Blood Group System

[[p.1]]
Blood Group System
5.1 ABO' BLOOD GROUP SYSTEMS AND ABO VARIANT
In 1875, Landoisfirst noticedclumping/agglutination when red cells of animals of one species is treated
with serum of another species.In human being the same phenomena was observed by Karl Landsteiner
in .1900 when he mixed red'cellsfrom one person with the sera from another person that lead to the
discovery of A, B, 0 group by Landsteiner in 1900, a year later, Decastillo and Sturli (students of
Landsteiner) discovered another group in human blood and that is AB. And thus the whole ofhuman
population was dividedin four groupson the basis of major blood groups to which theybelong.In 1910
Dungerin-and HirszfieldestablishedtheMendelian inheritance of blood group and was later confirmed
in 1924 by mathematician Bemstin.
Other systemsof blood group were'discovered after about 25 years of discovery of A, B, 0 system.
As hasbeenstatedaboveanindividual's bloodgroupis'determinedmainlybygeneticcontrolgoverned
by principles of Mendelian inheritance, supplemented by Landsteiner's law and regular presence of
ABH antigens in tissue cells and secretionsin the secretors.
Landsteiner law states that there is a reciprocal relationship between antibody present in serum/
plasma and antigen on his own red cells. In his experiment, he observed that a person's serum does not
contain the antibody against the antigenpresent on his own red cells viz. individual with 'A' antigen on
red cells will not contain anti-'A' in the serum, instead it contains anti-'B'. He further observed that two
antigens namely 'A' and 'B' were needed to explain four groups (A, B, 0 and AB) in whole human
population. Blood groups are the phenotypic expression of individual but the.genetic constitution or
genotype is expressed in a,different way (Table5.1).
Table 5.1. Phenotype and genotype
Name of phenotype
Genetic constitution or genotype
0
00
A
AAorAO
B
BB orBO
AB
AB
67

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68
Blood Banking and Transfusion
The group A is further subdivided in AI and A2 known as subgroups of A, and thereby increase the
number of phenotypes from four to six AI' A2, AJB, A2B, B and O. This lead to further experiment and
it was observed that anti-A obtained from serum of group B persons has two antibodies namely anti-A
and anti-A, and blood group Al has two antigens on red cells A and Al whereas A2 has only one antigen
namely A. So the distribution of antigens on red cells and antibodies in serum/plasma respectively
present in six blood groups (AI' A2, AI B, A2B, Band 0 can be plotted as follows (Table 5.2).
Table 5.2. Type of, antibodies in blood group
Antibody (agglutinin)
Typeof antibody
Antigen (agglutinogen)
Blood group
Anti-B
Usually IgM
A and At
At
Anti-B
Usually IgM
A only
A2
None
x
A,A} andB
AlB
'Anti-A and anti-At
Usually IgM
B
B
Anti-A, anti-A)
Usually IgG
None
0
AndB
Cross reacting
-
More weaker varients of A and subsequently for group B was discovered and were classified on the
basis of A and B antigens in the saliva of secretors. Mainly classified in AXlBX and AmlBm. The AmI
Bm type possess AlB and H while AXlBx possess only H in saliva. Weaker variants of A discovered till
now are A J, Aint,~,
A3, Ax and Am. Weaker variants of B so far reported are B3, Bx, Bm. But all these
subgroups or weaker variants reported so far except Ai and A2 are more of theoretical value.
The frequency distribution as observed in British population compared to Indian population for four
groups A, B, AB and 0 is given below (Table 5.3).
Table '5.3. Blood group frequency distribution
Blood group
British population
Indian population
A
41.7%
24.7%
B
8.6%
37.5%
AB
3.0%
5.3%
0
46.7%
32.5%
* Average percentage distribution, for details of regional and castewise distribution refer to works of Bhalla (1966)
quoted by Dr. H.M. Bhatia in procedures in blood banking and immunohaematology, published by Institute of
Irmnunohaematology, Bombay.
Another important blood group and more in Indian context is Bombay group discovered by Bhende
and Bhatia in 1952.
Salient features of this phenotype is absence of A, Band H antigens on red cells and serum/plasma
of these persons possess anti-A, anti-B and anti-H.

[[p.3]]
Blood Group system
69
The B0I11bay phenotype (Oh) individual are therefore devoid of antigens pres~nt
in ABO system
which can be observed in vitro by lack of reaction of Oh phenotype red cells with antisera A, antisera B,
antisera ~~B
and antisera H whereas a person or individual with 0 group does not exhibit reaction with
-A, -B, -AB, but reacts very strongly with -H and this indicates'clear differentiation between 0 and Oh
individual. The importance of Oh lies in multiplicity of antibodies in serum and for transfusion purpose
a Bombay phenotype individual can receive blood from another Bombay phenotype individual only."
The weaker variants of Oh are also present and are designated as Parabombay which are very rare. The
Oh individuals'are non-secretor ofABH substance and are mostly Le (a +b-), rarely Le (a-b-) Parabombay
phenotype are associated with depressed or abscence of H antigen on red. cells in weaker variant of A
and B and given notation of Ah Bh etc. In this phenotype probably a modifying gene plays part causing
abscence or depression of 'H' antigen on red cells but with no effect on salivary antigen. Red cells under
this classification are distinguished by their ability to partially express antigens A or B. They donot
necessarily have anti-H in their saliva or ABH substance in their saliva. So depending on group of blood
or as observed by reaction in vitro, notation, given are Am
h (weaker variant ofA with weak or negative H
on red cell and no ABH in saliva) Bmh etc.
The incidence of Bombay phenotype in India amongst Maharashtrian population is 1 : 13000 and 1
: 7600 in population of Bombay. It is observed also in Goanese and south Indian population. In north
India it has been detected in Bengal, Orissa, Bihar, Uttar Pradesh and Madhya Pradesh. Paraboinbay
type is very rare as frequency of weaker variants of ·'A' and 'B' itself are respectively 1 : 3300 and 1 :
9300. The weaker variants of A and B already described are due to rare alleles in ABO locus. Bombay
group is due to action ofmodifying or regulator gene which controls the expression ofantigen particularly
of H antigen on red cells and saliva. There are other ABO variants like CIS, AB and ABO variants due
to disease.
CISAB·
In this group the members who are AB inherits both A and B from one parent, the other parent is usually
'0' group. First described by Seyford et al, in 1964. In this phenotype B-antigen is very weak. The
condition is attributed to mutant gene producing a single enzyme.
ABO variants due to disease
It has been observed that certain disease condition has the capacity to influence or modify the antigens
particularly of A and B antigens.
Leukaemia for example reduces the antigenic strength of individuals of 'A' group. It has been observed
that in persons suffering from leukaemia and who were reacting strongly with anti-'A' before the disease
process initiated, exhibited very weak reaction with anti-'A' after the individual developed the disease
again during remission the reactivity with anti-'A' improved.
Acquired B antigen are usually of bacterial origin, it has been observed in persons particularly those
who are suffering from gut infection due to E coli. The ABH substance isolated from E coli 086 are
almost like, l\BH substance from human origin. E coli causes secondary infection in obstruction. In this
Bhcnde, YM., Despande, C.K. and Bhatia. H.lVI. et al. A "lVEtV" blood-group character related to the ABO system
in Lancet, J : 903. 1952.

[[p.4]]
70
Blood Banking and Transfusion
condition serum of the person though contains antibody in conformation with Landsteiner Law but the
red cells were agglutinated by anti-'B' sera and those who are secretors contain 'A' and H in saliva but not
'B', Though from above statements, it is clear that acquired 'B' is due to bacterial origin mostly gram
negative, but the bacteria responsible could not be detected in blood except in one case where proteus
vulgaris was detected. It is suggested that bacterial deacetylase and/or T and TK activation may play an
important role in such conversion of red cell antigens.
5.2 BIOC.HEMISTRY, GENETICS AND INHERITANCE OF ABO SYSTEM
Red cell antigens of ABO system
They are mainly A, Band H antigen which are protein in nature and various proteins are embedded in a
mozaic pattern without any fixed position on fluid lipid layer of cell membrane (Singer and Nicholson,
1972). But Marchase in 1979 proposed that spectrin forms the core of the matrix of closely packed units
of various forms viz. rod-like, globular etc. and they interacting with transmembrane proteins forms a
complex of fixed substrate and thereby allowing a restricted movement of the protein only on the cell
membrane. These proteins on red cell membrane are classified as :
(a) Perepheral which can be dissociated readily, and
(b) Integral that cannot be dissociated easily and has tendency to remain associated with lipids even
after drastic treatment.
The A,B and H antigen sites are greatest on band 3 of sialoglycoprotein and they are also found on
polyglycosilceramides and simple glycolipid. The number of A, Band H antigen sites varies in new born
and adults. These antigenic.sites are important because the antibody molecules gets attached to redcells
at these sites. In Al adults, it varies between 810,000 to 1170,000 and in newborn 250,000 to 370,000
whereas in A2 adults, it varies between 240,000 to 290,000 and in newborn 140,000. In B adults, it is
approximately around 750,000 and in newborn 220,000 to 320,000 ,A, B adult 460,000 to 850,000, in
newborn 220,000, A2B adult 1~0,000
to 140,000. H sites onO cells in newborn is 325,000 and in adult,
it is 1700,000 but in A, Band AB newborn, the site approximately around 70,000.
So, it has been observed that the order of reactivity of antigenic determinant is nil in Bombay
phenotype and progressively reduces as follows 0 > A2 > A2B > B > Al > AlB, therefore, 0 red cells
contains the numerically greatest number of H antigenic site.
.The ABH antigens are widely distributed they are even found in animals, plants and bacteria. In
human body apart from the red cells it is also found in saliva, fluid of psudomucinous ovarian cyst of
secretors, meconium of secretor baby, it was even discovered in Egyptian mummies, cornea and in the
tissue epidermal and epihelial cells, in spermatozoa. Amongst blood component AB and H were observed
on normoblasts, the A and B antigens occur on platelets, white cells and serum.
Biochemistry of ASH antigen
A tremendous volume of biochemical works were done to determine the chemical nature of AB and H
antigens and their genetical background. A simple and brief description is as follows.
1. Singer, S.l. and Nicolson, G.L. (1972). The fluid mosaic model ofcell membranes, Science, 175 : 720.
2. Marchase, VI: (1979). Functional proteins ofthe human red blood cell membrane : Semin Haemat, 16,3-20.

[[p.5]]
Blood Group System
71
A. Type 1 Chain
p1..3
Galactose attachment in Type 1 Chain
5
5
B.Type II Chain
131-4
c.
Galactose attachment in Type II Chain
3
2
3
2
p1-3 Unkage
3
2
3
2
P1-4Unkage
Gene
Unidentified
HGene
addition
ofL.Fucose
Le gene
addition
ofL.Fucose
CHOchain
(1)
(1)
(1)
Specificity
Precusor
H
;
(1&2)
A
AGans
(N acetyl 0
galactosamine
added)
BGene
(0 galactose added
to H activechain)
(1&2)
B
.SIMPLIFIEDREPRESENTATION OF FORMATION
A,B,H, Lea and Leb
c=:> 0 Galactose
<ll7LllJ> N acetyl 0 Galactosamine
<:;;:::> L Fucose
<IlJ.]]> N acetyl 0 Glucosamine
--i
Peptide backbone
Fig. 5.1.
(1)
(1&2)
}
Type
of Chain

[[p.6]]
72
Blood Banking and Transfusion
A, Band H antigens can exist as glycoprotein in secretion of secretors but in cell membrane, it
exists as glycolipid. The basic difference in chemical reactivity of glycolipid and glycoprotein respectively
shows that glycolipid are alcohol soluble and could be extracted by treatment with alcohol only.
Subsequently, it was observed that antigens are highly branched complex of glycosphingolipids - lipid
part of which remain attached to red cell membrane, whereas, protein part exposed above is responsible
for antigenic specificity. Glycoprotein are soluble antigen observed in at least 75 to 78%, individuals in
their tissues and body secretion. Glycoprotein are water soluble and specificity corresponds to individuals
own blood group. Their presence was detected in almost all body secretion except cerebrospinal fluid.
They have been traced to saliva, urine, bile, sweat, tears, serum and gastric juice. The persons in whom,
this antigens, called blood group substances are found are called secretors and whom, it is not found, are
called non-secretors.
The polysaccharide portion of glycoprotein and glycosphingolipid are similar and the chemical
constitution of two basic chain reveals presence of L-Frucose, N-acetyl-D-galactosamine, N-acetyl-D-
glucosamine, D-galJactose. The difference between type 1 chain and type II chain is that in the former,
the terminal galactose is bind by ~ 1-3 linkage, whereas in the later the galactose is bound by ~ 1-4
linkage. The antigenic specificity is determined by the terminal sugars viz. fucose when attached to the
basic chain the antigenic 'specificity is H, if it is N-acetyl-D-Galactosamine the specificity is A and if it
is D-galactose, the antigenic specificity is B. The attachment to basic chain is effected through enzyme
transferase, Precursor chain is substrate for H gene specified Fucosyl transferase. With addition of sugars
to basic chain a new structure is fanned. This acts as substrate for another enzyme transferase. For 'A'
specificity, Nacetyl-d-galactosamine is added to H chain by 'A' gene specified transferase and so on.
The.J\BO blood group system are inherited. The system consists of a single genetic locus from
where tJiree allelic genes namely A, B and 0 operates. Again A can be AJ and A2, genotype, phenotype
has been described earlier, following Table 5.4 expanded version.
Table 5.4. Phenotypes and genotypes ABO system
Phenotype
Genotype
A
AA and AO
B
BB andBO
AB
AB
0
00
AlB
AlB
A2B
A2B
Al
AlAI' A}A2, Al~'
A1A4, AIO
A2
~~,
A2A3, A2A4, AzO
A3
A3A3, A3A4, A3AO
A4
A4A4,A4AO
A2B
A3B
A4B
A4B

[[p.7]]
Blood Group System
73
A and B genes are condominant and 0 is an amorph (gene does not express phenoty pically). Since
each body cell has a pair of chromosome and each of which either carries or fails to carry one of the
above genes, the genotypic constitution will be represented as above in the Table 5.4. The thing that is
detected in blood are not gene but the product produced under appropriate genetic influence and that is
the observed trait or phenotype of individual. Inheritance of blood group as a result of mating between
parents is correct, It has been shown mathematically in the Table given below (Table 5.5).
All the alleles present in one locus may not have the same frequency because variations within
species may result from genetic changes which is not unnatural and this phenomenon is known as
mutation. This when occurs in any individual of a population ultimately gives rise to allelism and
subsequently through mating over a long period, new allele gets distributed into the particular population
group to which the individual originally belonged. The percentage of occurrence of all the genes of a
particular trait is called gene frequency and the sum total of which must be equal to 100%. From the
foregoing statement and the tables, it is thus clear that one of the three possible genes is transmitted by
each parent to their offspring through mating.
Combination of two separate genes respectively from each parent results in formation of genotypic
constitution which is reflected in anyone of four possible phenotypic constitution. This can be detected
in the reaction by treatment with antisera A, antisera B etc. The reaction exhibits dosage effect if the
genetic constitution of an individual homozygous. In homozygous individual viz. DD, the reaction will
be stronger with antisera D, then on heterozygous individual viz. Dd.
Antibody of ABO system
Anti-A and anti-B are present in blood when the corresponding antigen is absent on the red cells, i.e.,
they bears a reciprocal relationship - known as Landsteiner's law. Exception to this is very rare and
usually found in conditions those are physiologically not normal viz. hypogammaglobulinimia, chimera,
dispermy or in Am phenotype.
In new born and young infants, child develops antibody between 3 to 6 months and the limit of
normal range is reached by 5 to 11 years. So blood group antibody present in new born believed to have
I
been acquired from mother passively.
Table 5.5
Genotype
I
IT
ill
W·
mating
AAxBB
ABxAB
AOxBO
·AAxAB
A
A
A
B
B
0
A
A
B lAB
AB
AI AA
AB
A I AB
AD
AIAA
AA
B
AB
AB
B
BA
BB
o
BO
00
B
AB
AB
Offspring
100% AB
1. 25% A
1.25% 0
1.50% A
2.25% B
2. 25% A
2.50%AB
3.50%AB
3.25% B
4.25%AB
* On the top are genes transmissible by one parent and on left hand side by other parent.

