CHAPTER ONE

INTRODUCTON TO IMUNOHEMATOLOGY
 Learning Objectives
At the end of this chapter, the student will be able to:
– Describe overview of Immunohematology
– Discuss historical background of Immunohematology
– Discuss blood group genetics (Inheritance pattern of blood group)
– Describe blood cell antigens
– Explain characteristics of blood group antibodies and their
stimulation
– Elaborate antiserum properties and preparation
– Discuss antigen –antibodies interaction
– List and explain factors that affect invitro antigen antibody
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interactions
 Chapter outline

– Overview of Immunohematology

– Historical background of Immunohematology

– Blood Group Genetics (Inheritance pattern of blood group)
– Blood cell antigens
– Characteristics of blood group antibodies and their stimulation

– Antiserum properties and preparation

– Antigen –antibodies interaction
– Factors that affect Invitro Antigen antibody interactions

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 1.1 Overview of Immunohematology
Definition :
 Immunohematology is more commonly known as "blood banking".

 a specialized branch of laboratory medicine dealing with :

preparing blood and blood components for transfusion
selection of appropriate, compatible components for transfusion.

 Study of red cell antigens and their corresponding antibodies on

blood for resolving the problems of blood transfusions.
 deals with the concepts and clinical techniques related to modern
transfusion therapy.
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 Overview of Immunohematology…

 Immunohematology involves a wide variety of

procedures used in:
donor selection,

Collection, preparation, storage of blood,

component preparation and use,
 detecting antigen/antibody reactions

 More generally immunohematology refers to immunological

reactions involving blood components

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 Overview of Immunohematology…
 Blood transfusion: is the process of transferring blood or blood-
based products from one person into the circulatory system of
another.
 Needed when there is massive blood loss or
 Needed when the blood producing mechanism fails.

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1.2. Historical background of Immunohematology
• The era of blood transfusion began when William
Harvey described the blood circulation 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.
• Denys also performed subsequent transfusions using
animal blood, but most of them were unsuccessful.
 1.2. Historical Overview, cont’d…
• 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.
• Blood transfusions continued to produce unpredictable results,
until Karl Landsteiner discovered the ABO blood groups in 1900.
• Karl Landsteiner introduced the immunological era of blood
transfusion.
 1900s

• 1900-1901 Karl Landsteiner, Austrian

physician, discovers the first three
human blood groups, A, B and O.
• 1902 AB, added by colleagues A. von
Decastello and Stürli
• 1907 Richard Weil - crossmatching
between donor and patient
• 1939 Philip Levine and R.E. Stetson
described Rh Blood Group System
 1914

• 1914-1918: mixing with sodium citrate prevented blood from clotting

and can last a bit longer if kept in a fridge. (Lewisohn and Weil)

• Blood collected in a citrated flask….…...and immediately transfused.

 1960s & 1970s

• 1967: anti-D immunoglobulin commercially

introduced to prevent RhD HDFN
• 1971: hepatitis B surface antigen (HBsAg) testing
of donated blood begins in the UK.
• 1979: CPDA1 anticoagulant preservative, extends
whole blood and RBC shelf life to 35 days
 1980s

• Early 1980s growth of component therapy,

products for coagulation disorders and
• 1983 Additive solutions extend shelf life of red
blood cells to 42 days. .
• 1985 first HIV screening test licensed
1990s
• 1990 first specific test for hepatitis C
• 1992 donor blood HIV-1 and HIV-2 antibody
testing implemented.
• 1996 HIV p24 antigen testing of donated blood
shortens the window period.
• 2001 - South Africa becomes first country to
approve an artificial blood substitute for use in
transfusions.
 1.2. Historical Overview, cont’d…

• It became clear that the incompatibility of many transfusions was

caused by the presence of certain factors on red cells now known as
antigens.
• Based on this scientific approach two postulates were drawn

1. Each species of animal or human has certain factor on the red cell
that is unique to that species, and
2. Even each species has some common and some uncommon factor
to each other.
This landmark event initiated the era of science based transfusion
therapy and was the foundation of immunohematology as a science.
 Blood Group Genetics

 Genetics
– study of inheritance

 A blood group
– is a classification of blood based on the presence or absence of
inherited antigenic substances on the surface of red blood cells
(RBCs).

 Blood group genetics is concerned with the way in which

the different blood groups are inherited.

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 Blood Group Genetics…….

