Immunehaematology

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Imm.unohaematology
4.1 INTRODUCTION
In the earlier days around 15th and 16th century when blood transfusion was in experimental stage,
violent and fatal reaction were observed in the recipients of blood - interspecies or homologous. These
reactions were more fatal in former than in later and very often lead to death of.recipients. This was
clearly an impediment in safe practice of blood transfusion. With our present day knowledge we know
that this phenomena observed was due to red cell destruction sequel to immunological reaction in an
incompatible blood transfusion. But until 1900this problem remain unsolved. An assistant at the pathological
Anatomical Institute of Vienna "Karl Landsteiner" (Founder father of iriununohaematology) in his paper
of historical significance "On Agglutination of Normal Human,Blood - The agglutination of Human blood
by human serum ought to be called isoagglutination according to nomenclature of Ehrlic and Mongenoroth.
Shortly after my publication these two investigators reported experiments in which they injected blood
of the same species and thereby succeeded in preparing isoagglutinins and isolysin, that is the sera
which acts upon the Red blood cells of the same species". This discovery served as a landmark
development in the history of evolution of immunohaematology. The term immunohaematology can be
better explained through its reference to immunological reaction that involves the. blood component
(cellular and liquid). It is one of the oldest subspeciality of Applied immunology, but lacked recognition
and proper attention was drawn to this subspeciality only when Dr. Israel Davidshon presented
"Immunohaematology, A new branch of clinical pathology", in his presidential address to the American
Society of Clinical Pathologist in 1954. Till 1960 major emphasis was laid on RBCs only but after that
the immunology of other elements of blood viz., platelet, leucocytes, plasma etc. became the focus of
investigation. With the advent in basic sciences, biochemistry and cytogenetics, this distinct subspeciality
encompassed into its folds principles of genetics, anthropology, criminology and resolution of disputed
paternity. But in this chapter we will only deal with those aspects of immunohaematology relevant to the
clinical practice of safe transfusion of ·blood.
4.2 ANTIGEN
Antigen are substances which could be protein or non-protein and 'when introduced to animal host can
cause production of specific antibody, thereby inducing an immune response-cellular or humoral or both.
The antigens can react with elicited antibody or wit.h sensitised lymphocyte to induce immune response.
53

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54
Blood Banking and Transfusion
Haptens are known as incomplete antigen. It cannot elicit immune response but can react with
specific antibody, also it can block a reaction. Thus hapten have only the specificity and their usual
molecular weight is below 40,000. In blood banking red cell antigen are of special relevance other
components exhibiting antigenic property are thrombocytes, leucocytes and certain biological fluid.
Property of antigen
Chemically complete antigens are protein-glycoprotein and lipoprotein, pure carbohydrate acts as antigen
in certain animals viz. man and mouse. Lipids usually serves as hapten. The molecular weight of antigen
varies between 40,000 to 50,000.
Physical property is governed by criterias such as site of antigen, number of antigenic site, antigenic
determinant.
Site of antigen is determined by cell structure, size, shape and membrane condition etc. In blood
group ABO antigens are extramembraneous whereas Rhantigen are assumed to be intramembranous.
Number of antigenic site and closeness of these sites explains the diversity in behaviour of antigen.
By means of immune electron microscopy, it has been confirmed that ABO antigen occur in cluster
whereas Rh antigens are isolated. It has been estimated that 800,000. A sites are present on Al red cells
and about as many B sites on B cells and for number of H sites, it has been estimated twice as numerous
on 0 cells than on A or B cells. The RH antigenic site varies between 10,000 to 85,000 depending on
phenotypes.
Antigenic determinant is the particular site of an antigenic molecule that combines with specific
antibody. Using isomers of Tartaric acid as the haptens, antibody were produced in rabbits and the
resultant antisera were capable ofdistinguishing the isomers in serologic tests. This immunologic reaction
was beautifully demonstrated by Landsteiner.
Specificity is also very important property of antigen for which polysaccharide component of
antigen is responsible and form the basis of dividing human blood group in A, 13, O. "and AB, e.g.
erythrocyte of B-group will react specifically with anti-B and not with anti-A and vice versa.
Antigenicity gives us an idea about the potency of a particular antigen. It is the amino acid fraction
that determines the antigenicity in blood group antigen. It varies with type of antigen and also with
immunised host. We are more concerned with red cell antigen and it is said that blood group antigen are
far less immunogenic than microorganism. And again in blood group, it is red cell antigen which are
comparatively stronger and in order they can be placed as follows: A.B > RhD > Kell > c & E > Fya >
e· & C > JKa > JKb > S > P.
Red cell antigens are weakly expressed in new born they are weak expression in A and B and very
weak expression are of I, Lea' Leb and sd'. Fully developed antigen at birth are Rh, K, Fy, JK, MnSS, DI
and Sc system (Race and Sanger, 1975).
