ABO Blood Group Systems

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The red cells have antigens on their surface. The importance of
red cell antigens is manifold. Since the work of Landsteiner in
early 1900s, it has been recognized that knowledge and under-
standing of blood groups is essential for transfusion therapy.
HISTORY OF BLOOD GROUPS
The major events in history of blood transfusion are given as
follows:
1900 Karl Landsteiner (Austrian physician), discovered A, B
and O.
AB blood group was discovered in the year 1902 by A Decastello
and A Sturli.
1939/40: The Rh blood group system was discovered by Karl
Landsteiner, Alex Wiener, Philip Levine and RE Stetson.
1945: Coombs, Mourant and Race discovered anti-human
globulin (Coombs’ test) for incomplete antibodies.
However, the efforts to discover blood groups were started
much earlier. The events are given below:
1628: English physician William Harvey discovered the
circulation of blood.
1665–67: Physician Richard Lower (England) and Jean-Baptiste
Denis (France) recorded successful blood transfusion in animals
and report transfusions from lambs to humans.
After this, transfusing the blood of animals to humans
became prohibited by law, delaying the advances in transfusion
medicine for about 150 years.
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ABO Blood Group Systems
38

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ABO Blood Group Systems
39
1795: In Philadelphia, an American physician, Philip Syng
Physick, claimed to perform the first human blood transfusion,
although he did not publish this information.
1818: James Blundell, a British obstetrician, transfused human
blood to a case of postpartum haemorrhage. During 1825 to
1830, he did 10 transfusions, five of these were beneficial to
the patients, and he published these results.
1840: Samuel Armstrong Lane and Blundell, undertook first
successful whole blood transfusion to treat a case of haemophilia.
In 1900 and 1902 the major milestone was discovery of A, B,
O and AB blood groups as stated above.
Landsteiner received the Nobel Prize for Medicine for this
discovery in 1930.
In 1907 Hektoen suggested that the safety of transfusion
might be improved by crossmatching.
In 1912 Roger Lee (physician) from Massachusetts General
Hospital coined “Universal Donor” and “Universal Recipient”.
In 1939/40 another major blood group ‘Rh’ was discovered
followed by 1945, a test to detect ‘Incomplete Antibodies’ by
using anti-human globulin (Coombs’ test) was established.
History of other rare blood groups:
MNS system was discovered in the year 1927.
P Blood group system was also discovered in the same year as
MNS system, i.e. 1927.
Kell blood group was discovered in the year 1946 which was
named after Mrs. Kellner, mother of the first child to be affected
with HDN.
Duffy in 1950, Mr. Duffy was a hemophilia patient.
Kidd blood group was named after Mrs. Kidd whose serum
contained the antibody and antigen was named ‘JK’ after
woman’s child John Kidd, who suffered HDN.
Bombay blood group was discovered by Dr YM Bhende and
colleagues in the year 1952.

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Essentials of Blood Banking and Transfusion Medicine
RED CELL BLOOD GROUP SYSTEMS
In routine practice, it is necessary to determine the compatibility
of certain red cell antigens between the donor and the recipient.
Also understanding of other blood group systems such as Kell,
may be associated with both functional and morphological
changes in the red cells. Study of inheritance of blood group
antigens provides a greater understanding of the mechanism
of gene expression.
Amongst these, the two most important blood groups in
blood transfusion are:
• ABO system
• Rh system
International Society of Blood Transfusion (ISBT) has
instituted a numerical system of nomenclature to standardize
red cell blood grouping terminology. According to ISBT (2004)
around 30 blood group systems are known.
Different blood group systems
(Contd.)
Number
Name
Abbreviation
001
ABO
ABO
002
MNSs
MNSs
003
P
P
004
Rh
Rh
005
Lutheran
LU
006
Kell
KEL
007
Lewis
LE
008
Duffy
FY
009
Kidd
JK
010
Diego
DI
011
Cartwright
YT
012
XG
XG
013
Scianna
SC
014
Dombrock
DO
015
Colton
CO
016
Landsteiner-Wiener
LW
017
Chido/Rodgers
CH/RG
018
Hb
H
019
Kx
XK
020
Geribich
GH

