IMMUNOHEMATOLOGY

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 CHAPTER ONE
INTRODUCTON TO IMUNOHEMATOLOYG

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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. Efforts to
save human lives by transfusing blood

 Is the area of laboratory medicine dealing with the

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

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Immunohematology…

 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.

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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.

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Historical background…
 In 1667, jean Bapiste 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.

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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 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
 Physical expression of inherited traits,
 Determined by reacting red cells with known antisera
(Ab)

 Genotype
 Actual genes 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
 Genetic characteristics are controlled by unit factors that exist
in pairs in individual organisms
 each individual receives one unit factor from each parent,
 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.
 Example
 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.

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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.

 Its expression 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

 During the processes of heredity, the paired

unit factors separate so that the offspring
receives one unit factor from each parent,
 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 Homozygosity &
Hetrozygosity
Homozygous
• the unit factors that determine a particular trait are the
same,
• Genotype is made up of identical genes, such as AA,
BB, or OO,

Heterozygous.
• the unit factors that determine a particular trait are
different,
• Genotype is made up of different alleles from each
parent, such as AO, AB, or BO,

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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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Blood group Abs & their
stimulation

Blood group antibodies are classified into:

 Natural and

 Immune antibodies.

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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.

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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

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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.

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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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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.

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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

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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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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.

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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…
 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 against A2 cells, and

 1/16 against AB cells

- Anti-B should have a titer of at least 1/64

against B cells
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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

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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.

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In vitro detection of Ag and Ab
reaction

The presence of In vitro 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

 lattice formation.

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

 represents the physical attachment of Ab molecules to

Ags on the RBC membrane.

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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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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.

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 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.

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 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

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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.

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