Reticulocytes are strongly agglutin~ted
?y anti-A and anti-B, Red cell antigen A, AI' Band H have all
been demonstrated on erythroblast and D antigen on Pronormoblast.
4.3 ANTIBODY
Agglutinin or antibody are serum protein more specifically immunoglobin. In human being antibodies
can be natural or immune depending on its origin or source. Natural could be due to bacterial or of plant
origin because it has been observed that both may contain A or B antigen and as a result of immunisation

[[p.3]]
Immunohaematology
55
produces anti-A or anti-B of IgM type, 19A type and they occur without known antigenic stimulus,
Immune anti-Aor anti-B areproduced as aresultof irrununisation duetofoetomaternalleakage, incompatible
transfusion and they are acquired or incomplete antibody usually of IgO type. When red cell possess the
corresponding antigen the antibody are generally not produced'.
Antibody in new born infant - Normally human foetus does not began to produce significant amount
of antibody when in the mothers womb. Usually IgM is produced in utero and after birth since the IgM
does not cross placenta, it may be detected in the cord serum. IgG is' usually not detected and if at all
then this can beattributed to placental transfer. Antibody starts appearing in second month of intrauterine
life the IgM reaches adult level by 6 months to 1 year. Serum IgG reaches adult level between 6 to 7
years after birth and serum IgA reaches adult level by about 12 years after birth. Thus antibody level
increases between birth and puberty after that a constant level is maintained till old age and then it
gradually falls and may even reach the infantile level.
Red¢lls are destroyed by antibody in vivo this destruction may be either extravascularor intravascular.
The intKtvasc~lar
destruction is complement mediated and takes place within the vessel whereas
extravascular destruction takes place outside the vessel mainly in the spleen from where the deformed
and inelastic erythrocytes are removed.
4.4 IMMUNOGLOBULIN
Immunoglobulin are gammoglobulin fraction of plasma protin. Plasma proteins consists of albumin,
alphaglobulin, betaglobulin, gammaglobulin. The y (gamma) globulin are again subdivided in IgA, IgG,
IgM, IgD and IgE. The Immunoglobulin are protein and as such they are aminoacids linked by peptide
bonds. Basic structure consists of two heavy polypeptide chains and two light chains (Fig. 4.1). The
two heavy chains are held together by disulphide bonds, each chain has a molecular weight of 55,000
MW and consists of 440 aminoacids. Th heavy chains are placed parallel to each other.Each light chain
are small polypeptide chains and are attached to each heavy chain in the hinge region by means of
disulphide bonds. Hinge region is a coiled region in the heavy chain where the molecule is flexible i.e., it
can bend, open or close, the enzyms and alubmin acts in this region. The light chains can be either
Kappa (x:)or Lambda (A).In any given immunoglobulin molecule, they are either Kappa or Lambda but
their content may differ in any immunoglobulin class. Each light chain have a molecular weight of
22,500 Daltons and consists of 220 aminoacids. Heavy chains are structurally different from each other-
are designated by Greek letters as follows, IgA by ex (alpha), IgM by Jl (mue), IgG by "((gamma), IgD
by 0 (delta) and IgE by e (epsilon). Heavy chains are class specific. The antigenic difference in four
subclass of IgG namely, 101, ,02, )G3 and ,04 are due to structural difference in 'C' region of heavy
chain. The gammaglobulins are serum proteins and the antigenic differences between the members of
the same species that may occur are known as allotypes.
The allotypes are genetically controlled variations in aminoacid sequences found in the 'C.' region of
heavy chain and light chain and are not observed in all individuals.
The Isotypic antigenic determinant are found on all heavy chains and light chains (Kappa and
Lambda) in their constant region. The immunoglobulin isotypes namely IgG, IgM, IgA, IgD, IgE are
c.apable of combining with Kappa (lC) and Lambda (A.) light chains and forms at least 10 (ten) diffemt
composites present in all the individuals. Isoallotypic markers are those which are isotypic for one
subclass and allotypic for another subclass.

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56
Blood Banking and Transfusion
Abs __
I
x3
~
f
>•
FAB
l
I
I
Basic Structure of
Immunoglobulin Molecule
Abs - Antibody Binding site
V - Variable Domain
S-S • Disulphide Bond. L - Light
Chain, H - Heavy Chain
C • Constant Region, h - Hinge
Light Chain
Showing Km Allotype
Variable Domain
FAB - Fragment Antigen
Binding, FC-Fragment
CrystaUisable (Site for
Complement, Grn Allotype)
Heavy Chain
ShowingGm Allotype,
Hinge(h)
Region. Papain (pa)
And Pepsin acting
site (pe)
Fig. 4.1. Schematic represontation (not as per scale).
