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1. 1
CHAPTER ONE
INTRODUCTION TO IMMUNOHEMATOLOGY
By
Mohammed Hashim (BSc, MSc)
.

2. CONTENTS
Overview of Immunohematology
1.2 Historical background
1.3 Blood Group Genetics
1.3.1 Inheritance pattern of blood group antigen
1.3.2 Chromosomal assignment
1.3.3 Homozygocity and hetrozygocity
1.3.4 Genetic inheritance
1.4 Blood cell antigen
1.4.1 Blood cell antigen
1.4.2 Human leukocyte antigen (HLA)
1.4.3 Platelet antigen

3. 1.5 Blood group antibodies and their stimulation
1.5.1 Natural (non red cell immune) antibodies
1.5.2 Immune antibodies
1.6 Antigen –antibodies interaction
1.7. Antiserum
1.8. Invitro Antigen antibody interactions---
1.8.1The influence of antibody type on
agglutination
1.8.2 Methods of enhancing agglutination

4. Learning Objectives
At the end of this chapter, the student will be able to:
 Explain a brief history of Immunohematology.
 Discuss patterns of inheritance of A and B antigens.
 Describe the synthesis of H, A and B antigens.
 State the genotype of individuals with the Bombay
phenotype.
 State the characteristic genotype of secretors and non-
secretors.

5. Overview of Immunohematology
 Immuno hematology:-
 Is the area of laboratory medicine dealing with the general
procedures involved in collecting, preparing, storing and
transfusing blood.
 Is more commonly known as "blood banking“
 Deals with the concepts and clinical techniques related to
modern transfusion therapy.
 Refers to immunologic reactions involving blood components
 An application of the principles of immunology to the study of
 red cell antigens and
 their corresponding antibodies on blood for resolving the
problems of blood transfusions.

6. 1.2 Historical background
 The era of blood transfusion began when William Harvey
described the circulation of blood in 1616.
 In 1665, Richard Lower, successfully performed the first
animal-to-animal blood transfusion.
 In 1667, jean Bapiste Denys transfused, blood
from the carotid artery of a lamb into the vein of a
young man, which at first seemed successful. using
animal blood, but they were unsuccessful.
 Later, it was found that it is impossible to
successfully transfuse the blood of one species
of animal into another species.

7. …con’t
 Transfusions were prohibited from 1667 to
1818 Due to the disastrous consequences
resulting.
 In 1818, James Blundell of England
successfully transfused human blood to
women suffering from hemorrhage at
childbirth.
 Such species-specific transfusions seemed to
work sometimes but mostly the result was
death.

8. History of Blood...
•Experiments with blood transfusions have been
carried out for hundreds of years. Many patients
have died and it was not until 1901, when the
Austrian Karl Landsteiner discovered human
blood groups, that blood transfusions became
safer.
• He found that mixing blood from two individuals
can lead to blood clumping. The clumped RBCs
can crack and cause toxic reactions. This can
be fatal.
http://nobelprize.org/medicine/educational/landsteiner/readmore.html

9. •Karl Landsteiner discovered that blood clumping was an
immunological reaction which occurs when the
receiver of a blood transfusion has antibodies against the
donor blood cells.
•Karl Landsteiner's work made it possible to determine
blood types and thus paved the way for blood
transfusions to be carried out safely. For this discovery
he was awarded the Nobel Prize in Physiology or
Medicine in 1930.

10. 1.3 Blood Group Genetics
 Concerned with the way in which the different blood
groups are inherited
Chromosomes and Genes:
 The nucleus of each human body cell contains 46
small thread-like structures called chromosomes,
arranged in 23 pairs.
 The length of each chromosome is divided into many
small units called genes.
 Genes code for different inherited physical
characteristics, including blood groups.
 Each gene has its own locus, along the length of the
chromosome.

11. Genetics….
 Certain inherited characteristic can be represented by
a group of genes, and the locus can be occupied by
only one of these genes.
 Such genes are called alleles or allomorphic genes.
 Mitosis: While body cells multiply they do so by
producing identical new cells with 46 chromosomes.
 Meiosis: When sex cells are formed either male or
female, the pairs of chromosomes do not multiply but
simply separate so that each of the new cells formed
contains only 23 chromosomes.