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41
Indian blood group is named after the country, 4% Indians
from Bombay express this blood group. The antigen is encoded
by the CD44 gene located on chromosome 11p13.
It has two variants as shown below:
• IN a
IgG Antibodies
HDN No
No HTR
• IN b
IgG Antibodies
HDN No
Rare HTR
Importance of ABO Grouping
• ABO compatibility between donor cells and patient serum
is the essential foundation of pre-transfusion testing.
• It is the blood group system with expected antibodies.
• Whether they are IgG or IgM, ABO antibodies can activate
complement readily.
• This means that incompatibilities can cause life-threatening
situations (transfusion reactions).
• It is so important that a mistake in grouping and transfusing
can lead to grave complications.
ABO System
• It is based on the presence of two antigens on the wall of
RBCs: A antigen and B antigen. These antigens are formed
from a basic antigen called H antigen which is present in all
individuals with rare exception.
• ABO blood groups: A, B, AB and O.
• ABO system obeys Landsteiner Laws with regard to antigen
and antibodies.
Number
Name
Abbreviation
021
Cromer
CROM
022
Knops
KN
023
Indian
IN
024
Ok
OK
025
Raph
RAPH
026
John Milton Hagan
JMH
027
I
I
028
Gloside
GLOB
029
GIL
GIL
030
RHAG
RHAG

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Essentials of Blood Banking and Transfusion Medicine
Landsteiner Laws
Law 1: If an individual has an antigen on the wall of red cell,
the corresponding antibody should not be present in plasma.
Law 2: If an individual has an antibody in the plasma, the corres-
ponding antigen should not be present on the wall of red cell.
Blood Group Antigens
• The antigenic determinants or epitopes are small portions
of molecules recognised by antibodies. ABO antigens are
carbohydrate in nature. They are oligosaccharide chains
anchored to glycoproteins or glycolipids of the RBC mem-
brane.
• They are highly immunogenic. A and B antigens differ in
only the terminal sugar. There is terminal sugar N-acetyl
galactosamine in A group and terminal sugar galactose in
B group.
• There is no A or B antigen in O blood group. There is absence
of terminal sugars in O phenotype.
Antibodies
• In ABO system there are natural antibodies. They are present
in the serum/plasma. They are IgM type and have high mole-
cular weight and hence cannot pass through the placenta.
• These antibodies react well at 4°C, room temperature or at
37°C.
• Sometimes IgG anti-antibodies may be produced in O blood
group patients and sometimes by A2 blood group patients,
which can cause haemolytic transfusion reactions and
haemolytic disease of newborn.
• Antibodies of ABO blood group system react at a wide
temperature range and potentially haemolytic at 37 degree
C. Group O donors should always be screened for IgG
(hyperimmune) anti-A and anti-B antibodies which may
cause haemolysis, when group O whole blood is transfused
to recipients of A or B blood groups. These dangerous
O donors should be used for O recipients only or should be
used as packed red cells.

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ABO Blood Group Systems
43
The frequency of occurrence of ABO blood groups varies
from country to country and region to region.
Genetics
• The genes for ABO system are at three separate loci (ABO,
Hh and Se) which control the occurrence and location of the
A and B antigens.
• Three common alleles for ABO (A, B, and O) are located at
the ABO locus on chromosome 9. The genes occupy 9q34.1-
q34.2 has 7 exons and is of 18 kb. A and B genes differ in
7 nucleotides, difference is mainly in amino acids at 266 and
267 in exon 7.
• The A and B genes encode glycosyl transferase that produces
A and B antigens, respectively.
• The red cells of O person lack A and B antigens, but carry
abundant amount of H antigen, the precursor material on
which A and B antigens are built.
• The genes for two loci, Hh and Se (Secretor), are on the
chromosome 19 and are closely linked.
• H gene (19q13.3) transmitted in Mendelian Manner. Each
locus has two recognized alleles, one of which has no
demonstrable product. The active allele at the H locus,
H produces a glycosyl transferase that acts at the cellular
level to form H antigen on which A or B are built.
• The Se gene is directly responsible for the expression of
H antigen, which may then be modified to express A and B
on the glycoproteins in epithelial secretions such as saliva.
Eighty percent of the population are secretors. Secretors have
inherited the Se gene and express H antigen, which can be
converted to A and/or B if they also have the A and/or B gene.
TABLE 4.1: ABO blood groups: Antigens, antibodies and frequency of
occurrence
Name of blood group
Antigens
Antibodies
Frequency of occurrence
AB
A, B
None
3%
A
A
Anti-B
42%
B
B
Anti-A
8%
O
None
Anti-A, Anti-B
47%