The idiotypic antigenic determinant are genetic variants closely related to antigen combining site of
immunoglobulin molecule. The antigenic individuality of any immunoglobulin molecule is said to be
imported by idiotype. Anti-idiotypes have been demonstrated by injecting idiotypes.
In this connection it will beworth while to briefly discuss the immunoglobulin allotype because Gm
and Km are relevant to the transfusion practice.
Grubb and Laurel (1956) quoted by Boorman and Dodd (Ioc cit) observed that" very highly diluted
serum of sufferers from Rheumatoid arthritis agglutinated the Rh (D) positive RBC previously sensitised
by incomplete anti-Rh (D). They further observed that this agglutination can be inhibited by certain sera
and that the inhibitor is found located in gamma globulin fraction of the serum and Om (a+) was
designated for the sera having inhibitory property and Gm (a-) for the individuals having non-inhibitory
sera. Om are, therefore, polymorphic antigenic determinant located on the heavy chains of IgG The
agglutinating activity of sera as described before is due to anti-Gm acting with specific Om allotype/
antigen on anti-Rh (D) IgG molecule (incomplete) coating the RBC. In course of further investigation
various Immunoglobulin allotype was discovered and named accordingly, e.g., G1m, G2m, G3m, A2m,
Em and KIn. The former are allotype of heavy chain respectively, IgG 1, IgG2, IgG3, IgE and IgA2 and
the KInis for Kappa light chain.
The anti-Gm antibody are detected in children, multiple transfusion cases, occasionally iiipregnancy.
In children they are mostly found in thalasemics and exchange transfusion cases. They are also observed
in patients with Rheumatoid arthritis.
Immunoglobulin molecule on treatment with enzymes. viz. papain and pepsin breaks into fragments,
When treated with papain, it breaks into three fragments the two identical ones are known as fragment
antigen binding and consists of a part of heavy chain and complete light chain. Both heavy chain and
light chain consists of a variable region and a part of constant region. Variable region in both light and
heavy chain consists of first 110 to 120 aminoacids in a variable sequence. It is at this place the antigen
binding occurs in a hypervariable region within variable domain and are thus responsible for antibody

[[p.5]]
Immunohaematology
57
specificity. The Fe fragment is the third fragment and consists of heavy chain only. This region bears
the characteristic ofimmunologically defined classand is responsible for complement activity, placental
permeability and antihuman globulin activity and also the site for GM allotype.
When treated with pepsin fragmentation is different and unlike the Fab fragments ofpapain treatment,
it retains both the ability to bind antigen and cause agglutination. Here the Fab fragment is known as F
(ab') 2. The remainder of fragment consisting of two heavy chains breaks up into small fragments.
Basic structural unit of immunoglobulin is a monomer - IgG is a monomer. It is found in T
configuration in native state having dimension of 12.5 nmIll.7 nm that forms a basic unit. Arms carrying
light chain can swing 30° arc in any direction at the hinge region. Upon combination with antigen, it
achieves and retain a y configuration. It is the amino acid sequence in heavy chain that is responsible for
immunologic difference in 1, u, a, 0, E.
IgG
It is a monomer, structurally 1 heavy chain class. 75 is the sedimentation constant, molecular weight is
150,000 to 160,000 dalton, shows isoagglutinin activity for both complete and incomplete type, it can
fix complement, cross placenta, serological activity remains unaffectd after heating at 56°C. It is divided
in four subclasses - )'01' )'02' )'03 and )'04. Serum concentration of IgG subclass varies between 12 to
14 mg/mI.
(a) lGt : Gm factors present are I, 2, 3, 4, 17 and 22, it fixes complement, anti-A and anti-Rh are
dominant antibodies, half life 23 days approx. crosses placenta and relative serum concentration
is 70% approximately, it binds to macrophage, moderately susceptible to papain at hinge region.
Dominant antibody activity - ATS, ADS, anti-DNA, anti-RH cleavage.
(b) lG2 : Relative s~ruln
concentration is 23 to 28%, GM factor 23 to 28, Gm factor 23, half life
22 days, poorly activate complement, dominant antibody activity are anti-DNA and anti-dextran,
mayor may not cross placenta, binds macrophage, poorly susceptible to papain at hinge region.
(c) lG3 : Relative serum concentration 4 to 8%. Gm factors 5, 6, 10, 11, 13, 14, 15, 16 and 21.
Fixes complement, half life nine days, dominant antibody activity anti-Rh, strongly susceptible
to papain clevage.
(d) lG4 : Relative serum concentration 3 to 4%, mayor may not fix complement, half life 22to 23
days, dominant antibody activity anti-AHF, may not bind macrophage moderately susceptible to
papain clevage.
'
IgM
It is a pentamer, structurally J..l heavy chain class, sedimentation constant 19S, molecular weight 900,000
.dalton, does not cross placenta, fixes complement, heat labile, isoagglutinatinin activity for complete
antibody, serum concentration in mg/ml is 0.95 to 1.5, half life is 5 days in vivo, electrophoretic mobility
is between 'Y and ~.