12. …con’t
 During fertilization when the egg and sperm unite
the fertilizer ovum receives 23 chromosomes from
each sex cell.
 Half of these from the male and
 half from the female and thus will contain 46
chromosomes which arrange themselves in pairs in the
nucleus.
Genotype versus phenotype
 Phenotype
 Physical expression of inherited traits,
 Determined by reacting red cells with known antisera
 Genotype
 Actual genes inherited from each parent
 Can only be inferred from the phenotype .
 Family studies are required to determine the actual
genotype

13. 13
Table 1.1. The ABO phenotypes and their
corresponding genotypes
Phenotype Genotype
A AA, AO
B BB,BO
AB AB
O OO

14. …con’t
Punnet square
 Illustrates the probabilities of phenotypes from known or
inferred genotypes.
 Visually portrays the potential offspring`s genotypes or
the probable genotypes of the parents
• Two group A parents can have a group O child?
• The parents of an AB child can be A, B or AB, but not
group O.
A O
A AA AO
O AO OO

15. 1.3.1 Inheritance pattern of blood group antigens
 In most cases blood group antigens are inherited
with co dominant expression.
 The product of each allele can be identified when
inherited as a co dominant trait.
 If one parent passed on an A gene the other parent
passed on a B gene, both the A and B antigens would be
expressed equally on the red blood cells.
Recessive or dominant inheritance patterns
 recessive
 inheritance would require that the same alleles from both parents
be inherited to demonstrate the trait
 Dominant
 expression would require only one form of the allele to express
the trait.

16. Gene Expression
 Gene expression follows one of the three patterns
 Dominant:
 one gene is always expressed when found in
combination with a second gene
 In this case, there is a gene that is not expressed
 Recessive: silent gene can only be expressed if
two identical genes are present.
 Codominant: the expression of two different genes
that are inherited at the same loci on a pair of
chromosomes.
 With rare exceptions, blood group systems are
expressed as codominant characteristics. E.g AB

17. Cont..
 Amorph/Silent:
 genes that do not code for the production of any
detectable product
 These genes appear to be recessive
 Amorph combined with allele that produce
detectable product, the detectable product of that
allele is expressed
 This allele is not dominant over the amorph nor is
the amorph recessive to the expressed allele.
 A common example of an amorph is the gene
that codes for the O blood group
 When inherited in a homozygous state, two O
genes, produce no detectable product.

18. 18
1.3.2 Chromosomal assignment
Table 1.3. Chromosomal assignment of genes in blood
group system
Blood group system chromosome
 Rh---------------------------------------------------------1
 Duffy------------------------------------------------------1
 Gerbich---------------------------------------------------2
 MNS-------------------------------------------------------4
 Kell--------------------------------------------------------7
 ABO-------------------------------------------------------9
 Kidd------------------------------------------------------18
 Lewis-----------------------------------------------------19
 Landsteiner-Wiener-------------------------------------19
 Lutheran--------------------------------------------------19
 Hh---------------------------------------------------------19
 P-----------------------------------------------------------22

19. 1.3.3 Homozygosity &
Hetrozygosity
 Homozygous-
 Genotype is made up of identical genes, such as AA,
BB, or OO,
 Heterozygous.
 Genotype is made up of different alleles from each
parent, such as AO, AB, or BO,

20. 20
Table 1.3.Dosage effect on antigen
expression
Genotype Dosage effect on antigen
expression
Homozygous :MM Red blood cell tested with
anti-M : +4
Heterozygous :MN Red blood cell tested with
anti-M : +2

21. 1.3.4 Genetic inheritance
 Genes can inherit with each other depending on
whether they are inherited on the:
 Same chromosome (Cis) or
 Opposite chromosome (Trans).
 Trans interaction may weaken the expression of
one of the antigens encoded by the genes,
For example:
 The C and D genes of Rh system are inherited on
different genetic loci .
 When C is inherited in trans to D, it will weaken the D
antigen expression on the red blood cell.