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

Locus
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Location of a gene on a chromosome
 Blood Group Genetics…
Allomorphic genes (Alleles), and Polymorphism
• Each gene has its own locus, along the length of the chromosome.
• Variation of a gene that produces an alternative Phenotype
• Certain inherited characteristic can be represented by a group of
genes, and the locus can be occupied by only one of these genes.
E.g. A,B and O are alleles on ABO gene

Polymorphic;
 Having two or more genes at a given locus
 Some blood group are more polymorphic than others.
E.g. Rh system is highly polymorphic than ABO system

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 Blood Group Genetics…
• Mitosis:
– While body cells multiply they do so by producing identical
new cells with 46 chromosomes.
– The chromosomes are duplicated and one of each pair passes
to the daughter cells.

• Meiosis:
– The production of sperm or ovum cells (gametes)
– When sex cells are formed, the pairs of chromosomes do not
multiply but simply separate so that each of the new cells
formed contains only 23 chromosomes.
– During fertilization when the egg and sperm unite
• will be 46 chromosomes
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 Blood Group Genetics…

Genotype versus phenotype

• Phenotype
– Physical expression of inherited traits,
– Determined by reacting red cells with known anti-sera

• Genotype
– Actual genes inherited from each parent
– The genetic composition of a particular inherited characteristic
– 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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Punnet squares showing ABO inheritance
• Determine two group A parents child blood type?
• Can they have a group O child?
Yes. show how?
A O

A AA AO
5 minutes
O AO OO

What can be the parents of an AB child?

A, B or AB, but not group O
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 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.

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 / dominant inheritance patterns

Recessive
• inheritance would require the same alleles from
both parents be inherited to demonstrate the trait.
E.g. O gene

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
• 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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 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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 Homozygosity & Heterozygosity
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,

• When antigen density varies between cells of different individuals,

it is often due to the inheritance of the antigen expression.
• Brings about variation in strength of agglutination during
antigen -antibody reaction is known as dosage effect

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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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 Genetic inheritance
Genes can be 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,

E.g. 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

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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

• Independent assortment does not occur when genes are linked.

• These gene units are called haplotypes

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 Genetic inheritance….

Silent genes
• Genes do not produce a detectable antigen product and are called
"silent” genes or amorphs.

• Amorphs can result in an unusual phenotype called "null"

= antigen are not apparent.

• Null types caused by amorphic genes are rare

• Unusual phenotypes may also result from the Action of either

– Amorphic genes or
– Suppressors or regulator genes.

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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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 Red Blood cell antigens

• 30 blood group systems with more than 200 unique red cell Antigens

• Each system is a series of red cell antigens, determined either by a

single genetic locus or very closely linked loci.

• Only a few red cell antigens are erythroid specific (Rh, LW, Kell and
MNSs),

• These antigens protrude from the surface of the RBC in three

dimensional configurations.

• In biochemical terms these antigens may take the form of:

Glycoprotein, proteins, Glycolipids

• Some of the red blood cell antigens are more immunogenic

E.g. The D antigen within the Rh group system.
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 Human leukocyte antigens (HLA)

• On nucleated cells such as leukocytes and tissues

• Can readily provoke an immune response if transferred in to a
genetically different (allogenic) individual.

• Encoded by genes which are parts of MHC gene system on

chromosome 6 .
– Class I (A, B and C locus); Class II (DR, DP and DQ) and Class III
(complement proteins)

• The MHC region is called polymorphic

For example :
• 49 different alleles at the A locus and 97 alleles at the B
35 locus
 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 agns are the major cause of :

– Neonatal alloimmune thrombocytopenia,
– Post transfusion purpura ,
– It can also decrease the expected increment of platelet transfusion.

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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 stimulated is more appropriate.

– Because these Abs resulted from some kind of outside
stimulus

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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

– Do not react above the body temperature

– Most of these do not give rise to transfusion reactions.

– They are of high molecular weight

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• cannot cross the placenta
 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.
– Causes more serious transfusion reactions than the naturally occurring
ones.
– Can cross the placental barrier.

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1. Cold reacting
2. Good complement activator IgM Antibody’s
3. Pentamer
4. ABO, Ii, Lewis, MN, & P

1. Warm Reacting

IgG Antibody’s 2. Poor complement activators

3. Monomer
4. Rh, Kell, Kidd, Duffy, and Ss

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 The Anti-serum
• Purified, diluted and standardized solution containing known
antibody

• Used to find the presence or absence of antigen on cells

• Antiserum is named on the bases of the antibody it contains

Antiserum Contain Antibody

Antiserum Anti-A Anti-A antibody

Antiserum Anti-B Anti-B antibody
Antiserum Anti-AB Anti-A and anti-B antibody
Antiserum Anti-D Anti-D antibody
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 The antiserum…
• Anti-serum must:
– Be specific for the antigen to be detected

– Have sufficient titer to detect antigen

- 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

e.g. Anti-A1should agglutinate:

– A1 cells in 10seconds or less,
– A2 cells in 20sec or less, and
– A2B in 30 sec or less

• Avidity: able to agglutinate red cells quickly and strongly

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 The antiserum…
• Be free from haemolysins, fat and rouleaux
• Be sterile and clear
• be stable: Preserved with 1% sodium azide
• Have a marked expiration date, and

• Should be stored at 40C

• The manufacturer directions must be followed carefully.