JgA
Usually a monomer but can polymerize to dimer or trimer, structurally a heavy chain class sedimentation
constant varies between 7S to 98, does not cross placenta due to lack of placental permeability factor in
Fe fragment, does not fix complement, serum concentration is 2 to 4 mg/ml, half life is 6 days,

[[p.6]]
58
Blood Banking and Transfusion
electrophoretic mobility between yand ~, molecular weight 160,000 to 180,000 dalton, antibody activity
is usually incomplete.
fgO
Monomer, structurally 0 heavy chain class, serum concentration is 0.~03-0.3
mg/ml, some antibody
activity, does not cross placenta, complement fixation activity not definitely known, molecular weight
185,000 dalton, half life 3 days in vivo, electrophoretic mobility between "(and p.
IgE
Monomer structurally £ heavy chain class, serum concentration is 0.0001 to 0.0006, antibody activity "is
present, sedimentation constant 8S, mol weight is 150,000 to 200,000 dalton, heat labile, half life 2.5
days, does not cross placenta, fix complement,
IgG, IgM and IgA are more relevant to transfusion practice.
.4.5 ANTIGEN·ANTfBOD'!
REP~CTiOt~
It4 VITRO
Mainly five types of reactions are observed in vitro and they are :
1. Agglutination
2. Haemolysis
3. Precipitation
4. Inhibition
5. Complement fixation.
Before going into details of the reaction in vitro we must consider the factors that affects 01
influences the Ag-Ab reaction. First to be considered are the reactants i.e. antigen and antibody, followed
by temperature, time ofincubation, ionic strength, pH, centrifugation, antigen and antibody ratio and
zeta potential.
(a) Antigen: Red cells are the antigen:
1. A properly stored red cells at recommended atmosphere will show better reactivity.
2. Dosage effect are shown by ho~ozygous
cells.
3. Repeatedly washed cells will get rid of plasma that contains .fibrinogen, blood group
substance, anti-coagulant that hampers in the reaction.
4. Repeated washing prevents rouleaux formation,
(b) Antibody: They are found in the sera and the sera reacts variably at times depending on source
of origin, on storing condition (stored sera losses its reactivity). It also depends on titre and
avidity .ofantisera. Inhibition of reaction may be observed with high titre sera when excess of
antibody is present-Prozone phenomenon.
(c) Temperature: Depends on type ofantibody viz. Rh antibody usually IgG shows betterreactivity
around 37°C whereas IgM
acts better at room temperature and cold agglutinin around and
below 4°C.
(d) Incubation time: Average incubation time is around 60 minutes but it may differ depending on
when the equilibrium is reached in a reaction. Time may be decreased by addition of agents like
LISS.

[[p.7]]
Immunohaematology
59
(e) Ionic strengtb : Low ionic strength solution (LISS) acts by increasing the rate and amount of
antibody uptake. While using LISS 2 drops of cell pretreated with LISS to be added with two
drops of serum to provide the optimum condition for reactants. Ionic strength, if lowered from
0.17 to 0.03, the rate of association is enhanced.
(0 pH: Reaction shows better result when close to normal pH 'of blood, i.e., 7.42 and optimal
activity is observed between 6.5 and 7.5 pH, around 7 is,ideal pH value, below 6 and above 8
may be detrimental for the purpose of reaction.
(g) Antigen-antibody ratio: A dilute solution of cells will ensure proper saturationof antigen sites
with antibody molecules and thereby provide the optimum mixture of reactants. So a dilute
solution of 2 to 5% cells suspension, two volumes of which is used with two volumes of
serum.
(It) Zeta potential: Erythrocytes have negative electric charge that keeps the cell apart viz. like
poles repels.The electrical activity of the cloud of ions arounda cell suspendedin NaCl solution
is called zeta potential which keeps the cell apart and needs to be overcome or abridged by
antibody for agglutination to occur. Addition of high molecular weight substance such as 22%
BSA or enzymes increases the dielectric constant of medium by lowering the zeta potential.
This facilitatethe antibodyto abridgethegap betweenantigensand the mutualattractionbetween
antigen and antibody.
Agglutination
This is the property of erythrocytes to gather into clumps under condition of immune response and is
the end result of antigen antibody reaction. It occurs in two stages :
(a) .Sensitisation : A process in which antigen on red cell surface takes up the antibody. Red cells
are now termed as sensitised.
(b) Oumping: Collisionoccursbetweenthesensitisedcellsand theintercellularbridgesof antibody
causes the necessary cross linking to effect the agglutination. And what.we observe by naked
eye is an aggregate of cells bound together by antibody bridges - a lattice work,Agglutination
can be large enough to be visible by naked eye or weak enough and needs help of microscope
to observe it. Agiven antibody when causes agglutinationwith red cell is complete antibodyand
if it only unites with red cells and does not cause agglutination, it is called an incomplete
antibody.
