saliva and glycolipids in
plasma carrying free-floating antigens
 Type 2: Glycolipids and glycoproteins carrying
bound antigens on RBCs
• The presence or absence of the A, B, and H
antigens is controlled by the H and ABO genes.

• 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 indirectly controlled by the Se
gene.
• Se (secretor) gene
 Required to make A or B antigens in secretions
 Product is type 1 H antigen
 80% gene frequency

• H gene
 Closely linked to Se on chrom. 19
 Product is type 2 H antigen.
 Virtually 100% gene frequency (Bombay = hh).
RBC Precursor Structure

RBC

Glucose

Galactose
Precursor
Substance
(stays the N acetylglucosamine
same)
Galactose
 ABH Antigens

• Red cell Ag : Type 2 Chain

– 80% glycoproteins, 20% glycolipids

• Plasma ABH Ag : Type 1 chain

– Predominantly glycolipids
Formation of ABH Antigens
• The alleles “code” for blood type.
• What they REALLY “code” for is a specific enzyme.
• That enzyme creates specific antigens on your RBC.
• Glycosyltransferases: are enzyme that facilitate
the transfer of carbohydrate (sugar) molecules
onto carbohydrate precursor molecules
• Immunodominant sugar: is the sugar molecule that
complete the antigenic determinant when combined
with the precursor substance
• The O gene is a silent allele.
 H antigen

• The H antigen is the foundation

upon which A and B antigens are
built.
• H gene codes for an enzyme that
adds the sugar fucose to the
terminal sugar of a precursor
substance (PS)
Formation of the H antigen

RBC

Glucose

H antigen Galactose

N-acetylglucosamine

Galactose

Fucose
 A and B Antigen

• The “A” gene codes for an enzyme

(transferase) that adds
N-acetylgalactosamine to the terminal sugar of
the H antigen.

• The “B” gene codes for an enzyme that

adds D-galactose to the terminal sugar
of the H antigen.
 A antigen b antigen

RBC RBC

Glucose Glucose

Galactose Galactose

N- N-
acetylglucosamine acetylglucosamine
Galactose Galactose

N-
Fucose Galactose
acetylgalactosamine Fucose
As more A or B is made, less H remains.
• H amount: O > A2 > B > A2B > A1 > A1B
1. Hh gene – H and h 1. Controls presence of H,
alleles (h is an a morph) A, and B antigens on
both RBCs and in
Secretions

2. Se gene – Se and se 2. Controls presence of H

alleles (se is an amorph) antigen in the secretions

3. ABO genes– A, B and O 3. Inherit 1 gene from each

alleles parent that codes for an
enzyme that adds a
sugar to the H antigen
Gene Glucosyltransferase Immunodominant Antigen
sugar

H L-fucose H
L- fucosyl
transferase

A N- N-acetyl-D- A
acetylgalactosaminyl galactoseamine
transferase

B D- galactosyl D-galactose B
transferase
 ABO ANTIBODIES
• Generally IgM class antibodies
• Antibodies clinically significant, naturally occurring.
• Three antibodies: anti-A, anti-B and anti-A,B
• For Group A and Group B persons the predominant
antibody class is IgM
• For Group O people the dominant antibody class is IgG
(with some IgM)
• React best at room temperature (22-24oC) or below in
vitro.
• Activates complement to completion at 37oC
• Can cause acute Hemolytic Transfusion reactions
RBC Phenotype Frequency (%) Serum Ab

A 43 Anti-B

B 9 Anti-A

AB 4 --------

O 44 Anti-A,B
 ABO Blood Groups
Native
Type Whites Blacks Asians Americans

O 45% 49% 40% 79%

A 40% 27% 28% 16%

B 11% 20% 27% 4%

AB 4% 4% 5% <1%
 Group O
Group O (Genotype OO)
• The most common blood group across racial
lines
• “Universal Donor”
• Antigen : H
• Antibodies : Anti-A, anti-B, anti-A,B
• Lectin :Ulex europaeus +
 Group a
Group A (Genotypes AA or AO)
• Antigens: A, H
• Antibody: anti-B (primarily IgM).