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Essentials of Blood Banking and Transfusion Medicine
H Antigen
• The H gene codes for an enzyme that adds the sugar fucose
to the terminal sugar of a precursor substance (PS). The
precursor substance (proteins and lipids) is formed on an
oligosaccharide chain (the basic structure).
• The H antigen is the foundation upon which A and B antigens
are built.
• A and B genes code for enzymes that add an immuno-
dominant sugar to the H antigen. Immunodominant sugars
are present at the terminal ends of the chains and confer the
ABO antigen specificity.
• Lectin from Ulex europaeus is source of anti-H and can
detect H antigen.
TABLE 4.2: To show enzymes and sugars added in different blood
groups
Gene
Enzyme coded
Sugar added by enzyme
A
N-acetyl galactosamyl transferase
N-acetyl galactosamine
B
Galactosyl transferase
Galactose
H
Fucosyl transferase
Fucose
O
Defective glycosyl transferase
None
Fig. 4.1: RBC precursor substance

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ABO Blood Group Systems
45
A and B Antigens
The “A” gene codes for an enzyme (N-acetyl galactosaminyl
transferase) that adds N-acetyl galactosamine to the terminal
sugar of the H antigen.
Fig. 4.2: Formation of H antigen on RBC precursor substance
Fig. 4.3: Formation of A antigen with addition of N-acetyl galactosamine
to H antigen.

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Essentials of Blood Banking and Transfusion Medicine
The “B” gene codes for an enzyme D-galactosyl transferase
that adds D-galactose to the terminal sugar of the H antigen.
Association of Blood Groups with Diseases
‘A’ blood group is frequently associated with gastric carcinoma
and ‘O’ blood group is frequent in peptic ulcer patients.
Universal Donors and Recipients
• The earlier concept of ‘O’ blood group as universal donor
and AB blood group as universal recipient does not hold
good in recent years.
• ‘O’ blood group person has no antigens on the red cells but
has anti-A and anti-B antibodies in the serum. When given to
recipient, these antibodies can destroy some of the recipient’s
red cells. Hence, ‘O’ blood, better not to be given to A, B or
AB persons. However, washed ‘O’ red cells can be given.
• Earlier notion of AB person as a universal recipient, as they
have A and B antigen on the red cells does not hold good. A
or B blood groups will have antibodies against AB blood
group antigens and can destroy some red cells of the AB
recipient. Hence A or B blood groups better not to be
Fig. 4.4: Formation of B antigen with addition of galactose to H antigen

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ABO Blood Group Systems
47
transfused to AB blood group patient. However, AB plasma
can be given to A, B or O persons as it does not have any
antibodies. Hence, washed O red cell packs and AB plasma
are universal donors.
Bombay Blood Group
• In this blood group the red cells lack H, A and B antigens. It
was first discovered in Bombay (India) in 1952 by Dr YM
Bhende and colleagues.
• Symbol Oh denotes the phenotype, on routine ABO typing
tests Bombay blood group mimic those of group O persons.
• Oh serum contains strong anti-H, anti-A and anti-B
antibodies.
• When serum from the Oh is tested against group O red cells,
after immediate-spin strong agglutination and/or haemo-
lysis occurs.
Note: For details refer to chapter on “subgroups and weaker
variants of A and B blood groups and Bombay phenotype”.
Para-Bombay Phenotypes
Para-Bombay phenotypes have a non-functional H gene but a
normal Se gene and thus, will express A, B and H antigens in
their plasma and secretions.
The sera of Ah and Bh people contain anti-H in addition to
the expected anti-A or anti-B antibodies.
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.
• The secretor gene consists of 2 alleles (Se and se).
• The Se gene is responsible for the expression of the H antigen
on glycoprotein structures located in body secretions.
• If the Se allele is inherited as SeSe or Sese, the person is called
a “secretor”. 80% of the population are secretors.