[[p.8]]
74
Blood Banking and Transfusion
The blood group antibody can be mainly subdivided into Natural and Immune antibody.
Natural antibody are IGM in nature and usually acquired from surrounding environment i.e. plant,
bacteria etc. Examples supporting this view is that "laghorn chicks kept in germ free environment does
not develop any antibody", genetic factors seem to have no direct control over the titre of antibody but
the antibody making apparatus, seems to be more responsible. They display homogenecity in physical,
chemical property and serological specificity.
Immune. antibody may develop due to pregnancy, injections:
(i) With intention to immunise or produce strong blood group serum
(ii) ATS or horse serum
(iii) Influenza virus vaccine
(iv) Incompatible blood transfusion, plasma transfusion etc. Immune antibody shows marked
heterogenecity which is in contradistinction to natural antibody which displays homogenecity.
Anti-A
This is found in group 'B' and '0' individuals. It is usually IgM or partly IgM and partly IgG It has been
observed that after injection of 'A' substance, a group '0' individual develops additional IgG although
the serum initially contained IgM only and it can thus be concluded that development of IgG to anti-'A'
and anti-'B' or 'A' individuals.
.~
.• <ti'
The anti-'A' antibody is usually saline reacting and reacts well with subgroups Al and ~
but the
other weaker variants reaction is less defined. Minimal concentration to produce reaction/agglutination
is only 25 (twenty five) IgM anti-A' molecules per cell are required but for IgG anti-A' it was observed
that the number per cell approximates around 20,000. Economidou et at observed that the minimal
concentration in serum required for IgG is 0.2 ug/ml whereas for IgM anti-A', it is 0.01 Ilglml. Anti-'A'
can also occur as autoagglutinin and reacts usually weakly at room 'temperature. It mayor may not be
associated with autoimmune haemolytic anemia.
Antl-B
This antibody is found in group 'A' and group '0' individuals. Mostly of IgM but may be IgG or IgA
even. It reacts optimally in saline and exhibits reaction in vitro both by agglutination and haemolysis and
IgM reacts more strongly at room temperature and 4°C than 37°C whereas immune of IgG B shows
better reaction at around 37°C. Economidou et al. observed that monomers of IgM anti-'B' shows
equilibrium constant "36-170" times lower than whole IgM molecule providing support that native IgM
anti-'B' is attached to red cells by at least two binding sites. Anti-'B' can occur as autoagglutinin and may
show specificity of anti-B and reactive at temperature 4°C to 20°C only, in vitro.
1. Kabat, E.A., 1956. The Blood group substances, Academic Press, New York, p. 267.
2. Schiffman, a, Howe, C., 1965. The specificity of blood group cross reacting antibody. Journal of the
Immunology, 94 : 1907.
3. Lincoln, P.J., Dodd, B.E., 1969. Antigen-Antibody Study to Cross Reacting Antibodies in Group '0' Sera,
Immunology, 16, 301.

[[p.9]]
Blood Group System
75
o
2)H.OH
-.......-_~
OH
O-galactose
(terminal sugar
Moleculefor B specificity)
Fig. 5.2A and B.
1 H.OH
o
Crossreacting A and B
It is found mainly in group '0' sera.
The group '0' sera possess both anti-
'A' and anti-'B' agglutinins. The cross
reacting antibody reacts with both 'A'
cells and 'B' cells. Two different views
NHCOCH3
are held: (a) cross reacting antibodies
Nacetyigalactosamia
(terminal sugarmolecule
are directed not only against A or B
for A specificity)
groupings on the molecule but also
.1.-.-
--1
against some of common structure on
the molecule. If we examine the 'A' and 'B' sugar molecule we will find that they are almost identical
save for the difference in 2 position.
This view was supported by observation that antibodies are usually A preferring or B preferring
depending on whether they are produced by stimulation of 'A' antigen or 'B' antigen. Original theory was
postulated by Kabat (1956)1 and Schiffman et al. in (1965)2, further experimental observationby Lincoln
and Dodd (1969, 1974)3. (b) It could be due to presence of another antibody; anti-C (Winer, 1953)which
reacts with C antigens in A, Band AB group individuals. Group '0' individuals does not possess the 'C'
agglutinogen.
This phenomena of cross reactivity was studied further by Mutahashi and Ogata then by Mutahashi,
followed by Franks andLiske, by Bove et aI., Radic-lmrnunoassay by Holbum and many others. However,
Rosenfield first stated the possible clinical significance of cross-reacting antibody, that it is capable of
crossing the placental barrier more readily than the monospecific antibody may be due to·smaller size of
type of immuno-globulin molecule. So this cross-reacting antibody are more important from viewpoint
of Haemolytic disease of newborn and its possible aetiology.
5.3 ABO SYSTEM LABORATORY ASPECTS GROUPING, REAGENTS AND STANDARDISATION,
TIRTATION, MONOCLONAL ANTIBODY
The individuals blood group can be determined in the laboratory by a simple serological reaction
(Agglutination reaction). It can beeffected either by testing the individuals red cells with commercially
available standard Antisera 'A' andAntisera 'B'-cell grouping or direct grouping, or by testing individuals
serum with st.andard red cells i.e., pooled'A', pooled 'B',pooled '0'-
serum grouping orreverse grouping.
The practical uses of of blood grouping are following :
(a) Blood transfusion of safe order.
(b) In haemolytic disease of new born and some other disease condition to know their association
with blood groups.
(c) Forensic medicine-medicolegal and determination of probable paternity etc.
(d) Population genetics and anthropology.
Some precautionary measures to be adopted :
(a) Testing sample must be properly labelled and checked for any contamination.
(b) No sample to be accepted if it is supplied in a vial tube with m.outh open.
(c) Check the slides, tube, microplate before use for proper cleanliness and dryness.

[[p.10]]
76
Blood Banking and Transfusion
(d) Check the antisera to be used and used as per inserted instructions.
(e) Pooled cells for serum grouping should be fresh and mot more than six hours has elapsed after
its preparation.
(f) Pollution free atmosphere and recommended temperature must be maintained within laboratory.
ABO group are best detected at room temperature. At higher temperature, there might be
weakening of reaction.
(g) Laboratory should have adequate light facility and less congestion.
(h) Laboratory must have alternative connection for electricity which can be energised during
emergency.
(i) Adequate water supply.
G) When testing the serum should be added before and all the tubes rechecked again after serum is
added. After ensuring that serum is within the tube, slide, or microplate the test cells are to be
added. Optimal proportion of cell and serum is essential for a good reaction.
(k) Quality control of antisera is essential to avoid running individual control with each test.
(I) Negative results by naked eyes should not be taken as granted and must be confirmed under
microscope or repeat test.
(rn) Observation to be recorded inunediately and not delayed.
(n) Test sample to be preserved for seven days, if not at least for three days.
( 0 ) Report of the test has to .be initialled by the technologist who has done the test.
Test procedure
As described earlier two procedures are adopted :
(i) cell grouping
(ii) serum grouping.
Principle
The antigen (washed red cells from the test sample) and antibody (antisera or serum from test sample)
when mixed in an optimal proportion (cell: serum ratio) and incubated for a recommended period at a
recommended temperature (room temperature for ABO) under proper biochemical environment (ionic
strength and pH) will exhibit a proper reaction within the framework of law of mass action. Positive
reaction is denoted by agglutination or haemolysis and negative reaction. is denoted by abscence of
either.
Technique
1. Slide method (Fig. 5.3)
2. Tube method (Fig. 5.4)
3. Microplate
4. Gel technology
Requirement
I. Reagents:

[[p.11]]
Group B
serum
Group A
serum
Blood Group System
77
GroupO
GroupB
Group A
GroupAB
Fig. 5.3. Slide method for blood grouping.
TUBETEST
SLIDE TEST
Fig. 5.4. Grouping of blood - slide and tube.

[[p.12]]
78
Blood Banking and Transfusion
(a) Antisera A and A j
(b) Antisera B
(c) Antisera AB
(d) Antisera H
(e) For cell grouping the reagents could be Monoclonal or Polyclonal.
II. For serum grouping pooled cells A, B and O.
Ill. Physiological saline (N/tO sodium chloride) for preparing cell suspension.
IV. AB serum as control.
v. Material:
1. Slide or opaque tiles and sticks (small) for slide technique.
2. Precipitin tube of glass, round bottomed, 50 mm x 7 to 11 mm internal diameter, for
sedimentation tube tests.
3. 75 mm x 10 nun x 12 mm tubes for spin tube tests.
4. Racks having at least 50 holes arranged 10 holes in 5 rows.
5. Graduated pasteur pipette to deliver one or two standard volumes.
6. Microscope (monocular or binocular with built-in illumination preferably) adjusted for
magnification x 80 x 400 approximately.
7. Laboratory centrifuge for spintube test.
8. Microtitreplate technology:
(a) Microtitre plate 8 x 12 wells u or v bottomed.
(b) Reagents same as above.
(c) Semiautomated multi-channel pipettes'for delivering equal volume, centrifuge plate
on plate centrifuge,' plate shaker, reader for automatic detection of agglutination.
Magnifying lens, if automatic reader is not available.
Interpretation of result
It is done on the basis of positive reaction (agglutination or haemolysis) or Negative reaction (no
agglutination or haemolysis). All doubtful results where interpretation is difficult or in case of negative
reaction help of optical aid is essential.
All agglutination reaction is expressed by gradation of agglutination.
Graduation of agglutination
(i) ++++, also scored (+4) : Shows complete single clump.
(ii) +++, also scored (+3) : Visual agglutination but clump tends to break.
(iii) ++, also scored (+2) : Many fair large clumps with few free cells.
(iv), +, also scored (+1) : Agglutinates with more free cells.
(v) +,: Still smaller agglutinates, fine granular appearance visually small clumps of 10 to 15 cells
per field in low power.
(vi) + W : W weak agglutinates of 2 to 3 cells per low power field.

[[p.13]]
Blood Group System
79
(vii) ±: Very very weak reaction or tendency to agglutinate.
(viii) Negative: All cells are free macroscopically and microscopically.
Haemolysis
It is also denotes positive reaction haemolysis might be partial or total but it is to be interpreted as
positive irrespective of its status.
Advantages and disadvantages of different techniques - Slide method.
Advantages
Can be used as emergency method particularly in outdoor camps where centrifuge may not beavailable,
requires less time .and supposed to be cost effective.
Disadvantage
Not reliable for weak reactive antigen or low titred serum because of its less sensitivity. Dries up quickly
in atmosphere resulting in aggregation of cells very often misinterpreted as agglutination. Thus not
suitable for routine use in laboratory.
Test method
Advantage
Easy to perform, no fear ofquick drying, centrifugation possible as and when required and small amount
of reagents required.
Disadvantage
For confirmation of results cell grouping, followed by serum or reverse grouping are ideal for tube tests.
It is preferable that for each, test sample test done separately by different technicians viz. cell grouping
by one, serum grouping by another, and reading or recording of results by third person. This is done to
avoid bias. Moreover, requirement of material and equipment is more. So in terms of man, money,
material and minutes (time) it is less cost effective but in view of overall result oriented performance, it
is accepted as a standard for routine works in blood bank laboratory.
Microtitre plate technology
Though a recent advancement, it has many advantages over other two methods.
Advantages
Enhanced sensitivity, less reagent, less space, less time is required, plates are reusable. In one plate both
serum grouping and cell grouping can be performed, total test performed is 96 number. So as a whole
the method is more cost effective. The Ll-shaped wells widely used because of better resuspension of
samples whereas in V-shaped wells micro amount of reagents required.
For all the advantages described above the microtitre plate method is gaining popularity and it is
replacing the tubes gradually.