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 Antigen - Antibody interactions

The presence of Invitro antigen and antibody

interaction can be detected by:

– Hemolysis

– Precipitation

– Agglutination (Most commonly used)

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 Antigen - Antibody interactions…..

• Antigen-antibody reaction occurs in 2

stages.
• Sensitization and
• Lattice formation (agglutination).

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 Stages of Ag-Ab Reaction…
A. Sensitization-the first phase
– Physical attachment of Ab molecules to Ags on the RBC
membrane.
Antibody Coating RBC Membrane without Aggn
There is no visible clumping, needs incubation or
enhancement media

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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 (anti - M and anti - D react best at a lower PH)

– Temperature (Cold (4-10oC) or Warm (37oC)

– Time of incubation (15 to 60 minutes.)

– Ionic strength: Lowering the ionic strength of the test

system. (low ionic strength saline (LISS) is appropriate)

– Steric hindrance: mutual blocking

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– competition
 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 or agglutination.
• Lattice formation is a much slower process than the
sensitization phase.

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 Factor affecting the lattice formation phase

 Size of the Immunoglobulin : IgM is better

 Number of binding sites :
 IgG: Two binding sites (anti-D, anti-Jka, etc.)
 IgM: Ten binding sites (anti-A,B, anti-I, etc.)

 Location and number of antigenic determinants

 Antigen dosage: double and single dose

 Zeta Potential: should be reduced to increase aggltn

– RBC surface – negative charge
– Ionic solution - cations

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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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 The antiserum preparation
• The anti-sera used in Immunohematology are prepared in one
of the two ways:

– Animal inoculation: By deliberately inoculating animals

with an antigen

– By collecting serum from humans who have been

sensitized with corresponding antigens

• Serum also can be collected from known blood groups

individuals.

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 Methods of enhancing agglutination
• Centrifugation: Brings Abs and Ag’s into close proximity. (optimum centr.)

• Enzyme treatment: Enhance or suppress the reaction

– Ficin (Fish), Trypsin (Pig stomach), Papain (papya), and Bromelin (Pineapple)
 Treatment RBC with enzyme releases Sialic acid = decrease negative
charege of RBC = reduce zeta potential

– Removes glycoprotein from the membrane, Ab molecules can be easily

reacting with the Ag on the membrane surface

– Rh, Kidd, P1, Lewis, and I Ags enhanced by enzyme treatment

– Fya, Fyb, M, N, and S Ags can be destroy or depress by enzyme treatment

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Enhancing agglutination
• Colloidal media: is bovine albumin solution,
– Albumin, reduces zeta potential = promoting agglutination

• Antihuman Globulin Reagent

– Promote agglutination of red cells sensitized with IgG or complement.

• Low Ionic Strength solution (LISS)

– Decrease the ionic strength of a reaction medium and thus,
reduce the zeta potential

• Poly ethylene glycol (PEG)

– removes water

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 Review Questions

1. Who discovered ABO Blood groups?

2. What are the characteristic differences between Natural and

Immune antibodies?

3. How the anti-sera used in Immunohematology are prepared?

4. Mention how the presence of invitro antigen and antibody

interaction can be detected.

5. Mention the characteristics of Ideal antisera?

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 Review Questions

6. Define: A. Antigen B. Antibody C. Immunogenicity

7. Identify some characteristics of the IgG subtypes

8. What are the characteristic differences between Natural and

Immune antibodies?

9. Which classes of antibodies predominate during the primary

immune response and secondary immune response?

10. List the factors that affect antigen and antibody interaction

11. Discuss the methods that are routinely used in the blood banking
laboratory to enhance agglutination     reaction.

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 References

1. Immunohematology for medical laboratory

science students,Yayehyirad T. and Misganaw B.,
Upgraded lecture note.2008
2. Basic and applied concepts of
Immunohematology, 2rd ed. Kathy D.Blaney and
Paula R.Howard,2009
3. Transfusion medicine made easy for students of
allied medical sciences and medicine, Osaro
Erhabor and Teddy Charles Adias, 2012
4. Immunohematology: Principles and Practice
Quinley. 2nd ed.1998.
5. AABB Technical Manual .15th Edition.2005.