22. Genetic inheritance…
Linkage and Haplotypes
 In some blood group systems, the antigens are encoded
by two or more genes on the same chromosome.
 When genes are very close together, they are inherited
from each parent as a unit and are known as linked
 Independent assortment does not occur when genes
are linked.
 These gene units are called haplotypes
Silent genes
 In some blood group systems genes do not produce a
detectable antigen product and are called "silent” genes
or amorphs.

23. …con’t
 Amorphs can result in an unusual phenotype if passed on
by both parents.
 The phenotypes are often called "null" type because
expressions of the blood group system antigen are not
apparent.
 Rare gene must be inherited from both parents
(homozygous) to produce a null phenotype.
 null types caused by amorphic genes are rare.
 Unusual phenotypes may also result from the
action of suppressors, or regulator genes.

24. …con’t
 These genes (suppressor /regulator)
 act to inhibit the expression of another gene and, must
be inherited in the homozygous state to create this
effect. suppressor genes that affects the blood group
antigen are rare.
 Null phenotype therefore can be resulted of
either an amorphic or a suppressor gene.

25. 25
Table 1.4. Blood group genes that can result an
unusual phenotypes
Blood group
system
Amorph/Regul
ator gene
Phenotype
H h Bombay
Rh r/x0r Rh null
Kell K0 Kell null
Lutheran Lu/in(lu) Lu(a-b-)
Kidd JK JK(a-b-)
Duffy Fy Fy(a-b-)

26. 1.4 Blood cell antigens
1.4.1 Red blood cell antigens
 A unique set of red blood cell Ag is determined
through genetic inheritance.
 These antigens protrude from the surface of the
RBC in three dimensional configurations.
 As a result, they are accessible to Ab molecules for
agglutination reaction.
 In biochemical terms these antigens may take the
form of:
 proteins,
 Glycoprotein,
 Glycolipids

27. Red cell antigens…
 Some of the red blood cell antigens are more
immunogenic than the others
Example
 The D antigen within the Rh group system.

28. 1.4.2 Human leukocyte antigens (HLA)
 Is possessed by nucleated cells such as leukocytes and
tissues
 Can readily provoke an immune response if transferred
in to a allogenic individual.
 Encoded by genes which are parts of Major
Histocompatibility Complex (MHC) gene system
 The MHC system is important in the:
 recognition of non self ,
 coordination of cellular and humoral immunity , and
 graft rejection .

29. Human Leukocyte antigens…
 The MHC region is on chromosome 6 and is
divided in to three categories or classes :
 Class I includes the A, B and C locus,
 Class II includes the DR, DP and DQ
 Class III includes the complement proteins
 The MHC region is called polymorphic , because
there are so many possible alleles .
For example :
 At least 49 different alleles or possible genetic expressions
have been identified at the A locus.
 At the B locus 97 alleles are identified.

30. 1.4.3 Platelet antigens
 Platelet possesses inherited membrane proteins that can also
elicit an immune response.
 Platelet antibodies are less frequently found, because there is
less antigen variability in the population
 Antibodies to platelet antigens are the major cause of
:
 neonatal alloimmune thrombocytopenia,
 Post transfusion purpura ,
 It can also decrease the expected increment of platelet
transfusion.

31. 1.5 Blood group Abs & their
stimulation
Blood group antibodies are classified into:
 Natural and
 Immune antibodies
Natural / non red cell immune Abs
 Are RBC Abs in the serum of an individual that are
not provoked by previous RBC sensitization.
 The term non red cell immune have crept in to
modern use.

32. Natural antibodies….
Characteristics
 They are mainly IgM type.
 Exhibit optimum in vitro agglutination saline media
 complete antibodies.
 Optimum reaction at room temperature or lower
 cold agglutinins.
 Do not react above the body temperature
 most of these do not give rise to transfusion reactions.
 They are of high molecular weight
 cannot cross the placenta

33. 1.5.2. Immune antibodies
 Produced due to previous antigenic stimulation
either by transfusion or pregnancy
Characteristics
 Mainly IgG type
 Do not exhibit visible agglutination in saline, but in
albumin medium .
 Incomplete antibodies.
 Optimally react at 370C
 warm agglutinins.

34. Immune…
 Causes more serious transfusion reactions than the
naturally occurring ones.
 Can cross the placental barrier.