[[p.14]]
80
Blood Banking and Transfusion
Disadvantages
Initial expenditure is more, requires a well trained technician to interpret the result in absence ofautomatic
reader, variable electrostatic property with different plate manufactured by different manufacturer.
Procedural details
Slide method
(a) Take a microscopic slide or an opaque tile.
(b) Make a 20% cell suspension of test sample.
(c) For individual case adopt the following:
(i) Put one volume (0.03 ml is the standard volume) of antisera A, antisera B, and antisera AB
each on the slide or tile with help of pasteur pipette, wash the pipette with saline each time
before use.
(ii) In the second row put one drop of pooled A cell, B cell and 0 cell with the help of pipette,
wash pipette each time before use.
(iii) Put one drop of 20% cell suspension of test sample in the first row nearer to antisera A,
antisera B and antisera AB with pipette, wash pipette each time before use. Wash with
distilled water first followed by NIIO saline.
(iv) Put one drop of serum from test sample in the second row nearer to A cell, B cell and 0
cell, with pipette, wash pipette each time before use. Wash with distilled water, then followed
by N/IO saline.
(v) Mix the antisera with cell suspension in the first row using different stick for each mixing
and spread this mixture on the slide over 15 mm, take due precaution that no accidental
mixing or overlapping of another field takes place when all three antisera and cells are
properly mixed.
(vi) Same procedure has to be followed in the second row.
(vii) Rock and rotate the slide and leave the test for at least 2 minutes, at room temperature, then
rock again and look for the result of reaction.
(viii) Record interpretation of result immediately (consult the table given for interpretation).
Tube technique
The tube technique can be done by two methods: (i) Spin tube method, (ii) Sedimentation method.
1. Unknown test sample received should be centrifuged to separate the cell and serum, then separate
the cells, wash it with saline (N/IO) at least thrice.
2. 2 to 5% saline suspension of red cells are made with NIIO sodium chloride.
3. Pooled A, Band 0 cells are to be prepared or use from stock (prepared cells) not older than six
hours.
4. Antisera are to be serially arranged.
5. Tubes are labelled properly and put in the holes of the rack. Tubes meant for cell grouping
should have separate labell mark thenthe tubes meant for serum grouping.
~.
Prepare papain cystine solution or use from stock solution.

[[p.15]]
Blood Group System
81
7. For individual case arrange in a horizontal row of the rack five tubes for cell grouping and four
tubes for serum grouping.
8. Centrifuge is kept fixed at 1500 revolution per minute (to be used in emergency and spin
procedure).
9. Tubes used in spin method is 75 x 10mm x 12 mm round bottomed glass tube, in sedimentation
method the tubes used are precipitin tube (50 mm x 7 to 11 mm) in case of non-availability the
above mentioned, bigger size tube can also be used.
10. Adopt the following irrespective of the method (spin or sedimentation).
In the tubes marked for cell grouping first add antisera one volume (standard volume should be the
order of 0.3 ml) with the help of pasteur pipette which is to be washed with DIW or saline after each use.
The order of antisera are :
1. Anti A to tube 1
2. Anti B to tube 2
3. Anti AB to tube 3
4. Anti Al to tube 4
5. Anti H to tube 5.
In the tubes marked for serum grouping one volume of pooled cells in following order:
1. ' Pooled A cell in tube I
2. Pooled B cell in tube 2
3( Pooled 0 cell in tube 3
4. Pooled 0 cell with one volume of papain cystine in tube 4
5. Pasteur pipette to be washed after each use. Tenth hole in the rack is meant for Rh grouping.
After completion of first step as described above proceed to second step.
Take 6 (six) volumes of 2 to 5% cell suspension in graduated pasteur pipette and add one volume of
saline cell suspension of each of five tubes in tubes marked for cell grouping; the balance one volume is
meant for Rh grouping. Similarly take four volumes of serum in another graduated pipette and add one
volume to each four tubes meant for serum grouping. Both the pipettes are to be washed after use.
Now if the spin method is to be used which is generally used in case of emergency, all the tubes are
shaked gently at first to allow proper mixing of the contents and then left for 5 to 10 minutes at room
temperature (20°C to 25°C) for incubation and after that all tubes are placed in the centrifuge and 1500
revolution per minute in respect of each tube are given and the results read-agglutination, haemolysis or
negative.
For sedimentation method
(a) All tubes are tapped gently to allow proper mixing.
(b) Incubate at room temperature for 1 to I ~ hour.
(c) Observe result, against a well lighted background :
(i) For haemolysis supernatant fluid, haemolysis may be partial or total but interpretations to
be made as positive irrespective of grade.

[[p.16]]
82
Blood Banking and Transfusion
(ii) For agglutination look the cell button at the bottom of tube, by gentle tapping disperse the
agglutinate to know the grade and accordingly score them.
(iii) Negative reaction to be always confinned under microscope.
Running of control though advocated is not essential, if proper quality control of antisera and pooled
cells are maintained.
Table 5.6. Interpretatin of result
Cell grouping
Serum grouping
-A
-B
-AB
-A]
-H
Group
A cell
B cell
o cell
o pap
Group
+
+
+
+
W
AlB
-
-
-
-
AB
+
+
+
-
+3
A2B
-
-
-
-
AB
+
-
+
+
W
AI
-
+orH
-
-
A
+
-
+
-
+3
A2
-
+orH
-
-
A
-
+
+
-
W
B
+orH
-
-
-
B
-
-
-
-
+4
0
+orH
+orH
-
-
0
-
-
-
-
-
Dh
+orH
+orH
+orH
+orH
Oh
Haemolysis - H, W - Weak agglutination + Agglutination, No agglutination
Microtitre plate technology
This is a recent introduction and advancement in blood grouping procedure particularly in respect of
ABO and Rh grouping, and is very useful in places where large scale work is done in laboratory.
Antiglobulin tests has also been performed satisfactorily in some laboratory.
The microplates are injection moulded polyesterine plates approximately 12x 8 ern in size containing
96 wells arranged in 8 rows, from alphabet A to H vertically x 12 (numbered 1-12) horizontally arranged
wells. Wells may be If-shaped or V-shaped. Since electrostatic property vary with different brand of
plates preferably one brand of plate. may be choosen for use. The electrostatic effect may be eliminated
by addition of bovine albumin or Tween 20. Plates may be rinsed and wetted in Tween 20 shaken and
dried overnight at 37°C before use.
The 0.02% albumin in saline used as red cell diluent also is effective in elimination of static effect.
Diluents and wash solutions requirec
1. 0.02% albumin in saline is recommended as red cell diluent and wash solutions. Albumin limits
the electrostatic effect and prevent monolayering of red cells.
2. Test sample preferably to be collected in EDTA to avoid ABO haemolysin. Red cells used for
serum grouping also are best diluted in EDTA, microtitre plate use can bedone by employing:
(i) Saline method
(ii) Enzyme method
(iii) Indirect, antiglobulin method
However, commonest method in use is saline method and is described as follows :

[[p.17]]
Blood Group System
83
Procedure (Table 5..7)
I. Decide whether !J or V plates are to be used.
2. Select plates with low electrostatic property.
3. Red cells preferably from EDTA sample are washed in 0.02% Albumin saline solution.
4. Resuspended in either 0.02% Albumin saline or Jiss to make a 2 to 5% suspension.
5. For serum grouping the red cells are diluted with EDTA/Saline (0.02% Albumin saline).
6. For ~O,
Rh grouping in 'U' plates 2 to 5% suspension is acceptable but in 'V' plates further
diluted cell suspension of 0.1% to 0.2% are used.
Table 5.7. Procedure details
1
2
3
4
5
6
7
8
9
10
11
12
A 000
0
0
0
0
0 000
0
B
0
C
0
D
0
E
0
F
0
G
0
H
0
One drop of anti A + one drop of 2 to 5% cell suspension
One drop of anti B + one drop of 2 to 5% cell suspension
One drop of anti AB + one drop of 2 to 5% cell suspension
One drop of A pooled cell + one drop of sera from test sample
One drop of B pooled cell + one drop of sera from test sample
One drop of 0 pooled cell + one drop of sera from test sample
One drop of 0 pooled cell + one drop of P.C. + one drop of sera
Showing arrangement of individual test sample in the first row. A hole is left vacant and used
for Rh grouping (Anti D + cell suspension one drop each). Drops are equivalent to 25 ul volume
for each well and dispensed by microchannel dispenser. Plate used is 'V' bottomed well.
7. Shake for half a minute.
8. Incubate for 30 minutes to 2 hours at room temperature.
9. After incubation centrifuge the plate on plate centrifuge at 100 g for 40 seconds and examine
for agglutination either after agitation or after tilting the plate.
In fomier automatic reader is employed which is fitted with microcomputer and gives out the printed
report and also stores it. Reading done at 570 nm wavelength.
In later the whole plate is tilted at an angle of 60 to 70 and kept in same position till the negative
reaction begins to trail but the positive reaction remains as a discrete button to the bottom of the well.
Magnifying hand lens may be used to interpret the result. Interpretation of the result will be the same as
shown in the Table for ABO grouping.
Grouping of infant blood
There is a basic difference in grouping of adult person's blood and newborn baby or infant blood. From
our knowledge in blood group serology, we know that in infant, newborn or a baby.
(i) ABO antigens are not fully developed
(ii) Antibody are usually absent till a certain age. So in a newborn blood grouping which is usually
taken from cord, only cell grouping is done and not serum grouping.

[[p.18]]
84
Blood Banking and Transfusion
2
3
4
5
6
Well No.1 and 6
Well No.2, 3, 4, 5
Showing discrete button at the bottom (positive reaction)
Showing trailing of cells indicating (negative reaction)
Well of an u bottom microtitre plate at
approx 60· shows positive reaction
Well of an u bottom microtitre plate at approx
60 angle shows loose button and trailing of cells
Fig. 5.5. Agglutination - microtitre plate.
The antigens A and B can be detected long before birth, but at birth to distinguish between subgroups
of A is difficult viz. A) and A2, of course plant lactin for A) is better reagent for this purpose. The
strength of Al antigen rises gradually and by three years, it becomes steady..
Similarly 'B' antigen also gradually raises and becomes steady by three years.
H antigen is also expressed on foetal red cell but not as strong as 'A' and 'B' antigen; but at birth, it
is fairly well developed.
Fully developed antigen in newborn are Rh, K, Fy, Jk, MNSs, Oi, Do and Sc systems.
Weakly developed are I, Lea, Leb, Sd", Ch, Rg.
Antibody
In most infants, the anti 'A' and anti 'B
I are of IgM type and can first be detected by 3 to 6 months. At 3
months the antibody levels is only 25% high as compared to adult. It gradually increases after 6 months
and becomes steady by 6 to 10 years. At 3 months median anti 'A' titre in infant is 4 and at 6-9 months,
it increases to 16 and reaches maximum at around 8 years. So the antibody of IgM type, level is not
significantly higher and most of the antibody detected in cord blood are of IgG type and probably of
maternal origin. However controversy still exists but for practical purposes the antibody or agglutinin in
newborn is measured by screening of maternal serum at least for three months. Practical application of
this knowledge is relevant to haemolytic disease of newborn.
GEL TECHNOLOGY FOR BLOOD GROUPING
A recent advancement in blood grouping procedure where readyrnade monoclonal cells of anti-A, anti-
Band anti-D are provided. These monoclonal cell lines are provided in gel matrix contained within
microtubules. Control (ctl) microtube is for negative control and there are two other microtubules that
contains neutral gel are meant for reverse grouping, with AI cell and B cells. All these six microtubules
remains fixed in a card supplied by manufacturer. Additional materials supplied with the card are:
(a) Diluent - modified LISS for preparing red cell suspension.
(b) Test cell reagent: Al and B usually in 8% suspension (ready for use) supplied in vials often ml.

[[p.19]]
Blood Group System
85
Principle and procedure of test
Basic principle is the Saine i.e. detection of agglutination at the end of test. Negative result is indicated
by a compact button cells at the bottom of microtubule positive result is indicated by a red line on the
surface of the gel or if the agglutinates remain dispersed in the gel within the microtubule and they can
be graded according to pattern described. Agglutinated cells showing a definite form of red line at
surface of gel and which could not be dispersed by shaking is considered ++++.
Before conducting test the following essentials are to be observed :
1. identify the card with patient or donors name and registration number.
2. Dispenser, pipetor, pipette tips, working table, tubes all glass, centrifuge for cards of 6 tubes or
its multiples.
(a) 5% red cell suspension: Put 25 fl) of packed cell or 50 J.l1 of whole blood in 0.5 ml of
diluent (readymade) supplied by manufacturer in a all glass tube and then gently shake for
mixing.
(b) Plasma or serum for reverse grouping.
(c) Known positive and negative control.
After this pretest preparation now proceed t~ the test.
Take the identified card for the patient or donor, remove the aluminium foil and see that card attains
the room temperature before use and seal is not broken and tubules doesnot contain bubbles of air. After
removing the aluminium foil pipette 10 JlI of patient red cell suspension in tubes 1, 2, 3, 4 respectively
for group A, group B, Rh group or D, and fourth one is D control.
In second step put 50 J.lI ofreadymade cell AI in tube 5 and 50 J.11 of readymade cell B in tube 6. Now
centrifuge the card for ten minutes in the card centrifuge provided. Now after that bring out the card and
read for reaction.
~")toblems
in ABO
groupi~g
Problems in ABO grouping may be of variety and can be broadly subdivided:
(a) Test sample
(b) Technical
(c) Human error
(a) Test sample
It consists of two component important from blood bankers view and they are Red cells and Serum. So
we restrict ourself mainly to problems with these two components.
Red cell may give rise to rouleaux formation, problem due to weakening or loss of antigen,
Polyagglutinability, erythrocyte coated with antibody in vivo, in vivo contamination of test sample,
Chimera or in condition where more than one cell population is found, acquired ,:3.
Problems with cerum is mainly antibody related viz. unexpected antibody, loss or abscence,
autoantibody, antibody with haemolysin property.. serum may also give rise to problem, if there is change
in its biochemical nature.
Technical problems may arise due to material viz. equipment and glassware etc., reagents and
specimen.

