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Medical Laboratory Technology: Volume 1
principles of immunohaematology
Before going into the details of blood transfusion procedures, it is important to understand
some of the basic principles involved in immunohematology and get acquainted with the
commonly used scientic terms.
Immune System of Blood
Red cells carry red cell antigens on their surfaces, while the serum carries antibodies within
the plasma. В lymphocytes are responsible for the humoral immunity and provide primary
protection against circulating antigen. These В lymphocytes produce antibodies, which are
immunoglobulins (proteins) that react specically with antigen. Plasma cells are dierentiated
В lymphocytes and are the most ecient antibody-producing cells. If the serum contains
antibody to its cognate red cell antigen, they will react and the red cells will either agglutinate
or lyse. In our search for compatible donor’s blood, we look for the one which does not have
the red-cell antigen whose corresponding antibody is present in the patient’s serum.
red cell antigens
Foreign antigens, when introduced into the body, can provoke the formation of specic
alloantibodies. Antigens and their specic antibodies react with each other when they come
in contact. Red-cell antigens are the blood group factors which reside on the surface of the
red cells. They may also be present in various body uids and tissues. These antigens can be
proteins or glycolipids. So far more than 300 antigenic congurations have been discovered
on the surface of red cells; the biological role for many is being elucidated.
The antigens of red cells are inherited. The antigen composition of all blood groups, as
determined in the laboratory, is the phenotype, based on the serologic reaction of red cells
(haemagglutination) with the corresponding antibody. Thus, AA or AO antigens on the red
cells react equally with anti-Α and the cells will be grouped as A. The genotype, i.e., the actual
genetic makeup responsible for the phenotype (e.g., AA and AO) can be deduced by tracking
the family history of blood types or by molecular testing.
Inheritance of the antigenic character of red cells follows Mendelian laws. Homozygous and
heterozygous conditions of red cell antigens are assessed from genotypes. When the alleles
of a gene, which are inherited from the father and the mother, code for the same antigen and
thus are identical (e.g., AA), it is called homozygous and when it is dierent (e.g., AO), it is
heterozygous. Thus the basic human blood groups A, B, AB and О can occur in the following
genotypes: AA or AO, BB or BO, AB and OO. We have ignored subgroups for the present.
This indicates that the blood groups of A and В can be homozygous or heterozygous; AB is
always heterozygous and О is always homozygous.
The Mendelian law of inheritance is applicable in the transfer of the antigen from the
parent to the ospring. Each antigen is controlled by a gene, which is the unit of inheritance.
A and В antigens of ABO blood groups are allelic genes and are of equal dominance. Lack
of both the antigens—A and В—on the red cells, results in the О blood group. If the father
and mother contribute to identical alleles—A/A, B/B, O/O—the ospring are homozygous,
whereas if they are dierent—А/О, В/О, A/B—the ospring are heterozygous. Inheritance of
the antigenic characters of red cells is illustrated in the following two examples (Figures 15.3a
and b).
Antibodies in Serum
В lymphocytes and plasma cells produce antibodies in an individual, following antigenic
stimulation. The antibodies are immunoglobulins, present in serum, and can be demonstrated
Introduction to Blood Transfusion Therapy
Figure 15.3(a) Inheritance of red cell antigens (Example 1)
379
Figure 15.3(b) Inheritance of red cell antigens (Example 2)
serologically by reacting with their cognate antigen (immunologic reaction). Antibodies
against red cell antigens can develop naturally in an individual without transfusion or
pregnancy. These are called naturally-occurring antibodies (e.g., anti-A). On the other hand,
immune antibodies are produced when red cells harbouring foreign antigens are exposed to
an individual who lacks these antigens (e.g., anti-D).
Immune and naturally occurring antibodies dier in physical and chemical properties. The
naturally occurring antibodies are a mixture of IgM and IgG with large molecular weight
(900,000 Daltons) whereas the immune antibodies (IgG) are of smaller molecular weight
(about 150,000 Daltons). Hence, the immune antibodies are capable of leaking through the
placental barrier and entering into foetal circulation.
Antibodies stay in the plasma and form a part of the humoral system of the body. Hence,
for the identication of an antibody, either serum or plasma is required.