Agglutination follows the principle of the law of Mass action which states that matter is neither
created nor destroyed so in a chemical reaction, reactants are converted into products.but mass remains
the same. The whole process'of agglutination is expressed in terms of reversible reaction. Hughes-Jones
in 1963 described the equation as follows :
K.
Ag+AB ~
AgAb
K2
whereAg is antigen andAb is antibody,K) is association constant and ~ is dissociationconstant,Ag-Ab
is antigen antibody complex.

[[p.8]]
60
Blood Banking and Transfusion
By applying law of mass action we obtain:
(AbAg) =!L=K
(Ab) x (Ag)
K 2
where (Ab) (Ag) represents the concentration of reactants and products and K is the equilibrium or
combining constant. Therefore at equlibrium :
(AbAg) eq =K(A )
Ab (eq)
g eq
Therefore, according to equation we are to consider the red cell concentration, number of antigenic
determinant, antibody concentration, antigen binding site (viz. IgG has two binding site) before accounting
for any agglutination or degree of agglutination. Higher the equilibrium constant more amount of antibody
will combine with antigen, thus the equilibrium constant is the measure of goodness of fit between
corresponding antigen and antibody Agglutination is influenced by factors as described earlier.
Haemolysis
Erythrocytes are normally dispersed in plasma at an osmotic pressure of isotonic saline, i.e., 0.9% NaCl
solution. It has been observed that with addition of distilled water blood becomes translucent and this is
due to absorption of water by osmosis into red cells which swells, burst and .haemolyse and haemoglobin
from ruptured cells released into plasma. Red cells under certain conditions viz. change in environment,
heat, light, freezing, thawing, electric stimulation. Animal poison - snake venom and toxalbumin; proteolytic
enzyme, drugs, haemolytic bacteria and under conditions of immune response with which we are most
concerned releases the intracellular haemoglobin where the antibody concerned has the property of
haemolysin. This haemolysis to occur under condition of immune response requires presence of
complement. If the complement is inactivated by heating at 56°C for half an hour then haemolysis
doesnot occur and instead agglutination occurs.
Precipitation is the interaction ofsoluble antigen with soluble antibody in optimal proportion resulting
in formation of a visible complex, the precipitate. The immunodiffusion and immunoelectrophoresis
involves the principle of precipitation as the essential basic step.
Inhibition reaction
Cannot bring about anything visible to the naked eye. The soluble blood group antigen present in salivary
and other body fluid can effect the neutralisation of corresponding blood group antibody; 'to this known
indicator cells are added to bring about agglutination reaction and when no agglutination is observed, it
denotes presence of antigen and if agglutination is observed, it denotes absence of antigen. Commonly
used for finding out secretor status of an individual.
4.6 COMPLEMENT AND COMPLEMEN''" FIXATION TEST
Complements are serum protein, most of which are globulin, they are thermolabile and non-specific in
nature and this system of protein numbers between 11 to 17. They are designated by a bar, e.g. C I q.
They circulate in serum as inactive precursor and are activated in a very precise steplike chain of
biochemical sequence resulting in cell lysis mainly. In addition, it plays part in vascular permeability,

[[p.9]]
Immu~ohaematology
61
FactorD
FactorB
Properdin
ALTERNATIVE PATHWAY
IgM,IgG,IgA. IgE
Zymosan
Inulin
Bacteria
Fungi
I
0;4I
w
wC'[C6-C
9
zc(
~ tn
Membrane
m ~
I Edta Inhibited
:EO
I
~ ~
Cell lysis
C:I:
ILl CLASSICAL PATHWAY
~
Antigen-Antibody (lgM, IgGl _3)
U)
zoi=Z
CJoow
a::
f
C4a
w
CJcr...
c~
en
z0
~
I
>
C3a
i=
0c(
I
Csa
Fig. 4.2. C~II
undergoes lysis after reacting with all component of complement.
immune adherence, phagocytosis complement mediated, chemotaxis and release of histamine and
lysosomal enzyme. Complement system is an interdeperident enzyme system and succeeding complement
component is activated by its precursor through interplay ofactivator and proactivator. Thus the sequence
is a cascade and together with clotting, fibrinolytic and kinin generating system are triggered enzyme
cascade system.
It is now established that.complement system can be activated in two different pathways:
(i) Classical
(ii) Altemativee
A very simple statement of sequence involved as follows :
Three separate stages are involved in activation of complement and they are recognition stage,
activation stage and membrane attack stage.