[[p.20]]
86
Blood Banking and Transfusion
Human error such as incorrect labelling, incorrect interpretation of results may give rise to serious
problem.
Solving 01 problems
1. Check the label of the test sample for its correctness and whether it is contaminated or not,
whether anticoagulant is used or collected in plain vial, how old is the sample.
2. Check the reagents, i.e. Antisera and pooled cell, condition in which they are stored. If not
stored properly at recommended range, it may loose its potency, whether within the limit of
expiry and has passed the daily quality control check. Similarly pooled cell must pass the quality
control check.
3. Check the centrifuge, Thermostatic equipment and glasswares must be clean.
4. Ensure that procedure is followed correctly while doing a repeat test, viz. antisera is added first
in cell grouping and pooled cells in serum grouping to avoid accidental missing of the above in
any of tubes. Correctly incubated at room temperature and at 4°C for the recommended period.
5. Repeat test procedure is mandatory to detect the discrepency.
6. History of disease, if already not provided may be asked, when calling for a fresh sample to do
the repeat test. In leukemia 'A' antigens gets weakened. In AIHA and HDN, and incompatible
transfusion cells are antibody coated in vivo. Disease associated with para-proteinimia,
hypogammaglobulinemia, Agammaglobulinemia causes problem in grouping. Dual populations
of cells are presented in multiple transfusion, chimera and marrow transplantation. These are
few examples only.
7. Fresh samples collected from cord (anti-I may be detected by use of group 0 cord cell) :
(a) Mother,
(b) Aged individual,
(c) Patient or healthy indivudual,
(d) Collected in plain vial or in anticoagulant media,
(e) Multi-transfused patients.
All the above has to be ascertained to avoid any problem.
Solving problems with Red cells
(a)
Antigens in red cell, if weak or absent fails to agglutinate in cell grouping but serum grouping
may show presence of reciprocal antibody.
The presence of the antigens can be confirmed in secretors by detecting presence ofA, B and H
substances and in non-secretor cases, it is confirmed by absorption elution technique, e.g. to
detect weak anti-A, Incubate such cells with anti-A at room temperature for one hour to get the
antibody absorbed, after that absorbed antibody is eluated by placing the tube in serological
water bath at 56°C for at least ten minutes with intermittent shaking of tube. If this elute is
tested with 'A' cells it will agglutinate.
(b) Poly agglutinability: This condition is exhibited by Acquired B antigen, T and Tn or TK activation
or by Th. Acqired B antigen has already been described T, Tn, Tk and Thetc, are being described.
To differentiate the polyagglutination due to Acquired Band T, Tn, Tk, Th consult the following
(Table 5.8).

[[p.21]]
Blood Group System
87
Table 5.8. Showing Poly Agglutinability
Acquired B
T, Tn orTk
Antisera A, B, AB
Agglutination +
Agglutination +
Serumgrouping
Reaction with B cells
Agglutinate with all
(Adult human sera)
Strongly -i-ve, -ve with others
Cord serum
Variable depending on strength of
Anti T, TK and Tn are absent
antibody in sera
in cord sera
Serum with own cells
Fails to agglutinate
Fails to agglutinate
DCT
Negative
Negative
Lectins :
Dolichos Bi floras
T (-), TK (-), Tn (+), TH (-)
Araches Hypogea
(-)
T (+), Tk (+), Tn (-), Th (+)
Salvea sclerea
(-)
T (-), TK (-), Tn (+), Th (-)
ABH substance in saliva
Secretor shows presence
Variable secretors shows presence of
of 'A' and not B
respective blood group substance of
ABO in saliva and not related to T, Tk
o and Tn
T, Tk and Tn are called cryptantigens because in a red cell they remain normallymasked under
cover the membrane consisting of NANA (N acetyl Neuraminic acid) and sialic acid and is
carried on red cell of every individual. Further almost all of the adult human serum shows
'presence of antibody specific to this antigen particularly "T" and under certain conditions viz.
bacterial infection (pneumococci, streptococci, staphylococci, E. coli, clostridia, V. cholerae)
and viral infection (influenza virus) unmasking of antigens occur.
The unmasking occurs due to production of N acetyl neuraminase and sialadase particularly by
E. coli and these enzymes removes covering over "T" antigen. The condition in earlierdecade
was known as Hubner-Thomsen-Freidenrich phenomenon and now is is known as "T" activation.
These "T" activated red cells when mixed with A, B and AB of human origin will agglutinate
due to presence of "T" antibody in the sera. Difference exists between "T" and "Tn" activation;
Tn activated individual usually suffers from chronic anemia, leucopenia and thrombocytopenia
suggesting some fault in original cells i.e, stem cells and less sialic acid was observed in 'T'n
activated cells from start itself, whereas in 'T' activated cells sialic acid is denatured by bacterial
enzyme. Both T and Tn can be differentiated by treating them with Lectins like Araches Hypogea
and salvea sclarea (see Table 5.8).
Tk activation is associated with infection of Bacteroids Fragilis. Tk cells has normal amount of
sialic acid, demonstrated by its agglomeration with polybrene (negatively charged normal red
cells are agglomerated by polybrene whereas T activated cell with less or no sialic acid are not
agglomerated).
Other conditions ofpolyagglutinability : Tn activated red cells are not, agglutinated by lectin
vicia cretica but it agglutinates the T activated cells. Tn does not show anyreaction with polybrene
unlike Tk and Th. The Th shows agglutination with polybrene and vicia cretica whereas Tk

[[p.22]]
88
Blood Banking and Transfusion
does not show any reaction with vicia cretica. The Th is thought to be due to deficiency in
enzyme or enzymes required to produce 'T'.
Cad.: It is a low frequency antigen that shows polyagglutinability. It shows positive reaction
with lectin Dolichos biflorus, lectin cardioca; with polybrene also, it shows positive reaction.
Anti-cad was observed in high percentage ofhuman serum. The anticad and anti-A, were inhibited
by Nacetylgalactosamine may support the view that the antigen cad might be similar to A in
some respect.
Hempas stands for hereditary erythroblastic multinuclearity with positive acidified serum test
The red cells of these patients at a temperature around 15to,20° agglutinated by an antibody of
IgM type present may be anti-I, (a weak old antibody in the serum of normal subject). The
agglutination and lysis of hempas cells was described by Crookston et a1. in 19,69 and Lewis et
a1. in 1970.
"Nor", is also very low frequency red cell characteristic inherited as a dominant phenotype and
exhibits the polyagglutinable property. Nor red cells fails to react with lectin dolichos biflorus,
arachis hypogoea and salvea sclarea.
(c) Dual population of cells showing mixed field agglutination also gives rise to problem and this
phenomenon is 'observed in chimera, an organism deriving cells from two or more azygote
linkage, and in such cases the dual population exists throughout life and a proper history can
only be helpful in solving the problem.
Dual population of cells are also observed in multi-transfused cases where '0' group blood was
used as an universal donor for 'A' and 'B' group patient, in cases of marrow transplantation. In
both the cases again proper history will he helpful. Serum related problems are mostly antibody
originated or it could be due to extraneous causes.
Antibody related causes : Antibody related causes, loss or absence of antibody-
agammagloblinimia, hypogammaglobulinmia, auto-antibody viz, in auto-immune haemolytic
anemia which could be due to anti-lor anti-HI may cause problem in serum grouping, these"
antibody are cold reacting antibody causing problem in serum grouping. Haemolysis of the
cells could occur due to presence of complement and IgG type of antibody resulting release of
haemoglobin'which can be detected by centrifugation of supernatant fluid.
Unexpected alIo antibody can also create problem and in such cases screening for antibody by
submitting it to panel of test cells is helpful. Prozone effect may be observed with high titred
antibody, The same antibody at lower dilution shows negative reaction whereas at higher dilution,
it shows positive reaction.
Extraneous causes
Presence of large molecular weight substance, viz. Dextran, Fibrinogen, Polyvinylpyrolidone, then
abnormal proteins found in conditions of paraproteinimia - viz. multiple myeloma etc. and some acute
infective condition may give rise to rouleaux formation. Rouleaux formation could be dispersed by
addition of saline.
Presence of dust, debris, whartons jelly or clots due to unwashed cells and chemical contamination
etc. may cause problem, this can be eliminated by keenly observing the test sample while receiving.

[[p.23]]
Blood Group System
89
ABO reagents, poly clonal and monoclonal antibody, lectins, titration of .antibody,
standardisation of antibody
ABO reagents are the grouping reagents employed for diagnosis of the ABO blood group ofan individual.
The same definition could beextended for Rh grouping reagent and other blood group reagent. Here we
will mainly deal with ABO grouping reagent. Minimum basic criteria, a reagent must have its specificity,
titre, avidity and firmness to agglutination all conforming to International Standard and devoid of
hemolysin, irregular antibody and prozone effect. It must be sterile and free from any extraneous particles
organic or inorganic. Long durability within the recommended storage temperature of 4°C in general, of
course some are stored at -20°C in a frozen state viz. neutral AB serum etc.
Preparation of ABO reagents
These ABO reagents could be prepared from animal or human source and lectins. In the former source
reagents processed are usually of poIyclonaI in nature viz. anti-A, anti-Rh, until and unless it is further
processed by absorption/adsorption etc. to separate them.
Reagents processed from lectins seeds of plants, are mostly monoclonal in nature viz, Al from
Dolichos biflorus etc. of course rare exception of polyclonals are also found.
Now-a-days most of the reagents used are of monoclonal nature and large scale production are done
through Hybridoma technique.
In earlier days polyclonal antibody (responsive to different subgroups of one system say Rh, D, C, E
etc. or At and A2 i.e., it has multiple specificity until and unless separated or processed further) were
produced by immunising either animal or human being (voluntary human donor). Following is a brief
outline or steps.
Particular group substance of suitable strength, injected I.M. to the voluntary donor and observe
antibody titre after 10 to 15 days ~ Bleed the donor and blood collected without anticoagulant ~ Blood
allowed to clot at room tempe~ature
and then kept in incubator at 37°C for 1~2 to 2 hours ~ Overnight
storage at 4°C ~ Separation of clear cell free serum by centrifuging the overnight stored serum; at 4°C
-7 Destruction of complement by keeping the cell free serum at 560C for 30 minutes.
All these procedures are to be conducted under strict sterile conditions'. The serum after preparation
treated with sodium chloride, 1.6% and sodium azide 1 : 1000 and then colouring agents such as methylene
blue, acriflavin and eosin are added respectively for anti-A, anti-B and anti-ABo
Anti-H of human source can be obtained from persons having blood group A], B or A]B groups, or
it can be obtained from Oh phenotype persons. The former though commonly available the anti-H titre
in them is not very high and Bombay group is very rare. Other sources viz. animal (cattle and sheep) etc.
were tried but the lectins which are available were the easiest and cheap sources.
Lectins (Table 5.9) are sugar binding proteins which were first observed in plants so were named as
phytoagglutinin in earlier daysbut at later period, it was observed in bacteria, fungi and mammals. an
example is albumin gland of snails produce lectins capable of agglutinating human red cell antigen.
The chemical nature of Iectins are glycoprotein that agglutinate cells which is influenced by various
factors but mainly dependent on the terminal sugar which determines the specificity of lectin. The
agglutination produced could be of :
(a) Direct agglutination in saline media,
(b) Indirect" agglutination in presence of enzyme, and
(c) Nonspecific agglutination.

[[p.24]]
90
Blood Banking and Transfusion
Special characteristic
Table 5.9
Specificity
Lectins
J-------------=i
12. Roots of Ononis spinosa
Anti H
13. Fruit pulp of Clerodendrum viscosium
Anti H
14. Vicia graminea
Anti N
1. Dolichos biflorus
2. Viciacracea
3.Phaseolus limensis (Limabean)
4. Bandciraca simplifolia
5. Marasmius oreades (Mushroom)
6. Fornes fomentarius
7. Cytisus sessilifolus
8. Laburnum alpinurn
9. Lotus tetra gonolobus
10. Plasma of eel, anguilla anguilla
11. Ulex Europaeus (common gorse)
15. Bauhinea purpura
16.. Molucella laevis
17. lheris Amara
18. Hydated cyst fluid from sheeps liver,
goat and rabbit
19. Araches Hypogea (Peanut)
20. Dolichos biflorus
21. Vicia cretica
22. Salvea Sclarea
Anti A)
Anti A
Anti A
Anti 'B'
Anti 'B' like
Anti 'B'
Anti H
Anti H
Anti H
Anti-H
Anti H
AntiN
Anti N
Anti'M'
Anti P and PI
Anti T, TK and Th
Anti Trr and cad
Anti T and cad
Anti Tn
Nacelyel D-galactosamine specific, highly
. specific with high avidity does not react with
'0' or 'B' cells suspended in albumin or
enzyme unlike other, anti-A lectin are
particularly useful in separating A1in chimera
of Al and 0 cells
Oldest agglutin discovered
Nothing special
Contains three lectin BS-I, BS-n and BS-III.
BS-I is a family of five tetramaric isolectin
of which A4 is specific for N acetyl-D
galactosamine, ~4 is specific for D glactose,
B·S-II
is
specific
for
N
acetyl-D
galactosamine
Nothing special
Highly specific and able to demonstrate that
'B' is weaker in Ajfl, thenA2B no D galactose
specificity
Nothing special
Nothing special
L fucose specific
Less specific
Commonly used L fucosespecificeand more
useful in classifying '0' saliva
Nothing special
Nothing special
Most potent and also shows anti-T activity
reacts both with normal'N' and formaldehyde
'N' red cells
Nothing special
Also has anti-'A'· activity reacts only with
normal 'N' red cells
Not very specific

[[p.25]]
Blood Group System
91
Monoclonalantibody are monospecific antibody produced on commercial basis by hybridoma
technique. Originally described by Kohler and Milstein in" 1975. They immunised mice with sheep red
cells and then take out the spleen cells and found out the cells producing anti-sheep antibody. The cells
producing anti-sheep antibody were then fused with mouse myeloma cells and produced hybrid-myeloma
cells which grew indefinitely in vitro.After a few weeks, these cells were screened for antibody activity,
the choosen cells were further propagated and processed. The whole process is known as hybridoma
technique..To inject human antigens into mouse is known as xenogenic immunisation and was employed
.to produce monoclonal- antibodies reacting with HLA t -A, -B and -c. In this process human
lymphoblastoid lines were injected into mice.
Human
Mouse 'hybridoma is now employed to produce anti-Rho Human hybridoma technique has also been
developed.
Advantage
Monoclonal antibody used for blood typing are HN free and they act specifically on epitope and as such
will react only with particular epitope, not with adjacent one viz. epitopes of At and A2•
A continued supply line can be maintained from which high quality antisera can be produced.
Disadvantage
It recognises only a particular epitope and apparently fails to detect the related epitopes.
Some-high affinity monoclonal antibody were found to show cross reactivity which is unbecoming
characteristic of monoclonal antibody but then only some-of them do. This perhaps supports the old
observation that higher the affinity, more it tends to cross react.
.Neutral AS serum
It is prepared in a sterile environment and serum of AB blood group is separated from the clot after
overnight storage at 4°C. The serum is seitz filtered and ifis then stored in a frozen condition at -200C.
It has to be test checked before use for auto and cold antibody at room temperature and at 37°C, with
ABO group by saline and enzyme technique, I.C.T. and albumin replacement. If the serum shows rouleaux
formation, it is discarded.
Preservative is sodium azide for the neutral serum. It is mainly used as a :
(i) negative control
(ii) diluent to many of blood grouping reagent.
Pooled cells
Preparation described in Chapter Standardisation of ABO reagents and quality control of reagents.
ABO 'pooled cells
Each of the pooled cells are prepared by pooling equal volume of fresh blood at least thrice from each
confmned groups. Detail of procedure described and 2 to 5% suspension in saline, not to be kept more
than six hours at room temperature parameters to be checked are physical appearance, specificity, and
reactivity.