Immune antibodies, associated with infectious agents, do not interfere normally in the
blood banking procedures except in case of certain diseases such as Mycoplasma infection
which is associated with atypical pneumonia and cold-reacting antibodies. These coldreacting antibodies can agglutinate virtually all red cells at 4°C.
380
Most often, the antibody reacts specically with its cognate antigen that stimulated its
production. Occasionally, however, two antigens have certain chemical groups in common
and an antibody made against one of them will react to some degree with the other. This
apparent dual specicity is known as cross-reactivity.
Isoantibodies are produced in the same species as the antigen source. Anti-D (anti-Rh)
produced by an Rh-negative individual, after receiving Rh-positive cells, is an example
of isoantibody production. Heteroantibodies, however, originate in species other than the
source of the antigen. The antihuman globulin used in antihuman globulin reaction (Coombs’
reaction) is an example of a heteroantibody. It is made by injecting human globulin into
laboratory animals. Lectins are plant products with antibody-like characteristics. Dolichos
biorus and Ulex europaeus are plant lectins that recognize Al and H, respectively.
Medical Laboratory Technology: Volume 1
recognition of immunologic reactions of red cells
Agglutination of red cells, called haemagglutination, is one of the most common visible
antigen-antibody reactions observed in blood banking for blood grouping and compatibility
testing. The red cell antigen involved in this process is referred to as agglutinogen and the
antibody as agglutinin. Various agglutinins, however, require dierent conditions for the
manifestation of agglutination reaction. The natural antibodies like anti-Α and anti-B react
with the corresponding agglutinogens A and В in saline medium. This is in contrast to immune
antibodies like anti-D, which react preferentially in a protein medium (albumin) with heat
treatment (37°C). However, the current anti-D produced by some manufacturers (OrthoEthnor, Mumbai, India) reacts with D antigen at room temperature and without protein.
Temperature has been found to aect the antigen-antibody reactions in some cases. Some
of the cold-reacting antibodies like anti-M react with the corresponding antigen at low
temperature (4°C), while others react at room temperature, and a few react only at 37°C. The
reaction strength of cold-reacting antibodies decreases at higher temperatures. Many immune
antibodies may show no visible agglutination reaction under any of the above conditions.
They are detected only by the antihuman globulin test.
Haemolysis, or the destruction of red cells, is another way of recognizing the antigenantibody reaction. In this case, the specic antibody (called haemolysin) needs complement
which is present in the fresh serum. Some of the blood group antibodies are only agglutinins
whereas others may be agglutinins which appear to become haemolysins upon antigenic
stimulation; the laer, however, once again agglutinate in the absence of the complement.
Complement is thermolabile and is destroyed by heating the serum at 56°C for 20 to 30 min.
It may also be lost upon storage for 2 to 3 days at 4°C.
Stages of Immunologic Reactions
Immunologic reactions occur in two stages:
1. Binding of the antigen and cognate;
2. Manifestation of the above reaction, leading to haemagglutination, haemolysis,
precipitation or other visible reactions.
The completion of the rst stage in case of the haemagglutination reaction results in coating
of the antibody onto the red cells, a phenomenon known as adsorption. Thus, some of the
immune antibodies may not produce haemagglutination in the saline medium but will be
adsorbed on the red cells carrying corresponding antigen. This is called sensitization. The
sensitized cells are later recognized by their reaction with antihuman globulin.
The elution process can separate antibody aached to the red cell antigen. The sensitized
red cells, when heated to 56°C or chemically treated and centrifuged, lose their coating
antibodies. These antibodies can then be concentrated into an eluate.
Introduction to Blood Transfusion Therapy
381
For visible immunologic reactions, proper proportions of antigen and antibody are required.
An excessive amount of antibody might lead to the prozone phenomenon (Figure 15.4). In
this, an immunologic reaction will not be visible until the antibody is suciently diluted.
Hence during titration, a positive reaction is not seen in undiluted serum but is visible with
diluted serum.
Figure 15.4 Eect of excess of antigen or antibody on immunologic reactions. Appropriate proportion
(equivalence) reveals the best visible eect of haemagglutination
laBoratory methods in detecting antiBodies
Blood bank laboratory adopts two methods in detecting and characterizing antibodies:
1. Detection of antibody reaction in vivo: Direct Coombs’ test detects the presence of any
antibody (or a group of antibodies) that might have reacted with the red cells in vivo.