35. 1.6 Antigen - Antibody interactions
 When Ag and Ab combines, an immune complex is
produced.
 The amount of Ag - Ab complex formation is determined
by the association constant of the reaction .
 When the forward reaction rate is faster than the reverse
reaction rate Antigen-Antibody complex formation is
favored.
 Therefore a higher association constant influences
greater immune complex formation at equilibrium

36. Ag-Ab interactions…
Properties that can influence the binding of Ag
and Ab
 The goodness of fit (as a lock and key fit)
 complementary nature of the antibody
 size, shape, and charge of antigen

37. 1.7.The Anti-serum
 To determine a person’s blood type, some sort of
substance must be available to show what antigens
are present on the red cell.
 The substance used for this purpose is referred to as
anti serum.
 Is highly purified solution of antibody.
 named on the basis of the antibody it contains
For Example:
 Solution of Anti-B antibodies is called
anti –B antiserum

38. Anti-serum…
 The anti-sera used in Immuno hematology are
prepared in one of the two ways:
 By deliberately inoculating animals with an antigen
 By collecting serum from humans who have been sensitized
with corresponding antigens
 Anti-serum must:
 Be specific for the antigen to be detected
 Have sufficient titer to detect antigen
For Example
Anti-A should have a titer of at least
 1/128 against A1 cells,
 1/64 againstA2 cells, and
 1/16 against AB cells
Anti-B should have a titer of at least 1/64 against B
cells

39. The antiserum…
 Have certain avidity or strength of reaction with,
corresponding red cells
For example
Anti-A1should agglutinate:
 A1 cells in 10seconds or less,
 A2 cells in 20sec or less, and
 A2B in 30 sec or less
 Be free from haemolysins, fat and rouleaux
 Be sterile and clear
 Preserved with 1% sodium azide and be stable.

40. The antiserum…
 Have a marked expiration date, and
 Should be stored at 40C
 The manufacturer directions must be followed
carefully.

41. 1.8. Invitro detection of Ag and Ab
reaction
 The presence of Invitro antigen and antibody
interaction can be detected by:
 Hemolysis
 Precipitation
 Agglutination (Most commonly used)

42. The mechanism of agglutination
Agglutination:
 Visible clumping of particulate Ags caused by
interaction with a specific Ab
 Occurs in two stages:
 sensitization and
 lattice formation.

43. 43
Stages of Ag-Ab Reaction…
A-Sensitization-the first phase
 represents the physical attachment of Ab
molecules to Ags on the RBC membrane.

44. Factors affecting the sensitization
phase
1. The antigen - antibody ratio
For example : pro-zone phenomenon.
2. Physical conditions such as:
 PH,
 Temperature
 Time of incubation
 Ionic strength, and
 Steric hindrance.

45. 45
Stages of Ag-Ab Reaction…
B- Lattice formation – the second phase
 Is the establishment of cross links between
sensitized particles and Abs resulting in
clumping

46. Factor affecting the lattice formation
phase
 Cross linking is influenced by Zeta potential
 Zeta potential - is the difference in electrostatic
potential between the net charge at the cell
membrane and the charge at the surface of shear.

47. 1.8.1. The influence of Ab type
on agglutination
 IgM antibodies are more efficient than IgG or IgA
antibodies in exhibiting invitro agglutination
 IgG antibodies are less efficient due to:
 The deep location of the antigen
determinants and Restricted movement of the
hinge region causes them to be functionally
monovalent.

48. 1.8.2.Methods of enhancing agglutination
 Centrifugation
 Treatment with proteolytic enzyme,
 Use of colloids, and
 Addition of anti-human globulin (AHG) reagent.
 Others
 Poly ethylene glycol PEG)
 Low Ionic strength saline (LISS)
 Polybrene

49. 49
Review Questions
1. Define:
A. Antigen
B. Antibody
C. Immunogenicity
2. Identify some characteristics of the IgG subtypes
3. What are the characteristic differences between
Natural and Immune antibodies?
4. Which classes of antibodies predominate during the
primary immune response and secondary immune
response?
5. List the factors that affect antigen and antibody
interaction
6. List the methods that are routinely used in the blood
banking laboratory to enhance
agglutination reaction.