[[p.26]]
92
Blood Banking. and Transfusion
Requirement
(a) No haemolysis or turbidity should be detected on naked eye examination of freshly prepare.
sample for daily use.
(b) Reaction exhibited must be clear cut i.e, positive or clear negative reaction any ill definer
reaction raises doubt regarding the"quality,
(c) Must be specific against the concerned red cell antigen. Serum grouping when shows 'A' am
'AB' group confirmation for subgroup ~~l
and A 2 has to be done. Frequency of check to maintaii
the st.andard and potency-For the cell suspension every six hourly check is necessary,
an~
deterioration, a new 2 to 5% saline suspension has to be prepared. Pooled cells to be prepares
daily in the morning while starting the routine laboratory work and checked before use for al
the parameters as described above.
For Q papain and 0 saline both papain cystine and saline solution has to be checked before use
Papain cystine stock solution preserved at -20°C from where required amount for daily usage is brough
out and tested with anti-D for reactivity and specificity and titration by double dilution technique witl
~
anti-D is done while a comparison between newly prepared solution and old solution is also done anc
check comparative results for equal potency, the new should not show less than the old. Old solution i:
regarded as standard solution.
Saline required for daily use should be prepared freshly, if preparation is done in the laboratory 0:
otherwise, a fresh bottle of physiological N/IO saline bottle is to be opened for use. The content of the
bottle should be crystal clear, it must not be turbid, bottle must not contain any extraneous agent such a~
fungus etc. The stopper of the bottle should be tight and not loose and no crack detected in the bottle.
Antisera
-A, -B, ~AB,
-H.
Since Bereau of Indian Standards has not laid down any criteria or standard for antisera describec
above and in abscence of Bureau of Biologics, the manufacturer are to depend on phannacopias. Indiar
Pharmacopia mention only of anti-A, anti-B and anti-D. So they need consultation of other standarc
pharmacopias or produce antisera that conforms to the approved International Standard so that tes
results are not compromised and accurate inference could be arrived at. It is thus to the users benefit tha
every new batch on receipt should be checked for physical appearance, reactivity, specificity, avidity
and potency to detect that it qualifies for the standard or not. In case, it is prepared in the laboratory, the
reagents are to be standardised in the line described above.
Physical appearance
The antisera must be crystal clear and is not turbid, it must not contain any extraneous agent colour must
be approved colour asdescribed elsewhere in this chapter, any variation is disapproved.
Reactivity
Antisera produced or being checked must show clear reactions and not illdefined reactions, rouleaux
formation, gel formation; prozone effect, immune haemolysis are but few examples that interferes in
con-eet interpretationof results. A clear reactive agent should show firmness to agglutination, +4 at 1 : 4
dilution of serum at two minutes in room temperature. It ofcourse varies between +4 to +2 depending on
cell type.

[[p.27]]
Blood Group System
93
Avidity
Speed and strength of agglutination reaction is terrned as avidity (Table 5.10).
(a) Make 10% saline suspension of appropriate red cells.
(b) Take a clear glass slide.
(c) Put one volume of antisera to be checked on the glass slide and place one volume of 10%
suspension ofappropriate redcells prepared as above. Mix with a stic and spread out the mixture
in a circle about 25 mm in diameter. Rock and tilt the slide from side to side till agglutination
starts.
(d) Stop watch is to be started with rapid mixing of sera and cell suspension and stopped with first
appearance of macroscopic agglutination. Time interval recorded is the avidity of antisera under
test. For ABO reagents the test is done at room temperature.
(e) Perform each test twice difference in time between both the tests should not exceed more than
3 seconds.
Table 5.10. Avidity anti-A and anti-B sera
Serum
Phenotype of
Avidity
Serum
Phenotype of
Avidity
test cells
test cells
Anti A
A)
10 sees
Anti AB
A] cells
10 sees
A2
15 sees
A2B
16to 20 sees
A2 cells
15 sees
Anti B
B cells
·10 sees
B cells
10 sees
Thus the avidity varies depending on cell type and average is approximately around 13 sees.
Specificity
Antisera produced must be specific against appropriate red cell antigen.
Any particular antisera when tested, red cells, i.e., blood is to be collected from different donors and
appropriate red cells suspension in saline 2 to 5% is made· and equal volume of sera and red cells
suspension usually one volume of each are mixed at room temperature incubated and observed for
reaction.
Antisera A is tested with phenotype A), A2 and 0 cells. Antisera AB is tested with phenotype AI' A2,
B and 0 cells. Antisera B is tested with phenotype B, AI' AlB and 0 cells.
Potency
Any products manufactured should show the effectiveness and functional ability. Potency is a.measure
of the expected effectiveness and functional ability respectively ofproduct and its component. In antisera
standardisation, it is judged by titration of respective antisera with 2 to 5% of saline suspension of
appropriate red cell and compare the result with standard.
In titration procedure serial double dilution technique is followed. The reciprocal of highest serum
dilution that shows the detactable agglutination is expressed as the titre of antibody.

[[p.28]]
94
Blood Banking and Transfusion
Procedur8'I· details
(a) Level a raw of round bottomed glass test tubes and arrange them in a single raw of at least ten
tubes as shown in Fig. 5.4.
(b) Deliver one volume of ~/10
saline into all tubes except the number one.
(c) Now add one volume of serum under test to each first and second tube.
(d) Mix the content of the second tube thoroughly withthe help of clean pipette. After the mixingis
over transfer one volume of serum saline mixture to the third tube and again thorough mixing is
done. The serum dilution in tube number 1,2 and 3 respectively 1 : 1, 1 : 2 and 1 : 4.
(e) Same procedure is continued upto the last tube.The one volume of saline-serum mixture pipetted
out of last tube may be either discarded or saved for future use.
(f) Now add one volume of 2 to 5% saline suspension of appropriate red cells in each of the tubes
marked 1 to 10. Again mixing is done with pipette in each tube.
(g) Incubate at room temperature, for ABO reagents for 60 minutes..
(h) Read the result at the. end of incubation period by gently tapping the bottom of the tubes.
(i) Record result for each tube and the one which shows weakest detectable agglutination on visual
inspection gives the titre of antibody. In the Fig. 5.4, 1 : 512 is the titre of the antibody.Minimal
titre for ABO reagents after 60 minutes of incubation at room temperature should show (Table
5.11).
Stability
Antiseraproduced must be stable and the standard is two years from date of manufacture when stored at
2 to 8°C.
1:1
o
o
1:2
o
1:4
o
1:14
o
®
1:32
1:64
1:128
1:256
1:512
1:1024
•
-
Saline one volume
o- Antisera one volume
Agglutination
o - 2 to 5
% cell suspension
@- mix of antisera and saline
transferrea from previous tube
Cij]7+4
\If} +2
\JIV +3
Q7 +1
In figure above 1 : 512 is
the titre of antibody
W
-Week
\!J - Weakest detectable
o - No agglutination
Fig. 5.6.

[[p.29]]
Blood Group System
95
Table 5.11. ABO reagents titre
Serum
. Phenotype of red cells
Titre
Antisera A
Ai cells
1 : 256
A2 cells
1 : 128
A2 B cells
1 : 64
Antisera B
B cells
1 : 256
Al B cells
1 : 256
Antisera AB
AI cells
1 : 256
(Serumfromgroup '0')
.A2 cells
1 : 128
B cells
1 : 256
Antisera H
AI cells
1 : 1
A2 cells'
I: 8
o cells
1 : 16
Precaution
1. Antisera, if not in use, to be stored at 4°C or at recommended temperature to maintain the
standard.
2. Monoclonal antisera A, Band anti-AB are strongly haemolytic in presence of fresh serum and
this might be due to complement. So blood samples collection to be done ill EDTA or if it is in
plain vial add EDTA 0.1 mIlL at pH to 7.4
3. If colour.is added then approved and standard colour is to be used. Approved colour described
elsewhere in this' chapter.
Since we donot have any laid down national standard, it has to be compared with other standards
approved and internationally recognised viz. AABB, BOB, FDA and Paul Ehrlic Institute Standard; so
that test results are not compromised and standard maintained.
:5.4 SECRETOR STATUS AND \.BLOOD \GROUP SU.BSTANCE
Contrary to the belief held earlier that ABHantigens - blood group substances are restricted only to red
cell surface, there presence was detected in body fluids and secretions, in body tissues except eNS.
Amongst all the body secretions saliva is the richest source for blood group substance. The persons
whose saliva contains blood group substances are called secretors and in persons in whom it is lacking
.or contains only very trace amount are called non-secretors, and there approximate distribution in
population is 80% for the former and 20% for the later. They are inherited as mendelian dominant
character and controlled by a pair of allele Se and se genes that segregate independently ofother system.
Secretor locus was identified near Lutheran locus but there autosomal linkage is very rare. Genotypically,
they can be homozygous and heterozygous, e.g. Se Se, Se se, se see The first two are observed amongst
the secretor population whereas the third one amongst non-secretor population.
A.B~H.
antigens are well developed in saliva of newborn babies,' it was even detected during third
month ofintrauterine life. In adults the ABH substances in saliva are mainly produced by salivery glands
viz. submaxillary and submandibular glands.

[[p.30]]
96
Blood Banking and Transfusion
Biochemical nature of blood group substances on analysis reveals that they have two distinct forms :
I. Glycoprotein which is soluble in water and present in most of body fluids and is under control
of secretor gene.
2. Glycolipid which is soluble in alcohol, chloroform etc. This alcohol soluble form is present on
red cell and other tissues but unlike the previous one their genetic control is ill defined.
Secretor individuals of respective blood group contains H substance in addition, e.g., A group person
will contain A substance and H substance in their saliva. 0 group persons are divided in secretors and
non-secretor depending on the presence or absence of "U" in saliva. It has been observed further, that
ABH antigens are found in conjunction with Le and a detailed analysis of their different characteristic
like serological behaviour, biochemicalnature etc. points to four independent locus wich are responsible
for control of blood group substance and Lewis antigens on red cells, tissue and body secretions. FOQr
responsible genes are H. Lewis (Le), Secretor (Se) and ABO. The amorph genes are respectively h, le,
se, 0 and does not produce any detectable effect even in double dose. A summary of interrelation and
genetic control in respect of above is given in the Tables 5.12A and 5.12B.
Table 5.12A. Biochemical nature and distribution" of ASH
ABH
I
I
Glycoprotein
(water soluble specificity with anti-A
anti-B and anti-H) distributed on body secretion
saliva, serum, gastric juices, ovarian cyst
(pseudomucinous cyst adenoma), sweat, urine, tears, bile
and milk, semen amniotic fluid except in CSF
I
Glycolipid
(Alcohol soluble specificity with anti-A
anti-B and anti-H) red cells epithelial
cells except C.N.S. It is irrespective
of whether the individual is a
secretor of non-secretor.
Table 5.12. Genetic control of secretor and non-secretor, interrelation
1Ype
Distribution
Lewis
ABH
H
in population
Secretors
80%
Genotype - Le Le
ABH substance
HH
genotype
Lele
present in body
Se Se
Control gene - Le
Secretion in
SeSe
Red cells - Le (a-b+)
Le (a-b+) but absent
Control gene
Le (a+b-)*
in Le (a+1r-) ABU
Se
Substance in secretion
antigen present on
hh
...
_Leaand Leb
red cells in both
Non-secretor
20%
Genotype - Ie Le
ABH substance
Genotype-
Control gene -Ie Ie
absent in secretion
se se
Lewis substance
secretion
Control gene
absent in secretion
ABH antigen
hh
se
Redcells - absent on
Le (a-b-)
red cells
Bombay phenotypes are Oh Le (a+ b-) or oh Le (a-b-)

[[p.31]]
Blood Group System
97
Blood groupsubstances are mainly usefulfor following purposes:
(a) To detect blood group where it is difficult to ascertain.
(b) Voluntary donors are immunised to raise the antibody titre.
(c) Anthropological and immunochemical study. Demonstration of soluble substance in saliva as
evidence for inheritance. Lesser uses are preparation of antisera etc.
Determination of secretor status
As we know from above discussion that a person in most of the cases (about 80%) possess corresponding
antigens in saliva and body fluids which is of course dependent on respective individuals blood group
(ABO). Rtr
Principle
If saliva contains blood group substance, it will combine with antisera thus neutralising its property for
agglutination so that when respective red cells are added, it will not be agglutinated and absence of
agglutination can thus be interpreted as indicating the presence of antigen.
Requirement
1. Lab equipment viz. racks and tubes.
2. Saliva collected in a glass beaker after thorough washing of one's mouth and then denature it of
enzyme by heating for ten minutes in a boiling water bath. The whole amount of saliva
approximately ten ml is now filtered through a filter paper. The filtrate is collected in a test tube,
the amount of filtrate shouldnot be less than 2 ml. It is always necessary to centrifuge the saliva
at 3000 rpm for two minutes before rendering i~ to filtration. Filtrate if required can be preserved
at -20°C.
3. Red cells of different groups each washed three times and then made in to 5% cell suspension.
4. N/IO saline.
5. Antisera: Human antisera are the best reagents but due to very rare availability lectin antisera
are used.
Steps
(a) Filtrate of saliva is diluted to 1 : 2 with saline; undiluted saliva may sometime contain non-
specific glycoprotein.
(b) Antisera dilution : At first find out the final titre of respective antisera ABH by serial dilutior
procedure and then it is diluted with saline according to the titre desired and the titre chooser
are usually multiple of 8, e.g. 16, 32 etc. If the final titre of antibody in a particular antisera is
say 64 and desired titre is 1 : 8, then dilute one drop of antisera with 7 drops of saline (64 h
divided by 8; 64/8 = 8) will give 1 : 8.
or
Make serial dilutions of antisera A, Band H in separate row. Serial dilution is made with N/I(
saline in 1 : 2, 1 :'4, 1 : 16, 1 : 32 and upto 512 titrate againstA cells, B cells and 0 cells incubate
at R.T. for one hour.
Now select the tube preceding the one giving last +4 agglutination for each row. This is the
dilution of antisera to be used for test procedure.