The direct Coombs’ test demonstrates that in vivo coating of red cells by an antibody has
occurred. It does not identify the antibody responsible for the immunologic reaction.
It is a one-stage procedure. A positive Coombs’ test can occur due to blood group
incompatibility, may be drug-induced or observed in many clinical conditions.
2. Identication of the reacting antibody: If the direct Coombs is positive, antibody
identication becomes necessary. The unknown antibody in serum is reacted with a
group of red cells of known antigen phenotypes under dierent controlled conditions
to identify the antibody.
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Medical Laboratory Technology: Volume 1
human Blood group systems
The basis of human blood group system is the antigenic characteristics of red cells. The presence
or absence of antigen A and/or antigen В on the red cells determines the basic four blood
groups - A, B, AB and O. The presence of antibodies in the plasma follows Landsteiner’s law
which states that the corresponding antibody is never present in the serum of an individual
when the antigen is manifest on his/her red cells (Figure 15.2). The antibodies of the basic
ABO blood group system are naturally occurring.
It was later found that A and В are not the only antigens present on the red cells. There are
many other antigens, which are capable of producing immune antibodies in the recipient
when the laer is exposed to ‘foreign red-cell antigens’ (i.e., not those present on the recipient’s
native red cells) through transfusion. This observation led to the discovery of other blood
group systems.
Since the discovery of the ABO system, there have been over 300 blood groups systems
discovered so far, and more are added to the list all the time. Some of the common ones are
listed below while others are relatively rare.
Natural: ABO; P, MNSs, Lewis (Le), Lutheran (Lu), Ii
Immune: Rh, Kidd (Jk), Duy (Fy), Kell (К),
Note Ρ, MNSs, Lewis, Lutheran and Ii also act as immune systems.
Antigenic characteristics of red cells vary widely. The variations are not only due to
numerous combinations of antigens but also to their degree of dominance. For example, A
and В are co-dominant to each other while both A and В are created at the expense of O.
The more complex Rh blood group system has numerous antigens. Taking all systems and
type combinations into account, billions of dierent combinations of RBC phenotypes are
possible. However, only the ABO blood group system and the Rh system (D antigen typing)
are initially tested whenever blood is donated or administered. Others are identied only
when the recipient’s serum shows the presence of abnormal antibodies, or when phenotyping
of the individual’s red cells is of clinical utility.
Basic Blood group system: aBo
ABO blood group system is the most important of all blood groups because of the natural
presence of anti-Α and anti-B antibodies in persons who lack the corresponding antigen on his/
her red cells. In addition, transfusion of incompatible ABO blood groups can lead to potentially
fatal haemolytic transfusion reactions. A and В transferases (which move A and В antigen onto
the red cell surface) are in one locus on each copy of chromosome 9. These genes are alleles,
meaning that they are interchangeable at their chromosome location. Therefore, each of the
paired chromosomes carries any one of the copy antigen genes. A and В are relatively strong
antigens and serologically identied by commercially available anti-Α and anti-B typing sera.
Blood group О (or H) is dened, in part, by the absence of reaction for either A or В antigen, so
that О blood type implies no A or В transferases on either copy of chromosome 9.
This makes four major phenotype groups possible—А, В, AB and O. In addition, when
either A or В antigen is present on an individual’s red cells, the corresponding isoantibodies
(anti-Α or anti-B, respectively) will be absent from the serum; conversely, if an individual
lacks either A or В antigen, his or her serum will contain isoantibody to the missing isoantigen.
For practical purposes, О is considered to be non-antigenic. Therefore, a person who is AA
or AO will have anti-B isoantibodies in his or her serum, a person who is OO will have both
anti-Α and anti-B isoantibodies, and so on. The examples presented in Figure 15.3 will help
to explain the genetic behaviour of these red-cell antigens. Anti-Α and anti-B are bivalent
antibodies that react in saline at room temperature and are easily recognized. Occasionally,
Introduction to Blood Transfusion Therapy
383
however, the blood bank faces problems in their typing due to the presence of subgroups.
This is discussed later.
The distribution of the naturally-occurring anti-Α and/or anti-B antibodies is shown in
Table 15.1, along with antigens found on the red cells that determine the blood group.