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Essentials of Blood Banking and Transfusion Medicine
• Secretors express soluble forms of the H antigen in secre-
tions that can then be converted to A or B antigens (by the
transferases). Individuals who inherit the sese gene are
called “nonsecretors”. The se allele is 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.
• The Se gene codes for the presence of the H antigenin
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.
ABO Discrepancy
• An ABO discrepancy is said to exist when forward and
reverse typing do not agree.
• The patient’s serum contains ABO antibodies not compa-
tible with the antigens on red cells detected by forward
grouping.
• A and B antigen expression can be weakened in acute
leukemias especially the A blood group (acute myeloid
leukemias) or some carcinomas (stomach or pancreas).
Note: For details refer to chapter on errors and discrepancies
in ABO typing.
Rh System
• After A, B, H antigens, the D antigen of the Rh system has
the most significant implications in transfusion practice.
• Patients with D antigen are said to be positive.
• The Rh system also contains other clinically significant
antigens (C, E, c and e) and less significant antigens (variant,
cis products and G antigens).
• IgG antibodies can be produced in Rh system with immuno-
logical sensitization.
Note: For details refer to the chapter on Rh typing and weaker
variants of Rh system.

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ABO Blood Group Systems
49
Other Important Blood Groups
MNSs System
• M and N alphabets are derived from the word IMMUNE
and S from the name of place Sydney city where this blood
group was discovered.
• There are two loci: M/N and S/s. The antigens are M, N, S,
and s.
• Anti-M and anti-N are naturally occurring (IgM) antibodies.
• Anti-S and anti-s are IgG class antibodies as a result of preg-
nancy or transfusion.
P System
• This blood group was discovered by Landsteiner and Levine,
the gene is located on chromosome 22q11.2.
• P1 is the most common antigen which has variable strength
of expression.
• Anti-P1 may be naturally occurring.
• It is most often an IgM antibody.
Lutheran (Lu) System
• This system has a single locus on chromosome 19q13.32, has
19 antigens of which the antigens Lua and Lub are strong ones.
• The Lu(a) negative phenotype is very rare.
• Antibodies to Lutheran antigens are IgG type.
• The genes of the Lutheran group are linked to the genes
responsible for the secretion of ABH substances.
Kell System
• This blood group has four antigens at two loci: K (Kell) and
k (cellano), and Kpa and Kpb.
• The Kp(a+) phenotype and the Kp(a-b-) phenotype are both
rare.
• The Knull phenotype K-k-Kp(a-b-) is associated with chronic
granulomatous disease (CGD), an inherited defect in the
bacterial phagocytosis of neutrophils.
• Antibodies to Kell system antigens are IgG, named after the
family of the antibody producer Mrs. Kellner who suffered
haemolytic transfusion reaction and her baby had haemolytic
disease of newborn.

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Essentials of Blood Banking and Transfusion Medicine
• McLeod syndrome: In 1961, Allen and co-workers found
that one of their students, Mr. McLeod, had RBCs with weak
expression of Kell antigens. He had lacked Kx protein which
is important for expression of Kell antigen.
• These patients will have weak expression of Kell blood group
antigens and have acanthocytes, haemolytic anaemia and
elevated serum creatinine kinase. These patients have
muscular and neurological defects.
Lewis System
• This system focuses on a single locus with two antigens,
Le a and Le b on chromosome 19.
• These antigens do not form an integral part of the red cell
membrane, but are soluble antigens which may be present
in body fluids and secretions.
• They are adsorbed on to the surface of red cells if they are
present in the plasma in sufficient amounts.
• There are only three phenotypes: Le(a-b-); Le(a+b-); and Le(a-
b+). Lewis phenotypes may change during pregnancy.
• Lewis antibodies are IgM type.
Duffy System
• The Duffy system has single locus with two antigens Fy a
and Fy b.
• The only rare phenotype is Fy(a-b-), which has a higher
frequency in countries where there is a high incidence of
Plasmodium falciparum malaria.
• This phenotype gives a degree of immunity to the disease
because the malarial parasite requires Duffy antigens to enter
the red cells.
• Duffy antibodies are almost exclusively IgG type.
• This system is named after the family of the antibody
producer, Duffy.
Kidd (Jk) System
• The genes are on single locus, two-antigen system (Jka and
Jkb). There are four possible phenotypes: Jk(a-b-); Jk(a+b-);
Jk(a-b+); Jk(a+b+).