Classical
In classical, it follows EAC 142356789, i.e. erythrocyte antibody complement 1-9. In this pathway-
antigen-antibody (IgM, IgG I and IgG3) complex is formed by interaction of antigen with C1 fixing

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62
Blood Banking and Transfusion
, antibody described in Fig. 4.2 under an appropriate condition, probably through a conformational change
in FC fragment in antibody, C 1 thus gets bind. C 1 has three subclasses - C 1q, C 1r and CIs. The CIq
subclass takes part in the binding process leading to generation of C 1 esterase activity which can cause
cleavage in C1r and CIs protein chain. The complex thus formed is capable of interacting with next
component C4 and C4 is cleaved by C 1 acting as an enzyme resulting in release of C4a. The c4bC1
complex in presence of Mg+ acts on C2 and cleaves C2 forming complex the C1 C4b C2a and the
inactivated C2b is released. The C14b2a is unstable and acts on C3 through C3 convertse, C3a is released
and C3b gets attached to the complex C14b2a + 3a and forms complex C14b2a3b at membrane site
they again bind and cleave Cs into Csa and Csb.Csa is released and Csb gets attached to the complex
marking the end of activation stage. With the attachment of C5b to the complex membraneattack phase
starts and the pattern is continued through C6 to C9 and when the whole complex is formed i.e. EAC
142356789 sufficient damage has been done to cell membrane as to cause a hole of 0.8 to 1.0 urn in
diameter.
Alternative pathway
In this antigen-antibody complex though required but not essential to initiate the process and it may even
be started with bacteria, fungi, zymosan, inulin and variety of endotoxin can also start the process. It
avoids C 142 pathway of classical which is replaced by properdin factor D analogus to C 1 and properdin
factor B analogus to C2• Both the components are heat labile and they forms an unstable complex that
cleaves C3 in C3a and C3b of this C3a is released and C3b interacts with components of the alternative '
pathway.
Complement fixation
In complement fixation process complement gets bind to antigen antibody complex and results in lysis
of sensitised red cells there are two types of approach: (1) Concentrated fresh serum is source of
complement and the amount of complement .fixed is determined by titration of serum before and after
test or (2) the amount of complement fixed is determined by using dilution of serum that will provide
just enough complement for lysis, here the sensitised cells are added without further dilution. Incubation
of test material at 37°C for one hour. Control necessary for the tests. This test of eFT is rarely used in
immunohaemotology laboratory.
Use of complement is mainly diagnosis or assessment of'clinical activity in disease state viz. Ac.
glomerulonephritis, SLE, Membraneproliferative nephritis, Rheumatologic disorder, Haematologic disease
particularly in PNH (Paroxysmal nocturnal haemoglobinuria) where a membrane defect develops in
RBC and this renders them susceptible to complement mediated lysis, and also in pemphigus, herpes'
where immunofluoroscent analysis of tissue biopsy is employed for study of complement. Some inborn
error of complement system is also observed in thymic alymphoplasia, SLE particularly discoid lupus,
Frequent infections viz. repeated Nisseria infection, haemolytic uremic syndrome and hereditary
angioedema.
A schematic representation of classical and alternative pathway is given in Fig. 4.2.

[[p.11]]
Immunohaematology
63
4.7 IMMUNE RESPONSE
When a foreign protein enters the body and challenges the body two types of responses can occur:
(a) One that results in production of antibody, a highly specific protein that is ultimately released
into serum - Humoral immune response, we are primarily concerned with this in blood banking.
(b) Another type of response that refers ·to the production or synthesis of an immunologically
committed killer cells - Cell mediated immunity or delayed hypersensitivity.
Foreign protein are the antigens that evokes the immunological responsiveness and could be
heterologus antigen-microorganism etc., isologus antigen, i.e., from the same species viz. blood, Autologus
self antigen modified and no longer recognised as self.
Genetic basis
This system is mainly maintained by cells and tissue. At birth the pleuripotential stem cells generates (a)
Haemopoitic cells that differentiate into RBC, WBC and platelet, (b) Lymphopoitic cells differentiate into
T and B lymphocytes.
Immune response associated molecules are encoded by HLA. D region in short arm ofchromosome 6.
T lymphocyte forms more than 60% of perepheral blood. These lymphocytes after having been
originated in bone marrow are processed in Thymus and brought to maturity viz. T helper cells, suppressor
cells and killer cells. Helpercell promote activity ofT cell and B cells, helps in stimulation ofmacrophages.
Suppressor cells have inhibitory action over T and B cells. Cytotoxic or killer T cells kills the foreign
cells directly.
B lymphocytes tQO originates in bone marrow and consists of about 30% or more in perepheral
blood lymphocyte. Unlike the T lymphocyte, the B lymphocyte are not processed in thymus. B lymphocyte
differentiate into plasma cells responsible for producing specific antibody found in the form of IgM,
IgG, IgA, IgD, IgE and are therefore, easily identifiable in lymphatic system and perepheral blood.
Macrophages
Monocyte, other leucocyte or granulocyte all develops from bone marrow. Monocytes leaves the circulation
reaches the tissue where they spend several months and are converted to histiocyte or macrophages.