[[p.32]]
98
Blood Banking and Transfusion
Test
Set up test and control as shown in Table 5.13. For each test control is a must and controls are both positive
and negative control. A range of three rows of tubes each row containing three tube labelled A, Band H.
Stage 1
Table 5.13A.
A
B
H
0
0
0
2 drops of saliva
2 drops of saliva
2 drops of saliva
2 drops of diluted A
2 drops - B diluted
2 drops - H diluted
+ve
+ve
+ve
2 drops of - A
2 drops of - B
2 drops of - H
+2 drops group specific
+GSS
diluted +2 drops
substance
of O.S. (l\BH)
(positive control)
(positive control)
(positive control)
-ve
-ve
-ve
2 drops of N/S
2 drops of N/S
2 drops of N/S + 2 drops
2 drops of diluted - A
+ 2 drops of diluted - B
of diluted - H
(negative control)
(negative control)
(negative control)
I
Table 5.138. Interpretation
N/Secretor
Secretor
Raw of tubes
+4
+4
+4
Neg
Neg
Neg
o test
-
-
-
-
-
-
+ve
(Ner'
(Neg)
(Neg)
(Neg)
(Neg)
(Neg)
Positi ve control
+4
+4
+4
+4
+4
+4
-ve
Negative control
Stage II
Incubate at rOOID temperature for fifteen to twenty minutes, now add 2 drops of 5% cell suspension in
saline A cells to A row B cells to B row and '0' cells to H row·after adding 2 drops saliva to each tube as
described mix and incubate all tubes at R.T. for 60 minutes. Read macroscopically naked eye for
agglutination interpretation of result (Table 5.13B) is given for non-secretors and secretors separately in
respect of each tube alongwith control tube results. Negative control should show agglutination and
positive control should not show any agglutination. Inhibition is indicated by lack of agglutination, i.e.,
secretor individuals does not show agglutination, the blood group specific substance in saliva has
neutralised the respective antisera. But if there is agglutination in test row, it indicates absence of blood
group specific substance in saliva i.e., individual is a non-secretor. In Bombay phenotype all test sample

[[p.33]]
Blood Group System
99
will show agglutination, i.e. non-secretor. If a saliva shows inhibition with Band H, it indicates person
is a secretor of group B and likewise. A note of precaution is added that if antisera is already titred upto
required dilution, a further dilution is not necessary, e.g, anti-H lectin are usually supplied by manufacturer
1 : 16 and if desired titre of antisera to be used in test comesout to be 1 : 16, in that case further dilution
of the particular antisera is not required and it can be used neat or undiluted.
For quantitative estimation, we can undertake the serial dilution technique. In this method, titrate
serially diluted saliva with a standard antibody and standard cells. This method is employed when saliva
from different secretors are compared.
Standard serum used should show at least two macroscopic agglutination with standard cells and
have the titre of 32.
Standard cells are 2% cell suspension in saline ofA, Band '0' cell. First one volume of 1 : 2 diluted
saliva is taken and serially diluted in a row of precipitin tube say upto 1024. Next add equal volume of
appropriate standard antisera to each tube separately and incubate at RT for 30 minutes. After this, add
one volume of standard red cells of appropriate group is added to each tube and "incubate it for 60
minutes to 90 minutes and now look for reactions :
(i) inhibition (complete or partial)
(ii) agglutination and results are graded accordingly.
Complete inhibition: Secretors (having enough group specific substance).
Partial inhibition: Secretors but not having enough group specific substance.
Saline control is must and has to be included for each test series.
5.5 RH BLOOD GROUP SYSTEM, INHERITANCE ·AND GENETICS, LABORATOR.Y
DETECTION AND OR VARIANTS
The Rh blood group system is one of the important system because it has clinical significance, very
commonly encountered in haemolytic transfusion reaction and haemolytic disease of newborn apart
from many other conditions where it is implicated.
Rh system was first discovered following demonstration of an antibody in a '0' group mother who
delivered a dead foetus. Levine and Stetson in 1939 found that antibody present in the serum is due to
stimulation of antigenic factor present in the foetus.
Landsteinor and Weiner, while immunising rabbits with red cells from Rhesus monkey observed
that the serum ofso imrnunised rabbits can agglutinatered cells ofwhite human population and percentage
involved is around 85% and rest 15% of population doesnot produce any reaction. They named this
antibody asanti-Rh after the source of its origin and 85% of population showing agglutination reaction
was designated as Rh +ve and the rest 15% as Rh -ve, On further examination they found it to be
identical in activity with the antibody demonstrated by Levine and Stetson in 1939.
Subsequent follow up revealed that animal antisera demonstrated by Land Steiner and Weiner is not
the same as human anti-Rho Further work on this new antibody. revealed that it is not identical with the
anti-Rh rabbit origin. But then by this time the Rh system and designation was so much well established
that a new nomenclature is simply impossible to conceive and instead the anti-Rh of rabbit origin was
designated- as. LW (described later in this chapter) after the name of its discoverers and that has been
widely accepted.

[[p.34]]
100
Blood Banking and Transfusion
Rh blood group system is a very complex system and it is highly immunogenic particularly Rh 'D'
antigen. Any individual designated as Rh +ve usually denotes the presence of Rh 'D' antigen unless
specified otherwise. Because of its high immunogenecity, wider distribution and clinical significance, it
deserves a proper study of inheritance and genetics for a better understanding of the system.
The two theories of inheritance of Rh system are as follows :
(i) Weiner's one locus theory states that there is one gene and product of more than one specificity,
viz., one agglutinogen and three blood factors, i.e., Rho for the three antigen complex, 'hr', hr"
(see Table 5.14).
(ii) Fisher and Race theory proposes of three closely linked genes D-D, C-c and E-e. The C and c
are antithetical, E and" e are antithetical, whereas 'd' has not been detected as yet (see Table
5.14). The gene allelic to 'D' has been designated as 'd', known as amorph or silent gene. Thus
five antigens D, C, E, e, c, and eight haplotypes CDe, cDE, CdE, cDe, cdE, Cde, CDE, cde
(Table 5.14). Eight main haplotype of Weiner and Fisher, there interrelation given in Table 5.14.
Every human being will contain any two out of these eight gene complex so thirty six genotype
combination is possible. But the phenotype could be calculated by detection through antisera and will
depend on number and type of antisera"used. In average the distribution in white population is 85% for
Rh +ve and 15% for Rh -ve. The gene frequency were calculated statistically from observed phenotype
by Mourant in 1954, which varied widely amongst different population. Work of Mourant was mainly
restricted to some particular group of population mostly belonging to western hemisphere. In Indian
subcontinent, the incidence of Rh negative is 5% approximately, however, amongst different caste and
groups, this too varies, e.g. in Parsis, it may be as high as 15 to 170/0 and also found high in Chitrapur
Saraswat and Goanese Catholics. Pioneer work on Rh system in Indian population was done by Sanghvi
and Khanolkar (1949), Vyas et al. (1958) and Bhalla (1966). Badakare and Bhatia (1973). Percentage
distribution of different Rh genotype and phenotype in Indian population is given in Table 5.15.
Table 5.14
Short
Fisher and Race
Weiner
Reaction with antisera
notation
Gene
Antigen
Agglutinogen
Factors
Gene
-C
-E
-D
-c
-e
complex
-rh'
-rh"
Rho -hr' -hr""
Ro
cDe
c,D, e
Rho
Rho' Hr', Hr"
Ro
-
-
+
+
+
R1
CDe
C,D,e
Rbi
Rho' rh", hr"
R1
+
-
+
-
+
~
cDE
cDE
Rh2
Rho, hr", rh"
R2
-
+
+
+
-
R2
CDE
C,D,E
Rh2
Rho' rh", rh"
R2
+
+
+
-
-
r
cde
C, e (d)
rh
hr', hr"
r
-
-
-
+
+
r'
Cde
C, e (d)
rh'
rh
l hr"
r'
+
-
-
-
+
r
tl
cdE
c, E (d)
rh"
hr', .rh"
r"
-
+
-
+
-
rY
CdE
C, E (d)
rhY
rh', rh"
rY
+
+
-
-
-
(d) Stands for amorph and as much no antigen 'd' is expressed. In Weiner gene notation are always in italics.

[[p.35]]
Blood Group System
101
Table 5.15. Adopted from techniques in blood banking, edited byH.M. Bhatia (I.I.H., Technical Report
Series 3 : '1985)
-C
-<
-Do
-E
--e
Phonotype
Weiner
Average
Genetic
Consti-
RO
-rh' -hr' -Rh
rh"
-hr"
Fisher
frequency
Fisher-
tution
(D)
raee
much
symbols
-
+
+
-
'I-
ccDee
Rho
1.5
cde/cde
Ror
P
CDe/cDe
RoRo
+
+
+
-
+
CcOee
Rh, rh
30.0
CDelcde
Rl'r
CDe/cde
Rl'Ro
CDe/CDe
Ror,
0
+
-
+
-
+
CCDee
Rh l , Rb)
38.0
CDe/CDe
RI'R)
COe/Cde
Rt, ro
-
+
+
+
+
ccDEe
Rh2, rh
6.0
cDFJcde
R,Ro
eDE/cOe
R,r
S
eD/cdE
Ror"
-
+
+
+
-
ccDEE
Rho' Rho
0.5
eDE/cDE
R2'~
cDFlcdE
R2, r"
+
+
+
+
+
CcDEe
Rh,Rh2
15.0
CDe/cOE
Rl'~
CDE/cde
R2, R
cDElCde
R, r"
T
CDe/cdE
R2, Ro
CDE/cde
RO,Ry
cDelCdE
Rory
+
+
+
+
-
CeDEE
Rh1,oRhgz
0.25
cDFJCDE
R2Rz
CDFJcdE
Rzr"
cDElCdE
R1ry
I
+
-
+
+
+
CCDEe
RbI Rhz
1.0
COEelCOE
Rl Rz
CDFJCde
Rzr"
V
CDe/CdE
Rzry
E
+
+
-
-
+
CCnEE
RhzRhz
0.01
CDFJCDE
RzRzl
CDFlCdE
Rzry
+
+
-
-
+
Ccdee
rhrh
1.0
Cde/cde
r2r
N
+
-
-
-
+
CCdee
rhrh"
0.01
Cde/Cde
rr"
E
-
+
-
+
+
cedE
rh"rh
0.5
cdElcde
r"r
G
'\,
-
+
-
+
+
cccEe
rh"rh"
v.rare
edElcdE
t"
-I'
+
+
-
+
+
CcdEe
rhyrh
rare,
CdElcd
ryr
A
Cde/cdE
' "
rr
+
-
-
+
+
CCdEe
rhrhy
v.rare
CdElCde
ryr
,
T
+
+
-
+
-
CedEE
rh yrh"
v.rare
CdE/edE
rry"
I
+
-
-
+
-
CCdEE
rhyrhy
v.rare
CdElCdE
ryry
V
-
+
-
-
+
ccdee
rh
5.0
cde/cde
IT
E

[[p.36]]
102
Blood Banking and Transfusion
A third nomenclature was suggested by Rosenfield, Allen, Swisher et al. This nomenclature is a
numerical notation and free of genetic interpretation. Numerical notation is based on observed reaction
and phenotypes are expressed in terms of reaction. Positive and negative reactions are given equal
importance. The main symbol in this system is R followed by a colon, if there is a positive reaction the
number is listed after the colon and if a negative reaction then also the numberis listed but preceded by
a minus sign, e.g. CDe/CDe (Fisher), R) R 1 (Weiner) can be expressed as R : 1,2, 5, -3, -4 because in
this system 'D' is.listed as 1, C as 2, E as 3, c-4, e-5 and likewise.
But the CDE classification and nomenclature being easy to understand is widely used. InIndia also
the CDE classification is commonly used.
Distribution of different phenotypes detected by observing reactions with five antisera.
So from above, it is easy to understand that individuals have been broadly subdivided into Rh +ve
and Rh -ve according to reaction exhibited by cells with antisera having the similar primary Rh specificity.
Inheritance, genetics, antigens and antibody': The Rh gene complexes are inherited in a straight
forward way which is illustrated in mating below.
Homozygous father x Rh -ve mother.
First generation mating giving
rise to all- Rh+Ve offspring
Second generation mating
between Heterozygous
father and Rh -ve mother
offspring 500/0 Rh +ve and
50% Rh-ve
R,r
Fig. 5.7.
R1r
rr rr
'In the above model family can be expressed in terms of D antigen, e.g. DD (homozygous),
DD (heterozygous), Du, or absence of D in Cde type d being amorph phenotypically not expressed and
considered absent. It can also be expressed in terms of whole gene complex but for its high complexity
is avoided.
Rh antigens are mainly D, C, E, c, e and are well developed before birth particularly the tnt; the '0'
was even demonstrated on normoblast, it could not be demonstrated on spermatozoa, well developed in
cord cells. C, c, e, E, also could be detected in cord cells the later two were of course weakly present in
cord cells.
Variants of Rh antigens and in conditions where they are suppressed or absent are described later in
this chapter, Rh antigens are integral part of membrane of R.B.C. and structure is not definitely known
but it is presumably a protein with lipid with a mol weight around 28,000 and is destroyed by heating at
56°C. Not found on leucocyte platelet and other body fluid.