Table 15.1 Antigens and antibodies in ABO blood groups and its frequency (%) in population
Blood group Antigens on red cells Antibodies in serum Frequency (%) in population
Indian* White Black
A A Anti-B 20 40 27
В В Anti-A 43 11 20
0 Neither A nor В Both anti-Α and anti-B 30 45 49
AB A and В Neither anti-Α nor anti-B 7 4 4
*Asiatic
Antibodies present in an individual are not inherited. They develop after antigenic
stimulation. The blood group antibodies, anti-Α and anti-B, are not detectable at birth, rather
they are weakly detectable at the age of 3–6 months and gain maximum strength at the age of
5 years. Anti-Α and anti-B in cord blood or neonatal serum is usually of maternal origin. The
titre of the natural antibodies increases through adolescence and then gradually decreases
with aging. Why the body is stimulated to produce antibodies to the missing A or В antigens
is not completely understood. It is generally considered that these antibodies arise as a result
of antigenic stimulation from the ABO-like substances so widely distributed in nature and
somehow cause a natural sensitization. Due to the delayed appearance of A and В antigens in
the newborn, the potential for false negative reaction in the newborn exists.
The frequency of ABO blood groups in Indian population as compared to the white and
black races is shown in Table 15.1. The table, however, is more representative of North Indian
population (Asiatic). Blood group О is more common than blood group В amongst South
Indians (Asiatic).
Subgroups of ABO blood group system
Ordinarily, lile diculty is encountered in ABO typing. There is, however, a more common
and potentially serious situation arising from the fact that subgroups of agglutinogen A exist.
These are called A1, A2 and A3. The most common and strongest-reacting of these is A1, which
comprises about 80% of blood group A and AB red cells, with A2 cells comprising most of
the remaining 20% of red cells. A2 is troublesome because it is sometimes so weak that some
commercial anti-Α serums fail to detect it. This might cause A2B to be falsely typed as В or
A2O to be falsely typed as O. This situation, however, improves if one uses the present-day
monoclonal antibody typing sera. The main importance of the A2 subgroup is that persons
with A2 sometimes have antibodies to A1, the most common subgroup of A. Presence of anti-Α,
in serum of A2 or A2B individuals are usually not clinically important but might occasionally
produce blood bank typing problems.
Antigen A primarily exists as a strongly reacting antigen while A2 is a weakly reacting
antigen found in a few people. This, therefore, divides groups A and AB into the following
subgroups:
A
Thus, with the consideration of subgroups of A, six main groups (phenotypes) of the ABO
system become possible:
A
, A1, B, A2, and A2B
1
, A2, Β, A 1,B, A2B and О
1
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The combinations of red-cell antigen and the antibody present in the serum in all the above
six groups are shown in Table 15.2. Other rare subgroups of A also exist. Subgroups of В have
been reported but they are also very rare.
The subgroups are important in blood transfusion. As evident from Table 15.2, anti-A
may
1
be present in A2 person and also, weaker subgroups can be mistyped as O. If this mistyped
group О blood is transfused to group О recipient, whose serum contains anti-A, a transfusion
reaction might occur.
Table 15.2 Antigens and antibodies in ABO blood groups (including the subgroups)
Blood group Red cell antigen Antibody in serum
A
1
A
2
В В anti-A
Α, Β Α
Β Α2Β anti-A1 (*)
Α
2
O neither A nor В anti-A, anti-B
(*) Not always, but occasionally found.
A
1
A
2
Β None
1
anti-B
anti-B
Occurrence of A2 blood group is rare. The A2 antigen may give only a weak reaction with
the usual anti-A serum. A special agglutinin obtained from seeds (‘lectin’) with the specicity
of anti-A1 agglutinates all A1 cells but not A2 cells. Thus in reverse grouping, if the serum of
an A blood group individual shows the presence of anti-A activity (by agglutinating A1 cells),
the presence of an A subgroup may be suspected.
Other Variants in ABO Blood Group System
Variations in the ABO blood group system are occasionally seen that defy the general reactions
shown in Table 15.2. These are alternative blood groups. Some of these are listed below:
• Existence of subgroups.