A subgroups
o A1 (80%) and A2 (~20%) most important
o Monoclonal anti-A agglutinates both types well
o 1-8% of A2 and 25% of A2B form anti-A1
• Common cause of ABO discrepancies
o Dolichos biflorus lectin: A1 RBCs +, A2 RBCs –
WEAK A SUBGROUPS

1. A3
React weakly
2.Ax with Anti-A rgt.

3.Aend
4.Am Do NOT weakly
5.Ay react with Anti-A
rgt.
6.Al
 Group b
Group B (Genotypes BB or BO)
• Antigens: B, H
• Antibodies: Anti-A (primarily IgM)
• Lectin: Bandeiraea simplifolica +
B subgroups
– Usually unimportant and less frequent
WEAK B SUBGROUPS

1. B3
2.Bx Do NOT weakly
react with Anti-B
3.Bm rgt.

4.Bel
 Group ab
Group AB (Genotype AB)
• Least frequent ABO blood type
• “Universal Recipient”
• Antigens: A and B (very little H)
 Can be further subdivided into A1B
or A2B
• Antibodies: No ABO antibodies
 Bombay Phenotype (Oh)

• Genotype: hh
• Antigen: NO ABH antigen
• Antibodies : Anti-A, anti-B, anti-H
• Lectin :Ulex europaeus -
Classificatio Genes Glycosyltransferase RBC Presence Abs
n Ag of Present
Presen ABH
t Subs.
Classic hh/ None None None Anti-A,
Bombay sese Anti-B,
Anti-H

Para hh/Se A &/or B transferase Weak Yes Weak

Bombay to anti-H,
None Anti-A/B

H-partially Weak A &/or B transferase Weak None Anti-H,

def. Variant Anti-A/B
hh/se
 Abo grouping
Forward blood grouping/Cell Typing
Determination of ABO antigens found on patient red blood cells using reagent
anti-sera.
*Anti-A: Blue, Ditrypan Blue
*Anti-B: Yellow, Acriflavin Yellow

Patient Red Cells Tested With

Patient Anti-A Anti-B Interpretation

1 0 0 O

2 4+ 0 A

3 0 4+ B

4 4+ 4+ AB
Reverse Grouping / Back Typing
*Patient Serum tested with 2-5% known red cell suspension
*Confirmatory grouping

Patient A1 Cells B Cells Interpretation

1 4+ 4+ O

2 0 4+ A

3 4+ 0 B

4 0 0 AB
 TYPE O vs. BOMBAY
FORWARD TYPING BACKWARD
TYPING
Anti- Anti-B Anti- A Cell B O
A H Cell Cell
Type 0 0 4+ + + 0
O

Oh 0 0 0 + + +
 ABO DISCREPANCIES
• ABO discrepancies happen when there is no
match in results between forward and reverse
grouping.

• ABO discrepancies are usually technical in

nature and can be simply resolved by
correctly reporting the testing and carefully
checking reagents with meticulous reading and
recording of results.
 Technical Errors
• Clerical errors
– Mislabeled tubes
– Patient misidentification
– Inaccurate interpretations recorded
– Transcription error
– Computer entry error
• Reagent or equipment problems
– Using expired reagents
– Using an uncalibrated centrifuge
– Contaminated or hemolyzed reagents
– Incorrect storage temperatures
• Procedural errors
– Reagents not added
– Manufacturer’s directions not followed
– RBC suspensions incorrect concentration
– Cell buttons not resuspended before grading agglutination
 ABO DISCREPANCIES
• Problems with RBCs
– Weak-reacting/Missing antigens
– Extra antigens
– Mixed field reactions

• Problems with serum

– Weak-reacting/Missing antibodies
– Extra antibodies
 ABO DISCREPANCIES
• Group I discrepancies
• Group II discrepancies
• Group III discrepancies
• Group IV discrepancies
UNEXPECTED MISSING
REACTION REACTION
CELL TYPING GROUP IV GROUP II
SERUM TYPING GROUP III GROUP I
 Group I discrepancies
Weak/Absence of Antibody Rxtn

• Age - newborns/elderly
• Hypogammaglobulinemia
• Agammaglobulinemia
• Chimerism
• Often shows NO agglutination on
reverse groupings
*Most Common discrepancy
 Group II discrepancies
Weak/Absence of Antigen Rxtn

