IMMUNOHEMATOLOGY

CHAPTER
ONE INTRODUCTON TO
IMUNOHEMATOLOYG
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.
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1.1 Overview of Immunohematology

Immuno hematology:

 is more commonly known as "blood banking“

 deals with the concepts and clinical techniques

related to modern transfusion therapy.

 Is the area of laboratory medicine dealing with the

general procedures involved in collecting, preparing,
storing and transfusing blood.

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Immunohematology…

 refersto 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.

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 1.2 Historical background
 The first suggested case of ‘’transfusion “was the
rumored to have been given to pope innocent VIII in
July 1492.

 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.

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Historical background…

 In 1667, jean Bapiste Denys transfused,

 bloodfrom 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.

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Historical background…

 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.

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Historical background…

 Karl Landsteiner

 discovered the ABO blood groups in 1900,

 introduced the immunological era of blood transfusion.

 It became clear that the incompatibility of many

transfusion was caused by the presence of
certain factors on red (blood) cells now known as
antigens.

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 Historical background…
 Two main postulates were drawn:
1. Each species of animal or human have certain
factors on the red cells that are unique to that
species, and
2. Each species have some common and some
uncommon factors to each other.

 This landmark event initiated the era of science

based transfusion therapy and was the foundation
of immunohematology as a science.
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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.

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Blood Group ..

 The length of each chromosome is divided into

many small units called genes.

 Genes code for different inherited physical

characteristics, including blood groups
Blood Group Genetics…

Allomorphic genes (Alleles),and Polymorphism

 Each gene has its own locus, along the length of

the chromosome.

 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.

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Blood Group Genetics…

 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.

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Blood Group Genetics…

 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.

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Blood Group Genetics…

Genotype versus phenotype

 Phenotype
 Physicalexpression of inherited traits,
 Determined by reacting red cells with known antisera

 Genotype
 Actualgenes inherited from each parent
 Can only be inferred from the phenotype .
 Family studies are required to determine the actual genotype .

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Table 1.1. The ABO phenotypes and their
corresponding genotypes
Phenotype Genotype

A AA, AO

B BB,BO

AB AB

O OO

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Blood Group Genetics…

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 .

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Table1.2. Punnet squares showing ABO
inheritance

• 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

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1.3.1 Inheritance pattern of blood group Ags
 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.

 Ifone 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.

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Inheritance pattern…

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.

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Inheritance pattern…

 O gene is recessive, since it is expressed only

when both parents contribute the O allele.

 The product of an O gene however, does not

affect the membrane proteins.

 Itsexpression is termed as amorphic (a gene that

does not express a detectable product) rather than
recessive.

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 Inheritance pattern…

Mendelian principles:
law of independent segregation

 refers to the transmission of a trait in a predictable

fashion from one generation to the next.

 Independent assortment is demonstrated by the fact

that blood group antigens inherited on different
chromosomes, are expressed separately and
discretely.

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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
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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,

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Table 1.3.Dosage effect on Ag 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

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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).

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Genetic inheritance…

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.

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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

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Genetic inheritance….

 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.

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Genetic inheritance…

 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.

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Genetic inheritance…

 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.

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Genetic inheritance…

 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.

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Table 1.4.Blood group genes that can
result an unusual phenotypes
Blood group Amorph/Regulator Phenotype
system gene

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-)
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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.

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Red cell antigens…

 In biochemical terms these antigens may take the

form of:
 proteins,
 Glycoprotein,
 Glycolipids

 Some of the red blood cell antigens are more

immunogenic than the others
Example
 The D antigen within the Rh group system.

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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.

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Human Leukocyte antigens…

 The MHC system is important in the:

 recognition of non self ,

 coordination of cellular and humoral immunity , and

 graft rejection .

38
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

39
Human Leukocyte antigens…

 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.

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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.

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Platelet antigens…

 Antibodies to platelet antigens are the major

cause of :
 neonatal alloimmune thrombocytopenia,

 Post transfusion purpura ,

 Itcan also decrease the expected increment of

platelet transfusion.

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1.5 Blood group Abs & their stimulation

Blood group antibodies are classified into:

 Natural and

 Immune antibodies.

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1.5.1. 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.

44
Non-red cell immune 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.

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Non-red cell immune antibodies…

 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

46
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.

47
Immune antibodies…

 Optimally react at 370C: warm agglutinins.

 Causes more serious transfusion reactions than

the naturally occurring ones.

 Can cross the placental barrier.

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1.6 Antigen - Antibody interactions

 The binding follow the law of mass action and

is a reversible process.

 This union complies with the principles of a

chemical reaction that has reached equilibrium.

 When Ag and Ab combines, an immune complex

is produced.

49
Ag-Ab interactions…

 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

50
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

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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.

52
The antiserum…

 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

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The antiserum…

 The anti-sera used in Immunohematology 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

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The antiserum…
 Antiserummust meet certain requirements to be
acceptable for use.

- The antiserum has two be specific: does not cross

react, and only reacts with its own corresponding
antigen,

- Avid: the ability to agglutinate red cells quickly and

strongly,

- stable: maintains it specificity and avidity till

the expiry date.

55
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

56
The antiserum…

 Be free from haemolysins, fat and rouleaux

 Be sterile and clear
 Preserved with 1% sodium azide and be stable.
 Have a marked expiration date, and
 Should be stored at 40C
 The manufacturer directions must be followed
carefully.

57
1.8. In vitro detection of Ag and Ab
reaction

The presence of Invitro antigen and antibody

interaction can be detected by:

 Hemolysis

 Precipitation

 Agglutination (Most commonly used)

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The mechanism of agglutination

Agglutination:

 Visible clumping of particulate antigens caused by

interaction with a specific antibodies

 Occurs in two stages:

 sensitization and

 lattice formation.

59
Stages of Ag-Ab Reaction…
A. Sensitization-the first phase

 represents the physical attachment of Ab

molecules to Ags on the RBC membrane.

60
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.

61
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

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Factor affecting the lattice formation
phase

 Cross linking is influenced by Zeta potential

 Zeta potential

 isthe difference in electrostatic potential between the

net charge at the cell membrane and the charge at
the surface of shear.

63
1.8.1. The influence of antibody 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.

64
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

65
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.

66
References

1.Immunohematology for medical laboratory science

students,Yayehyirad T. and Misganaw B., Upgraded
lecture note.2008
2.Basic and applied concepts of Immunohematology,
2nd ed. Kathy D.Blaney and Paula R.Howard,2009
3. Blood banking and transfusion medicine: basic
principles and practice. Christopher D.Hilliyer et al.,
2nd ed.2007.
4.Safe blood donations, Module 1 WHO.2002

67
References…

5.Screening for HIV and other infectious agents,

Module 2, WHO. 2002
6.Blood group serology. Module 3 WHO.2002
7.Guidelines and principles for safe blood transfusion
practice, Introductory module. WHO 2002.
8.Immunohematology: Principles and Practice
Quinley. 2nd ed.1998.
9.AABB Technical Manual .15th Edition.2005

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