• Defective blood group О with only anti-B (and low or no anti-A) is occasionally
found. This may be due to very weak or abnormal A antigen present on the red cells.
Similarly the presence of only anti-A (and low or no anti-B) in blood group О also
exists.
• А, В and AB blood group individuals occasionally develop an insignicant anti-Η;
this may prevent these recipients from receiving group О red cells, which have the
Η antigen.
• Red cells of Bombay blood group lack A, В and Η antigens (H antigen is normally
present on the red cells of all ABO blood groups). Hence, they develop anti-Η along
with anti-A and anti-B. Presence of anti-Η prevents these group О variants (Oh) to
receive normal group О blood.
Landsteiner's rule regarding the absence of corresponding antibody in the serum, when
the antigen is manifested on the red cells, holds generally true. Exceptions, however, do
exist, with the presence of antibodies in the plasma against corresponding antigen on the
red cells.
Plant Agglutinins for ABO Blood Group
Some plant products are found to have properties like anti-A, anti-B and anti-Η. These
are called lectins. The most important is the puried extract of seeds of Dolichos biorus,
Introduction to Blood Transfusion Therapy
385
containing a phytohaemagglutinin, which agglutinates only human red blood cells that are
A1 or A1B. It is often sold under the name of anti-A1. Other lectins include products from Ulex
europaeus with anti-Η properties. These are used as reagents (antibodies) in the blood bank.
rhesus (rh) Blood group system and immune antiBodies
The Rhesus (Rh) blood group system is clinically the second most important blood group
system in humans because of the two following reasons:
1. Аn Rh-negative (i.e., D negative) individual if transfused with one unit of Rh-positive
(i.e., D positive) blood can form anti-D antibody in 20–80% of instances.
2. Haemolytic diseases of the newborn occurs as a result of Rh incompatibility of the
mother (antigen negative) and the foetus (antigen positive), which is not uncommon.
Unlike anti-Α and anti-B antibodies, which are naturally occurring antibodies, anti-Rh
(anti-D) does not develop without an immunization stimulus. The D-antigen, like any other
red-cell antigen, is inherited; however, anti-D develops in a D-negative individual receiving
D-positive red cells. AU D-negative individuals, however, do not respond to the stimulation
with D-antigen and only 20-80% of them produce anti-D. There are many other blood groups
that behave like the Rh, but no other blood group antigen has comparable immunizing
potential.
It may be appropriate to recall at this point the history of the discovery of Rh blood group
system. The presence of Rh antibody was rst suspected in a woman whose foetus had died
of haemolytic disease. The discovery of the Rh blood group system by Landsteiner and
Wiener in 1940 not only explained the reason for the death of the foetus but also provided
reasons for many unexplained strong transfusion reactions (and occasional deaths) following
blood transfusion even when the patient was given the same ABO blood group (Figure 15.5).
Figure 15.5 Immune reaction of Rh antigen: (a) Rh-positive blood given to an Rh-negative individual
does not cause any reaction in the rst transfusion, (b) but causes an adverse reaction
in the second transfusion, (c) which can be fatal in subsequent transfusion
Landsteiner and Weiner transfused the red cells of a Rhesus monkey (Rh) into a rabbit
(Figure 15.6). The rabbit produced antibodies (anti-Rh) that were capable of agglutinating the
red cells from Rhesus monkeys and also the h uman red cells of 85% of the human population
in the United States. These were designated as Rhesus-positive individuals. The remaining
15% of the human population whose red cells did not react with the rabbit antisera (anti-Rh)
were called Rhesus-negative individuals. Having Rh (D) antigen on red cells makes a person
Rh-positive, irrespective of the major ABO blood group. Thus a person belonging to any
of the ABO blood groups can be either Rh-positive or Rh-negative. The inheritance of Rhantigen is independent of ABO antigen. The Rh blood group is a highly complex system in
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Medical Laboratory Technology: Volume 1
which more than 110 antigens have been identied; however, in routine blood bank practice
one deals with ve basic antigens.
Figure 15.6 Discovery of Rh blood group system: Red cells of rhesus monkey (Rh antigen) when
injected into the rabbit lead to the production of antibody against rhesus monkey red cells
(anti-Rh) and also agglutinated the red cells of 85% of the white population (called Rhpositive) and failed to agglutinate 15% of the remaining population (called Rh-negative).