The blood monocyte and tissue macrophages together makes a mononuclear phagocyte system and
they takes part in phagocytosis of bacteria. Phagocytosed bacteria are usually digested. Through
phagocytosis macrophage performs very important function, both in cellular and humoral immune
response by way of handling and processing the antigen for presentation to lymphocytes, it also removed
the degraded cell products or altered cells.
Immune response as has been stated earlier is due to stimulation by invading antigen which sets in
motion the cellular elements described, takes part in different phases in the process, to elicit the immune
response. Type of antigen may play part in determining the nature of immune response, e.g. simple
antigen like those expressed on erythrocyte may evoke humoral response whereas complicated antigen
like viral, bacteria infected cell may evoke cellular response. Haptens which are incomplete antigen are
not capable of evoking immune. response and to induce immune response from hapten, it has to be
attached to a carrier molecule like bovine serum albumin.
The B-Iymphocyte developing from bone marrow are called perepheral cells and thymus processed
T lymphocytes are called central cells. B cells responsible for IgG synthesis may be directly stimulated

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64
Blood Banking and Transfusion
by contact with antigen. Acting as "Helper cells", T cells and macrophage together process the antigen
and present it to B cells or T cells may release some soluble factor which may stimulate B cells. So the
cooperation between B cells (peripheral) and T cells (central) is essential to start the process of eliciting
an immune response.
We are more concerned with immune response by erythrocytes. In vitro study by Archer in 1964
and subsequently by others through different method demonstrates that basic mechanism involved
are:
(a) Specific antibody sensitises the erythrocytes - Target cell.
(b) Sensitised cells gets attached to FC receptors of effector cells - Leucocytes having cytotoxic
activity viz. lymphocyte, monocyte-rnacrophages, neutrophill.
(c) Phagocytosis and cytolysis of target cells. In vivo this phenomenon of erythrophagocytosis
was reported in AIHA, IIDN and paroxyoumal nocturnal humglobinuria (Pf'JII).
However, present day concept revolves around, I-ILAD/Dl~
(D-relaled) closely linked loci found in
the short arm of chromosome No. 6 in man-Ml-lC. '1\\'0 gene products of ~.1HC
biochemically
distinguishable known as class I and class II gene products plays a major biological role in regulation of
immune response. This is believed to be executed through anyone or both of "one signal" and "two
signal" model governed by laws of self and non-self discrimination.
One signal model proposes that B cells can be triggered by antigen. Antigenic determinant that cross
links immunoglobulin receptor are placed near to each other on the. surface of B cells. In this model T
cell is believed to act as regulator either by suppressing.or stimulating the function of J3 cell.
In two signal model, it is proposed that B cells are to be delivered with t\VO specific signal:
(a) delivered by binding of antigen to immunoglobulin receptor.
(b) delivered by either of T-cell product or macrophage product.
Specific antibody produced by T helper cells are cytophilic for macrophages. Macrophages nOVJ
processes antigen for presenting it to B cells which in t.urn proliferate, clonal expansion takes place and
differentiation of clone to antibody 'secreting cells.
.Self-non self discrimination
The first law states that any mechanism of host defence against infecrious agent which has the capacity
to destroy macromolecules requires a recognitive component that can distinguish self from non-self.
Second law states that in the immune system antigen specific cells must have at least two states of
responsiveness to antigen an initial i-state in which eelIsare unable to exe~ute
their effector function and
an effector e-state in which cells are able to execute their effector function.
Immune response in general can be divided into two primary and secondary. Primary immune
response occurs as a result of first exposure to the antigen and in this type of response" antibody (Ig~lf,
usually) level is not high and the detectable response usually takes 7 1:0 15 days to occur.
Secondary immune response occurs when the host is re-exposed to the particular antigen also
involved in primary response. In this case antibody (IgG), usually shows remarkable rise within a day or
two of second exposure and this higher level is maintained with slow fall over a period of months.

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lmmunobaematoloqy
65
Anamnestic response
This is a type of hightened secondary response as a result of subsequent challenge by an antigen. Here
the response to the stimulus is brisk and antibody level reaches higher in a shorter period. This phenomena
of recollection reaction is called anamnestic response. And it thus shows that body's immune system is
equipped with "immunological memory". Recent observation has shown that after the primary stimulus
some plasma cells reverts to small lymphocytes. Anamnestic response is thus a resultant effect of a
combined action of small lymphocytes (memory cells) together with primed cells.