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ABO Blood Group Systems
51
• Jk(a-b-) is a rare phenotype.
• Antibodies to the Kidd antigens are almost exclusively IgG.
• Incompatible transfusion or pregnancy can lead to the
formation of antibodies to all these blood groups, if the
recipient/mother lacks the relevant antigen.
LABORATORY TESTING: ABO BLOOD GROUPING
Forward Grouping
Reaction of patient red blood cells is tested with reagent anti-A
and anti-B and anti-AB antiseras by the following methods:
• Slide method
• Tube technique
• Matrix gel card method
Slide method: Always confirm the results of slide with tube
test.
Fig. 4.5: Blood grouping by slide method
Procedure
Place 1 drop of anti-A and anti-B reagent separately on a labeled
plane glass slide or concavity slide. Add 1 drop of saline diluted
red cell suspension to each. Mix the cells and reagent with a
slick or a glass rod using different ends. Tilt the slide and look
for agglutination after 2 minutes at room temperature. Record
the results.

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Essentials of Blood Banking and Transfusion Medicine
Tube technique
• Prepare 2–5% suspension of RBCs.
• Take 3 test tubes.
1st test tube
2nd test tube
3rd test tube
1 drop of anti-A
1 drop of anti-B
1 drop of anti-A,B
+
+
+
1 drop of cell
1 drop of cell
1 drop of cell
suspension
suspension
suspension
Centrifuge at 1000 rpm × 1 minute.
Observe for agglutination.
Matrix gel system for ABO and Rh confirmation: Forward
grouping
Matrix gel system has the following:
• Neutral gel in microtubes.
• Preservative—0.1% sodium azide.
• Store the gel cards at 4–25 degree C, do not freeze.
• Wash the cells twice and prepare cell suspension of 2–5%.
• Add 25 µl of cell suspension to all the tubes.
• Centrifuge 1200 rpm × 10 min.
Fig. 4.6: ABO blood grouping and Rh typing by tube technique

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ABO Blood Group Systems
53
• Observe for agglutination.
• Clear line on the matrix gel surface: Agglutination (positive).
• Compact cell button at the bottom: No agglutination (negative).
Reverse Grouping
Reaction of the patient’s serum with known group A and
group B cells is tested and the procedure is as given below.
• 50 µl of serum to A and B microtubes.
• 25 µl of known A cells and B cells.
• Allow the cards to incubate for 10 min at room temperature
(18–25 degree C).
• Centrifuge at 1200 rpm × 10 min.
• Observe for agglutination.
ABO forward (cell) grouping reaction with result:
Sl No.
Anti-A
Anti-B
Blood group
1.
++++
 Negative
A
2.
Negative
++++
B
3.
++++
++++
AB
4.
Negative
Negative
O
Reagents: Anti-A, anti-B, anti-AB
ABO reverse (serum) grouping reaction with result:
Sl No.
A cells
B cells
Blood group
1.
Negative
++++
A
2.
++++
Negative
B
3.
Negative
Negative
AB
4.
++++
++++
O
Reagent cells: Pooled A cells, B cells, O cells.
Fig. 4.7: Matrix gel card method, forward and reverse grouping

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Essentials of Blood Banking and Transfusion Medicine
Strength of reaction
Comments
4+
Agglutinated red blood cells form a band at the
top of the column
3+
Most agglutinated red blood cells remain in the
upper half of gel column
2+
Agglutinated red blood cells are observed
throughout the length of the column. A small
botton of red blood cells may also be visible at
the bottom of the gel column
1+
Most agglutinated red blood cells remain in the
lower half of the column. A button of red blood
cells may also be visible at the bottom of the gel
column
±
Most agglutinated red blood cells remain in the
lower third part of the column.
Negative (0)
All the red cells pass through and form a compact
button at the bottom of the gel column
Mixed field
Agglutinated red blood cells form a band at the
agglutination
top of the gel or dispersed in the matrix and non-
agglutinated red blood cells form a compact
button at the bottom of the gel column
H
Hemolysis of red blood cells