Rh-Antigen
There is a considerable controversy over nomenclature of the Rh genetic apparatus between
advocates of the English Fisher–Race CDE–cde nomenclature and the American Wiener’s
Rh–hr labelling. The corresponding names of each of the antigens in the two systems are
given as follows:
Wiener Fisher–Race
Rho D
Rh‘ С
Rh” E
h’ С
hr” E
The Fisher-Race nomenclature, which is easy to follow, is currently adopted in most
laboratories. Hence, in our subsequent discussions we will only refer to this system.
The Rh blood group system is controlled by ve co-dominant closely linked allelic genes
that go together in three pairs—Cc, D and Ее. Presence of ‘d’ antigen cannot be proved
because anti-d has not yet been discovered. Thus, the absence of ‘D’ antigen is considered
as equivalent to the presence of ‘d’ antigen on the red cells. Persons whose red cells possess
‘D’ antigen, irrespective of the presence or absence of other Rh-antigens, are designated as
Rh-positive. Those whose red cells lack ‘D’ (or Rho) are designated as Rh-negative. Anti-D
was readily provoked and identied by its reaction with the red cells bearing D antigen (Rhpositive). Hence, D was the rst antigen discovered. The other four major antigens С, E, c, e,
though present in every individual in some combination, do not frequently produce strong
reacting immune antibodies. Since every person has two chromosomes, contributed by the
two parents, everyone has two Rh alleles to decide Rh-related red-cell antigen. They may be
identical or dierent—CDE/CDE or CDE/cde, for example. Fisher-Race visualized three pairs
of closely linked allelic genes giving rise to eight possible antigen combinations:
Introduction to Blood Transfusion Therapy
387
Dee, DCe, DcE, DCE, dce, dCe, dcE, dCE
Rh-positive genotypes Percent distribution
CcDe 34.7%
CCDe 19.3%
CcDEe 13.2%
ccDEe 11.5%
Others 21.3%
Inheritance of Rh group is independent of the ABO group. Approximately 95% of the
Indian population are Rh-positive and 5% are Rh-negative. The corresponding gures for
Caucasians are 85% and 15%. Of the Rh-negatives, the frequency of cde is higher, while the
other types (Cde, cdE, CdE) are rare. The presence of all the aforesaid Rh-antigens (C, c, D, E
and e) can be demonstrated by the haemagglutination reaction of red cells. This is shown in
Table 15.3.
Table 15.3 Identication of Rh-antigens by haemagglutination reaction of red cells with known
antibodies
Known reagent antibodies Specimen No. 1 Specimen No. 2 Specimen No. 3
anti-C + + –
anti-c + – +
anti-D + + –
anti-E – – –
anti-e + + +
Antigenic character CcDe CDe cde*
*Anti-d does not exist and the presence of ‘d’ is indicated by the absence of ‘D’
Rh-Antibody
Rh-antigens lack corresponding naturally-occurring antibodies in the serum. Therefore,
when antibodies (anti-Rh) appear, they are of the immune type and are result of sensitization
caused by Rh-antigenic stimulation on receipt of red cells by an Rh-negative individual.
This might happen from transfusion or in pregnancy. It is now well documented that red
cells from the foetus can reach the blood stream of the Rh-negative mother. In this way the
mother can develop Rh antibodies against the Rh-antigen of the foetus. One exception to this
occurs when the mother’s serum contains antibodies against the ABO group of the foetus.
For example, if the mother is group О and the foetus group B; in such cases, the foetal red
cells are apparently destroyed in the maternal circulation before Rh sensitization can proceed
to signicant extent, although this does not always happen. Rh incompatibility was a major
cause of blood transfusion reactions, although these reactions are usually considerably less
severe than ABO-incompatible transfusion reactions. Rh antibody transfusion reactions might
occur by transfusion of donor blood containing Rh antibodies or by previous sensitization of
a recipient, who now will have the antibodies in his or her own serum.
Rh-antigen may be typed using commercial antiserum. Preliminary screening is performed
only for antigen D, which establishes a person as Rh-positive (D-positive) or Rh-negative
(D-negative). If a person is Rh-negative, further studies with antiserum to other components
of the Rh group may be done, depending on the situation and the individual blood bank. In