Immunological tolerance and enhancement
The phenomena of failing to respond to antigenic stimulus by production of antibody under certain
circumstances is known as Immunological tolerance. This tolerance could be induced by exposure to
antigen during foetal life. But later on, it was further detected that for a life long tolerance to be developed
it requires a permanent graft which is observed in twins, each having cells derived from two distinct
zygotes-chimeras primitive cells of one twin takes root in the other and continue to produce red cells of
the genotype of original donor. The blood fanning tissues which gets exchanged during foetal life is
being carried in the blood of twins till their death without any ill effect i.e., each twin has a dual
population of cells but each is tolerant of the other. This was demonstrated beautifully by Owen in 1945
in. case of bovine twins and by Dunsford et a1. in 1953 in human twins. Apart from aforementioned
permanent chimerism, temporary chimerism can be induced in a bone marrow graft recipient. Tolerance
canalso be induced by injecting large dose ofantigen under certain circumstances in adult. Immunological
enhancement can be .induced by vaccination against infectious diseases. Many theory have been
propounded to explain this phenomena but are not found convincing.
~uppression
of ilnmune response
Administration of just sufficient antibody can suppress the response of antigen probably through a
feedback mechanism by controlling the rate of production of antibody. A competition for available
antigen between circulatingantibody and the antigen receptor sites on the plasma cells mayberesponsible
for control.
Juppression of Rh immunisation by injection of RhO antibody to the mothers at risk is a good
example, 25 Ilgof antibody is sufficient to neutralise the RhO positive cells of 1 mI.It can be calculated
at equilibrium when the antibody is distributed within a space about twice as great as plasma volume..
Cross reactivity
Cross reactions has been observed with antibody stimulated by one antigen, with that of a related
antigen having identical antigenic determinants, This cross reactivity is influenced by : (i) avidity of
antibody for related antigen, and (ii) time for how long the immunisation process is being continued. A
good example is 0 group sera which after an immune response will contain antibody that may cross
react with either or anyone of A or B antigen. In Rh system also we may get cross reacting antibody.
Immune response to blood group antige:r:
Study of ABO antibody reveals that human antibody for ABO antigen can be divided into natural and
immune. The natural derived antigen for A, B are present in microorganism, plant, gut flora diet etc. The
anti-A and anti-B agglutinins etc. are of low titred immune antibody as experimentally it could not be

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66
Blood Banking and Transfusion
demonstrated in animals kept in a germ free environment. Blood group substance can neutralise them
and they are of IgM class whereas anti-A, anti-B present in 0 group serum are usually of IgG type but
may be of IgM type and also IgA in few cases. They crossreacts with A and B and subgroups of A,
normally not detectable by anti-A. Haemolysin are usually associated immune antisera and haemolysins
were demonstrated in Anti-A both of IgM and IgG type but not in IgA.
Immune anti-A and anti-B are usually found in cases of foetomatemalleakage, or due to previous
isoimmunisation.
The Rh antibody ~ay
be found in both IgM and IgG form. The whole human population can be
divided into two :
(i) Responders: Responding by immune response to Rh.
(ii) Non-responders fails to produce immune response and one third of Rh negative fails to produce
immune response.
It has been further observed that in ABO incompatible haemolytic disease of Newborn, the Rh
antibody is less likely to occur as a result of Rh immunisation. ABO incompatible HDN thus in most
cases protects the foetus from Rh immunisation.
Cross reacting 'antibodyhas been observed in serum of individual belonging to '0' genotype and
they may crossreact with C or c and E or e.
Further rea.ding recommended, Articles and JOurnals
1. Antibody dependent cell mediated phagocetosis and cytolysis, Dr. (Miss) S.C. Gupte, VoL 20, No.3,
"Immunohaematology Bulletin", May-June 1989, published by I.J.H.
Books
1. Basic and Clinical Immunology, Fudanbergh, Stitis, Caldwell, Wells, 1976, Lange Med. Publication.
2. Blood Group Serology, Boorman, Dodd and Lincoln, Churchill Livingstone, sixth edition, ·1988.
3. Blood Transfusion in Clinical Medicine, P.L. Mollison, seventh edition,
1983~
Blackwell Scientific
Publication.
4. Clinical Diagnosis and Management by Laboratory Method,- Todd, Sanford and Davidshon, Second
Indian edition, 1989, edited by I.B. Henry; Published in India by A.J.T.B.S.
5. Fundamentals of Immunology, William C. Boyd, Fourth edition, 1966, IntersciencePublisher:
6. Immunobiology, R.A. Good and D.N. Fisher, 1911, Hospital Practice Company, Sinasur Associate-Inc.
20 Second Street Conn.. 06905, USA.
7. Introduction to Transfusion Medicine, Z. Bharucha and D.M. Chouhen, First edition, 1990, D.K.
Publishers, Bombay-400033.
8. Immune System, Rodney E. Longman," Academic Press Inc., Blaircourt .Brace Jovanovich, New York,
Tokyo, Toronto.
9. Textbook of Blood Banking Sciences, C.M. Zmijeswski and It:E. Haeslet; 1982 edition, Appleton
Century Crofts.
.