• A & B Subgroups
• Leukemia, Hodgkins
• Acquired B phenomenon
• BGSS (blood group soluble substance)

*The least common

 Acquired B Phenomenon

Cause: there are two causes of

acquired B phenomenon:

In vivo, patients with bacterial

infections and often cancer of the
colon or rectum may develop a
false B-like antigen.
The mechanism: The bacterial produce a
deacetylase (enzyme) which chemically alters the
terminal sugar of A antigens (N-acetyl-D-
galactosamine) into D-galactosamine.
Because the terminal sugar of the B antigen is
galactose, anti-B antisera will cross react with
the B-like D-galactosamine antigen. Because of
this, in vivo, only group A people can develop an
acquired B-like antigen. The condition is
transient and disappears when the infection is
cured.
 Acquired B
• Bacteria (E. coli) have a deacetylating
enzyme that effects the A sugar….

Group A Acquired
individual B
Phenotype

N-acetyl galactosamine Galactosamine

now resembles
D-galactose (found
Bacterial enzyme in Group B)
removes acetyl group
 Another mechanism
• In vitro, blood specimens can get an acquired B-
like antigen if they are bacterially contaminated.
This is because the membranes of some bacteria
(e.g., E. coli and P. vulgaris ) have determinants
which are chemically similar to the B antigen.

• In this case, anti-B antisera is actually reacting

with the bacterial antigens which have attached to
the red cells.

• In vitro, both group O and group A cells can

acquire the B-like antigen. Note: most examples of
acquired B phenomenon detected in the blood bank
happen in vivo to group A people only.
 Typical reaction pattern

• The reactions with anti-B are weaker than

expected (e.g., 1+ or 2+). The patient's
autocontrol is negative even though anti-B
is present (patient is group A).

• The patient's own anti-B will not recognize

and agglutinate the B-like antigen, but
everyone else's anti-B (including the typing
sera) will.
 Resolution of acquired B
• Check the past records in case the patient is a known group A.
• Check the diagnosis for bacterial infection (with or without
Cancer of the colon or rectum).
• Test the red cells with anti-B reagent acidified to pH 6.0
• If using human polyclonal reagents, redo the ABO group using
monoclonal anti-A and anti-B typing sera, which may resolve
the problem.
• If using monoclonal reagents, redo the ABO group using
human polyclonal anti-A and anti-B typing sera.
• Do autologous control it should give negative result.
• Try secretor status studies (usually not necessary). If the
patient is group A and a secretor, he will secrete A and H
antigens only.
 Group III discrepancies
Unexpected Antibody Rxtn

• Autoantibodies
• Increased globulin content and
fibrinogen
• Plasma expanders
• Wharton’s Jelly
• Rouleaux
 Group IV discrepancies
Unexpected Antigen Rxtn

• Polyagglutination
• Cold Reactive Antibody
• Unexpected ABO isoagglutinin
• Antibodies against acriflavine
 Polyagglutination

• Polyagglutination can occur due to

exposure of hidden erythrocyte Ag. (T
antigen) in patients with bacterial or
viral infection.

• Bacterial contamination in vitro or vivo

produces an enzyme that alters and
exposes the hidden Ag. on red cell
leading to T activation.
 Exercise

Example Anti-A Anti-B A1 cells B cells Tentative group

#1 +4 - +1 +4 A
#2 +4 +4 +2 - AB
 Exercise

Anti-A Anti-B A1 Cells B Cells

4+ 1+ 0 4+
Anti-A Anti-B A1 cells B cells Autocontrol

+4 - +1 +4 2+
Example Anti Anti A1 B Px History Tentative
-A -B cells cells group
#1 +4 - - - Newborn A

#2 - +4 - - Elderly B or AB
 Review

• Predominant immunoglobulin of ABO

antibodies:
a) IgM
b) IgG
 Review

• Blood group of an individual with aquired B

phenomenon:
a) A
b) B
c) O
d) AB
 Review

• Blood group antibody associated with

sever HTR:
a) ABO
b) Rh
c) Kell
d) Kidd
 Review

• No agglutination with anti-A, anti-B and

anti-H:
a) O
b) Kell
c) Bombay
d) Lutheran