[[p.37]]
Blood Group System
103
Five antigens were studied and on average the antigenic site for Rh on red cells,
Antigen
'D'
c
C
E
e
Range
9,900 to 33,~OO
sites per red cells which varies according to different genotype
37,000 to 53,000
Huges-Jones quoted
70,000 to 85,000
by Mollison in
13,400 to 14,500
'Blood Transfusion in
18,200 to 24,000
Clinical Medicine" (loccit)
The amount of antigen, we detect by antisera can thus be influenced by antigenic sites, dosage
effect, position effect viz. cisposition and transposition of a gene and antisera specificity.
In dosage effect, e.g. DD homozygous will react better and stronger with antisera than Dd
heterozygous.
It has been also observed that E is better expressed in genotype CdElcde than on CDElcde presumably
because of suppressing effect of 'D' in later-CIS effect, In transposition it has been observed that in a
cde/cDe combination cde has inhibiting effect on cDe and 'D' is not expressed fully but when 'D' segregates
independently. away from cde and in an opposite chromosome cde inhibitory effect on CDe is not
pronounced-transeffect.
RH antibody
·Very soon after its discovery Rh antibody were demonstrated in milk of women, containing Rh antibody
in their serum; incomplete anti-'D' in aminotic fluid of foetus, who developed HDN at a later stage. Anti-
c and anti-E could be 'demonstrated in ascitic fluid. The Rh antibody mostly immune in nature can be
produced as a result of immunising effect viz. pregnancy (HDN), haemolytic transfusion reaction or by
intentional immunisation of volunteers. In rare occasion anti-E and anti-c was found in naturally occurring
form. Therefore, most of Rh antibody require potentiation before detection and only few could be
demonstrated by saline technique, Incomplete anti-Rh antibody could be potentiated by high protein
media, 'enzyme or by antiglobulin, Different procedure to detect Rh is as follows. Rh genotype and
phenotypes can be determined by antisera and it is done in laboratory with the help of five antisera anti-
D, C, E· and .anti-c and anti-e and its main utility lies in paternity test, medicolegal cases, and
anthropological study,
As it has has already been stated earlier that'D' is the most antigenic of all and so the 'D' antigen and
corresponding antibody is clinically more significant and for clinical purpose, it is not essential to know
whether the antibody is pure 'D', pure 'C' or pure E or ·itis a mixture of antibody. Rather identification of
antibody is more important so in routine laboratory work detection of 'D' is done with anti-D' sera. All
persons who failed to show agglutination are considered Rh -ve persons. The cells of such persons
should be subjected to Du detection test by anti-globulin test and serum of such person are to be tested
by a panel of blood samples at" least with three following types, rr, R 1, R J and R 1, R2 by saline method
and other methods to detect "incomplete antibody.
Techniques that are routinely followed in any blood bank laboratory is to detect Rh antibody are:
(i) saline method by slide or tube technique and if it fails, then
(ii) other procedures to detect incomplete antibody.

[[p.38]]
104
Blood Banking and Transfusion
Requirements
1. Slide or tube (kahn or precipitin tube)
2. Anti-D sera
3. Anti-C, E, e sera and c sera
4. Rh view box in case slide method is done
5. Centrifuge machine
6. Serological water bath
7. Incubator
8. Cell suspension in saline (commonly used) or LISS
9. Magnifying lens
10. Microscope
1]. 22% bovine albumin
12. Enzyme-Papain cystin commonly used
13. Coomb's sera
In any of the technique, we choose to employ it is essential to have control both positive anc
negativecontrol and we must be choosy for reagents since the polyclonal antibody are now-a-days almos
replaced by monoclonal antibody we can choose from any ofthe following anti-'D' reagentofmonoclona
nature:
(a) IgM anti-D
(b) IgM and IgG anti-D
(c) A blend of IgM anti-D and IgG anti-D polyclonal
The source for IgG anti-D polyclonal are usually human serum.
The later two has the advantage of Du detection by indirect Coomb's test (leT).
Controls
Three controls are used :
1. Positive control: Known 'D' positive cells preferably from ORlr andif it is not available OR)R
red cells can be used with standard anti-D' sera.
2. Negative control : Known 'D'negative cells again preferably 'O' negative cells. with standan
anti-tO' sera.
3. Negative controlon cells under test: In this control the anti-serum is replaced by a bland serun
such as AB serum. So this control contains unknown red cells with a bland serum and 22t?
bovine albumin is added to it. It is customary to include this control in test with slide, enzyme
technique, cord, cells when tested; it can be omitted in tube technique but it is better "if it i
included.
TEST
SUdF)
1. Place one drop of anti -RhD on a labelled slide test.

[[p.39]]
Blood Group System
105
2. 22% bovine albumin added one drop on the slide labelled control.
3. Cell suspension in saline, 40 to 50% - one drop from such suspension of unknown cell on both
test "andcontrol slide.
4. Both cell and antisera cell and control is mixed thoroughly with the help of a clean stick using
separate ends of stick for mixing test mixture and control mixture. Both mixtures are spreaded
evenly, at least covering an area of 15mm.
5. Place it over Rh view box kept at a temperature of 45°C, so that slides in contact are maintained
at a temperature of 37°C. The slides are gently rocked over the lighted view box for 2 to 3
minutes and observe for agglutination.
Positive test is indicated when agglutination is observed over test slide and no agglutination on
control slide.
If agglutination is found on control slide the test is not considered valid and repeated again. If no
reaction is observed in ~y
of slide test, indicates Rh negative but then a Rh negative sample has to be
confirmed by Du test 'and antibody. detected by treating with panel of cells as described earlier in this
chapter. Slide test is 'an emergency test and done in places where centrifuge are not available and also
done in outdoor camps. For routine laboratory work, it is not commonly employed because ofunreliability
insofar reaction is concerned weak reactions may miss Rh and quick drying may lead to wrong inference
of the test.
Tube test
RBC are washed at least twice with saline before being subjected to the test. In a standard technique,
two different saline antisera D is used, two precipitin tubes are taken placed in two rows and labelled (i)
-and (ii) and then one unit volume of antisera D from two different brand is dropped respectively in the
tubes of first row and of second row and it is also confirmedthat antiseras are really present in the tubes
and any tubes are not missed in the process. Now take one volume of 2% saline suspension of unknown
cells and add them to the tubes in first row and second row. Run positive control and negative control
alongwith test. The important thing to be strictly observed is that rinsing of pipettes with normal saline
and distilled water after each single use. Incubate at 37°C for 1Y2 hours to 2 hours. At the end of incubation
period, tap the bottom of tube gently and carefully observe for macroscopic agglutination and grade
according to the reaction.observed viz, +4, +3, +2 etc. In case of doubt take the help of optics and if it is
negative, proceed for further test of detecting Du, While making inference for the test compare with the
controls before arriving to a decision. The tubes in the second row serves as an additional confirmation,
if it tallys with the result of first row and further excludes any doubt the avidity and specificity of
antisera used.
Modified tube technique: There are three different types of modified tube techniques and each one
is improvisation over the other described successively in order as below:
(a) One unit volume of antisera D of saline reacting type is taken in a precipitin tube, to this is ,
added one unit volume of 2-5% cell suspension in own serum or AB serum and then 220/0
bovine albumin" one drop is added further to this mixture and incubate at 37°C for one hour,
results read after mild centrifugation. Negative control seen simultaneously consists of one unit
volume of 2 to 5% cell suspension with one drop of bovine albumin.
(b) In this method cells suspended in saline are used and the cell serum mixture containing one
drop of each respectively is incubated at 37°C for one hour. The anti-D sera used containing

[[p.40]]
106
Blood Banking and Transfusion
incomplete antibody. After incubation is over, one drop of 30% bovine albumin is allowed to
fall slowly by the side of the tube in such a way that it doesnot disturb the cell button. The test
is now incubated for a further period of half an hour at 37°C,. Results read after this period, the
agglutinate thus formed by this process is firm and tightness is not easily breakable the positive
results, therefore, will invariably be macroscopic, the negative results are to be continned
microscopically. Negative control consistsof cell 'and bovine albumin. This is a more sensitive
technique than the previous one.
(c) All the methods described aboveare known as sedimentation tube technique but in case of
emergency, one drop of anti-RhD sera is taken in tube labelled test and to it and.one volume 5%
cell suspension in saline, incubate for 10 minutes at 37°C and theri centrifuge the tube atl000
rpm. for one minute then gently resuspendthe cell button by tapping and observe for agglutination.
Negative control consists 5% of cell suspension in saline with one drop of 22% 'bovine albumin.
This method is known as spin tube technique. Negative results are to confirmed under microscope.
Positive test: Agglutination in tube labelled test and smooth cell suspension in the control.
Negative test: Smooth suspension of cells in both tubes, i.e., test and control.
Agglutination, if observed in control tube will indicate invalidity of test and the whole procedure is
repeated.
False positive results may be observed if:
(a) Reagent, i.e., anti-D sera may contain antibody of different specificity-this can be avoided by
using .antiseraof two different brands as described above in determination of Rh by standard
technique.
If the reagent is from human origin or contains human serum poly-agglutinable red cells which
are usually surface altered may react with anti-T and anti-Tn and 'cause agglutination. Reagent
may be contaminated with bacteria etc. may lead to false agglutination.
(b) Cells may sometime cause rouleaux formation if not washed properly before use,
(c) Cells having an immunological coating may show false agglutination even after washing.
False negative results are obtained:
(a) if antisera is inadvertantly missed furing the procedure
(b) if cell suspension is heavy and more than recommended
(c) deterioration of cell surface antigen on long storage
(d) prozone effect
(e) agammaglobulinimia and leukaemia -,
(0 incorrect incubation, temperature and time
(g) poor interpretation of result
Enzyme technique
Enzymes like trypsin, ficin, bromelin and papain are used to detect Rh antigen on red cells. For this
purpose enzyme solution has to be prepared easiest is papain and solution is made with saline whereas
ficin and bromelin are respectively dissolved in hydrochloric acid solution of N/20 strength and phosphate
buffer. Underlying biochemical process is that proteolytic enzymes as described above exposes the Rh
antigen and this in turn reacts"with incomplete antibody. Papain is commonly used, with papain we can
do the test by two methods :

[[p.41]]
Blood Group System
107
"(i) two stage technique
(ii) one stage papain cystein technique
Latter is preferred in most laboratory, ten grits of papain powder is dissolved in 250 ml of phosphate
buffer p~
6.2 and constantly shake while mixing is continued at R.T. for 15 minutes, Centrifuge and
deposit has to be discarded. Take 4.85 gm of cystin hydrochloride and dissolve in 25 ml of phosphate
. buffer. Supernatant of papain solution is 'now mixed with cystin hydrochloride solution and made upto
1000 mI. To test the Rh, add one volume ofabovepapain cystin mixture. with one volume of antisera. To
this add one volume Of2 to 5% cell suspension in saline incubate at 37°C for 30 minutes and read the
result.
·Anti human globulin m.ethod
Employed mainly to detect Du and antibodies of Rh system. Indirect test is employed to detect Du
antigen, 'full name of test is indirect Coomb's test.
(a) Red cells are washed at least thrice and a 5% cell suspension in N/IO saline is made.
(b) A drop of anti-Rh D serum is taken.in a tube labelled test.
(c) Add it drop (one unit volume) of 5% cell suspension to tube labelled test.
(d) Mix and incubate at 370C for 30 minutes.
(e) Centrifuge at~OOO
rpm for 1 minute.
(0' .Look for agglutination, and ifobserved then interpreted as Rh +ve for D' and need not proceed
ftwrtheL
.
(g) If no agglutination is observed or in case of any dubious reaction. Wash the cells again for three
to four times with saline,·supernatant is discarded after last washing and deposit of cells at
bottom is preserved.
(h) One unit volume of the cells at bottom of tube is pipetted out and transferred to another tube,
Containing one unit volume of AHG reagent, the cell and reage~t
is mixed thoroughly and
centrifuged at 1000 rpm for oneminute.
(i) After that read the result. Agglutination indicates positive for Du.
(j) In case of negative test confirm the validity of test procedure by adding one drop of known. IgG
sensitised cells again centrifuge and look"for agglutination. Presence of agglutination indicates
that the test is valid.
Essential in the procedure is running of control that consists. 'D' ,-ve cells and BSA one unit volume
of each. The control runs through all steps in the technique. Antisera '0' used for On testing may be any
one" of the following polycional anti-D-human ·origin, IgM and IgG' anti-D morioclonal reagent or
polyclonal anti-D (human origin) blended with IgM anti-D monoclonal.
.Better is to follow the i~struction
given in the insert with antisera 'D' vial.
Other antibodies
Apart from Rh 'D' antibody other antibodies, anti-C, anti-E. then anti-c and anti-e, the latter antibodies
are less commonly encountered 'd' being amorph, no anti-d has yet been encountered.
Anti-'D' : Mostly observed in people who are Rh negative. If any individuals lacks 'D' antigen. the
serum shows presence of anti-D', There c~uld
also be a mixture of anti-CD or anti-Dfi, this has to be

[[p.42]]
108
Blood Banking and Transfusion
distinguished from anti-D'. In such cases seras arc treated with phenotypes of standard cells and reactions
observed will help In distinguishing the different antibody or a mixture of antibody.
Standard cells used from following phenotypes for detection of antibody are. R]RI' R2~~
R.Rz,
RoRo' IT, r r, r"r (Table 5.16).
Anti-C is a pure form and unmixed with any 'other antibody is very rare and occasionally observed in
C negative individual and may occur in both complete and incomplete type, former is more common.
Anti-E is commoner than anti-C and commonly found in' E negative individual, can be detected by
papain enzyme. It has less clinical significance.
Anti-c : Commonly found in D +ve individuals in a mixture form because c antigen is comparatively
weaker antigen and repeated exposure fails to produce anti-c.
Anti-e : Very rarely encountered because 'e' antigen is a weaker antigen but because it is a common
antigen so individuals who lack it are very few as such frequency of.anti-'e' is also very less. There are
mixtures such as anti-CD, anti-CDE and anti-DE, they are not very common but occasionally observed.
In some cases. mixture containing all three antibody. may be observed.
.
Table 5.16. Detection of Rh· antibodies
Antibody
-
Phenotypes
R]RI'
R2~'
R1R2,
RoRo,
IT,
r'r,
rr
anti-D
-
(+)
(+)
(+)
(+)
anti-C
(+)
(-)
(+)
(-)
anti~E
-
(-)
(+) )
(+)
(~)
anti-c
-
(-)
(+)
(+)
(+)
(+)
(+)
(+l
anti-e
-
(+)
(-)
(+)
(+)
(+)
(+)
(+)
anti-CD
-
+
+
+
+
-
(+)
-
anti-DE
-:-
+
+
+
+
-
-
(+)
Variants of Rh antigen
The common Rh antigen are 'D', 'C', 'E', c and e and 'd' being amorph no expression of antigen on cell
surface, apart from the antigen described, there are other allelic antigen or variants some of which have
clinical importance and some are only of academic interest. They are described briefly asunder,
'0' variants and Du
Du : This is a weak expression of'D' antigen. Cells fails to agglutinate with anti-Rh '0' sera should not
be declared Rh -ve ~s it could be a Du and is best detected by AHG test. Du follows mendelian dominant
type ofinheritance and it has been observed more commonly in negro population than in white population.
The Du has been. further subdivided into high grade and low grade Du. The clinical significance in
transfusion practice is that Du though less immunogenic because of less antigenic sites can" stimulate an
anti-D in Rh negative recipient. Also Du infant can suffer from 'haemolytic disease of new born, the
proc.ess involved is same as in case of other Rh HDN. Haemolytic transfusion reaction may also occur as

[[p.43]]
Blood Group System
109
a result of Du blood transfusion to a recipient having anti-D' in his or her serum. And, therefore, for all
practical purposes, Du positive individuals are to be considered as Rh negative as recipient and Rh
positive as a donor.
.
.
D variants: It has been observed that many persons who ..are 'D' positive possess an anti-D, this
phenomenon was explained that'D' antigen"normally consists of a group of antigen. 'D' antigen mosaic
and in any individuals who is 'D' positive contains all the component part of mosaic they"are designated
as RhA, RhB, Rhc and Rho.Any positive individuals lacking any ofthe component part will be considered
as D variants and although behaves as D-positive usually possess an antibody that may be formed against
the missing fraction, They are usually detected when various anti-D sera do not exhibit pattern ofreaction
leading one to infer that there could be antibody of different 'D' specificity which is possible, if consider
'D' antigen as a mosaic or complex and the antibodies of different specificity are due to different part of
mosaic. It was also"observed that an Rh positive antigen lacked all of the four component parts, Du
individuals sometimes lack one or more of these fractions.
Clinically significant membersthat deserves attention are antigen Goa, Gonzales an antigen ofcategory
IV, commonly found in blacks and may cause mild haemolytic transfusion reaction. D
W is another one
that belongs to category V, commonly found in blacks forms anti-Rh",
Suppres.1ion
"~D-"
: It was observed by Race and Sanger (1950-51) in the product of consanguinous marriage, they
found that sample of blood lack all representation of C, E and c, e. This very rare gene complex was
designated as -D-. The red cells appears to have very large amount ofD antigen and antigenic site ranges
between 1,00000 to 2,00000 or above. They agglutinates In saline medium even by some incomplete
anti-D sera. -D- are highly immunogenic because of lack of-restraint on immune response due to absence
of C, E and d, e and thus may present with transfusion related problem. D is another rare gene and
followed discovery of anti-Evans antibody in evans family who possessed D antigen product of a very
rare gene D, similar but.not identical to -D- and they can be distinguished by anti-evans antibody only
which fails to react with D. D on rare occasion causes haemolytic transfusion reaction.
C variants
Commonly observed variants ofC and CW,·~o.
CW was first described by Calender and Race in 1946. The antibody agglutinated blood. in about 2%
white population who were C positive. Antigen was designated as CW and thought to be a product of
allelic gene CW to C and c but later on demonstrated that a whole Rh gene complex can include. CW, e.g.
CW cde. In rare occasions anti-C" has caused HDN and transfusionreaction CO a weak form of C is
produced by rG genes and discovered by Rosenfield and Levine around 1961. The other less important
variants are cd, ce and CV, CD.
E ·variants
Commonly observed variants ofE and EW, Ell and e",
E
W antigen was discovered by binding of antibody in a haemolytic transfusion reaction case by
.Greenwalt and Sanger in 1955 and it is found to be product of EW gene included in CDE complex the
affected chid was .CDelcDEw~
EW cell reacts specifically with anti-E'" but may be agglutinated by some
of anti-E sera. EU variant was discovered in 1.950 by Cepperline, Ikin and Mourant. The antigen was

[[p.44]]
110
Blood Banking and Transfusion
included in CDE complex as cDEu, EU is an allele at E locus and may be a depressed antigen like Cu.
They agglutinate some anti-E sera only and not .all of them are agglutinated.
eS
: Commonly found in blood of black population originally designated VS by Sanger and his
colleagues in 1960 after the name of patient in whose blood, it was discovered. e" are usually found in
association with other antibody.
DrS : The eS and hrs are not the same but due to superscript S in bots the cases the confusion may
arise. It was discovered in a Bantu women by Shapiro and his colleagues in 1960. The antibody was
designated as anti hroS antigen.
The cells in these cases lacked a component of hr'' (e) antigen, the component named hr" antigen.
Less common variants ofE-e are hr", et and ER apart from all these variants some compound antigens
like rG, rM, v or ces and depressed antigens like RN and (c) D (e). Rh33 or Ro Hir are described and are of
academical interest only.
(-1-) Rh null is a clinically significant member of Rh system which is responsible for Haemolytic
anemia in individuals who possess them. In 1961 Vos et al. discovered it by observing that all Rh
antibody failed to react with a particular sample of blood from an Australian aboriginal woman and they
deduced that it could be most probably dueto failure in synthesis of the Rh pr~cu_rsor
like in Bombay
Phenotype. Rh null are, therefore, of two types" one is regulator type and theother is amorph type where
the null gene r is located at CDE locus and in case of mating between two such persons offspring are
bound to be r r, this is very rare types whereas in regulator type regulator gene is suppressed by two xo
genes and substrate could not be produced leading to non-production of Rh antigens. Normally Xi cell
- prepares the substrate on which Rh genes act to produce Rh antigens and Lw genes act toproduce Lw
antigens. Rhnull peoples who inherits two XOr genes are also found to be lw negative, so -a relationship
may possibly exist between genetic pathway and biosynthesis of Rh null, Rh and Lw antigen. As the
name infers red cells of Rh null type lacks in antigen and .thus the lipoproteins necessary -to maintain
integrity of cell membrane. Cells are, therefore, defective and leaky giving rise to haemolytic anemia
characterised by high reticulocyte count, abnormally' high foetal haemoglobin, decreased cell survival,
occasional stomatocytes commonest antibody encountered in Rh 'null peoples are- anti-Rh-29 and is
usually observed when Rh positive blood is transfused, in some cases of pregnancy also it has been
observed, peculiarity of this antibody is that it does not react with Rh null cells but reacts with all other
cells. Rh null recipient. should, therefore, be transfused preferably with Rh null blood.
5.6 LWANTIGEN
At the beginning of Rh system, it has been hintedthat anti-Rhesus antibody from rabbits and guineapig
source used by Landsteiner and Weiner in 1940 are not same as human anti-Rh (0) and it has been
observed that this antibody has a different specificity. The antigen thus discovered is named LWantigen
after its discoverer. Similarity in behaviour of human anti-RhD and anti-LW supports the view that a
possible relationship might exist between them as has been hinted while discussing Rh null.
LW genes are inherited independently of Rh gene irrespective of their close association with each
other. LW antigen is believed to be ahigh frequency antigen and is present on most human RBC. LW
antigen activity is stronger in Rh positive cells and it can be observed from the reactivity exhibited by.
weak anti-LW with Rh positive cells, it agglutinates the Rh positive cells but not all the Rh negative
cells. So Rh negative cells, contains very less LWantigen that very often fails to react with weak anti-
'LW.

[[p.45]]
Blood ·Group System
111
LW antibody are known as anti-L\V and can be recognised ·by their reaction with both Rh positive
and Rh negative cord cells where they reacted equally and strongly. Anti-LW is usually of IgM in n~ture
but sometimes can be IgG also anti-Lw is found as warm autoimmune antibody in some cases ofAlHA.
Four phenotypes have been described in order of decreasing antigenic strength of LW· antigen.
LW)
LW+ and D positive
LWz
LW+ and D negative
L~3
LW+ and could be D positive or D negative
LW4
LW+ and could be D positive or D negative
LW3 is the commonest amongst all phenotype. LW serum used for testing purpose must be specified
or otherwise it may show varied reaction.
It has been suggested that LW is formed by action LW gene or Rh antigen..
Subdivisions of LW can be distinguished by their reaction. Anti-LW produced by LW3 reacts with
LW., and LWz but not with LW3 and L\V4. Anti-LW formed by LW4 will rea~t
with LW3, LWz, L'W1•
Ne3
: A new antigen experimentally shown to be a part of LW system was first observed in Finnish
population and consists of approximately around 6%, but lesser in other Europeans. An antithetical
. relationship exists between anti-Ne" and anti-Lw, (anti-Lwproduced by LW3 peoples). Ne" an allele at
LWfocus is redesignated as LWB and accordingly anti-Ne" is called anti-LWB and anti-Lw produced by
.Lw3 peoples are called anti-Lw", LW4 cells donot show any reactivity with either anti Lw" or anti-Lwf
shows results identical to Lw in respect to there reaction with D positive or D negative cells respectively
in adult and cord blood. First example of anti-Lw'' was a weak IgG
5.7 OTHER BLOOD GROUPS
Apart from ABO and Rh system, other blood groups were also discovered which are of course of lesser
significance but because of their complicity in cross-matching and haemolytic disease of new born ar i
in autoimmune haemolytic anemiathey ~~serves
attention ofblood bankers. They are again some antigens
known to occur on red cells of large majority of population but"they differ from all known. blood group
system and are termed "high frequncy antigen" or .public antigen. Some antigens occur in very low
frequency and restricted to some particular group or family and not related to any established system and
they are known as "low frequency antigen" or private antigen.
A short account of important ones follows but for detailed account interested reader may consult the
book - Blood Group in Man by R.R. Race andRuth Sanger (Table 5.17A).
MNSS
·MNSS is one of the important system .because it ~an
cause cross-matching problem, haemolytic disease
of new born and also transfusion reaction. MNSS system is considered as one of important parameter in
determination of probable paternity. It has also significant association with some disease viz. Ankylozing
spondylitis.
Discovered by Landsteiner and Levine (1927-28), the MN system has two antibodies, anti-M and
anti-N, thus dividing human population in following groups:
(a) Red cells agglutinated only by anti-M
(b)
Red cells agglutinated only by anti-N
(c) Red cells agglutinated by both anti-M and anti-N

[[p.46]]
112
Blood Banking and Transfusion
Three groups are thus ~1,
N and ~I~
respectively, and this forms basis of different anthropological
and medicolegal study because treated with antisera they show dosages effect (Table 5.17B).
Table 5.17A. Other blood group system
MNSS
PLewis
Ii
Duffy
Kid
,yt
Lutheran,
Rogers,
Kell
Xg
Sd
Ina
Chido
Di
Bg
Less important
in blood banking
J,
Auberger (Au)
Dombrock (Do)
Wright (We)
Colton (co)
Sciana (se
(Sm-burell)
High frequency antigen
!
Vel (Cea)
Gerbich (Ge)
Gregory (Gya)
Holly (Hya)
Applethaith (Ata)
lra
Lan
Low 'frequency antigen
J,
Swann (SW 8)
'Radin (Rd)
Peters (Pta)
Table ·5.178. MNs reaction
Phenotype
.:Genotype
Dosage effect
I'. M
MM
Stronger reaction with anti-M
2. N
NN
Stronger reaction with anti-N
3. MN
MN
Weakerreactionwith either anti-M or anti-N because of heterozygous nature
55 system
In 1947, Walsh and Montogomery found a new antibody, at first they could not find out any relation of
this antibody to any of the known system at that time. They sent the serum to R.R. Race and Ruth Sanger
who discovered that though it is a separate antibody but it was recognising anantigen closely associated
with M and N. The new antigen is termed 'S' and association was proved mathematically' by Race and
Sanger.
In 1951 Levine et ale discovered another antibody and' observed that it detects an antigen closely
resembling S. This gene product they observed is produced by a gene allelic to 'S' and they termed it as
s. It, was thus inferred that a pair of gene Ss exists which tho.ugh separate from M and N but are very
closely associated which was experimentally shown by Race and Sanger in 1419 samples drawn from
English population. Samples were tested with anti-M, anti-N and anti-S, and s result oftheir observation
(Table 5.18).

[[p.47]]
Blood Group System
113
Table 5.18. Frequency of MNSs system
Positive with
All salnples
M samples
MN samples
N samples
Anti'S'
55%
72%
56%
31%
Anti's'
89%
78%
92%
97%
So from above result \\'e can find M is more closely associated to'S' whereas N is more closely
associated with s. From the above observation, we can infer the association between MN and Ss system.
Like MN the SS system also divides population into S5, Ss and SSe
The anti-S and anti-s are more active at 37°C and they are also responsible for transfusion reaction
and haemolytic disease of new born. Cross-matching' problem are rare with S, s antibodies.
. Following discovery of Ss Weiner, Unger, Gordon and Cohen described antibody anti-U which they
observed agglutinates blood in white population but not in Negros and they inferred "that it could be
probably due to pair of allelic genes Uand u where U is ~ominant
to u.
Relative frequency of two types of S-s- in random Negros recorded by Allen, Madden and King also
by Francis et al., about 84%are U- and 16% U+. S-S-U- samples appears to have.normal MN distribution
American Negros and U- is more frequent in Africans than in American Negros.
Rare alleles at MNSs locus
A. N2 : A we~
N antigen controlled by third allele a.t MN locus first noted in association with a
weak. M in two family only. It is often associated with positive DCT test.
Na: yery rare and can be differentiated by ·anti-NA only. Found in Melanesians mainly.
-8. M variants :
(i) Mg: Anti-Mg antibodies are not uncommon and the anti-M and anti-N activity lacking or
may be absent causes discrepancy in family study and inpaternity 'study, MgM parents
may be mistaken as MM and i