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Basics of Immunohematology

This document provides an overview of basic principles of immunohematology, including definitions of antigens and antibodies, characteristics of antigens, immunoglobulins, genetics basics, and common serological techniques. It defines key terms like antigen, antibody, immunogenicity, genotypes and phenotypes. It also describes the stages of antigen-antibody reactions and factors that affect these reactions.

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74 views

Basics of Immunohematology

This document provides an overview of basic principles of immunohematology, including definitions of antigens and antibodies, characteristics of antigens, immunoglobulins, genetics basics, and common serological techniques. It defines key terms like antigen, antibody, immunogenicity, genotypes and phenotypes. It also describes the stages of antigen-antibody reactions and factors that affect these reactions.

Uploaded by

microwstuff
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Basic Principles of

Immunohematology
Teaching Aims
• To understand the basis of Immunology
and genetics which are closely involved in
the understanding of blood group serology
Antigen and Antibody
• Antigen:
A substance that on introduction give rise to a
formation of antibody that reacts specifically
with the same antigenic substance.

▪Antibody:
A gamma-globulin product of an immune
response, (also called immunoglobulin) that
reacts with antigen against which it is
stimulated.
Characteristics of antigen
• Chemical structure of antigen
– Antigens composed of oligosaccharides tend to
stimulate IgM type of antibody production
– Antigens which are primarily protein in nature,
produce IgG antibody
• Degree of foreignness
• Number of antigens introduced
– Higher the dose, greater the antibody production
• Route of administration
– Intramuscular or subcutaneous route more potent
Immunogenicity

• Ability of an antigen to stimulate the


production of its corresponding
antibody in a person who lacks the
antigen

• Rh D most potent antigenic


• Fya is poor antigenic
Immunogenicity of antigens
Antigens System Immunogenicity (%)
D 70
C 0.2
c Rh 04
E 3.3
e 1.1
K 10
Kell
k 1.5
Fya Duffy 0.6
Jka 0.1
Kidd
Jkb 0.03
S 0.08
MNS
s 0.06
Immunoglobulins
▪ Five types, viz. IgG, IgM, IgA, IgD & IgE.
▪ Blood group antibodies are mainly confined to IgG &
IgM.
▪ IgG is a smaller molecule, with a pair each of heavy
& light chains of amino acids.
▪ IgG antibody can just coat but not agglutinate the
cells.
▪ IgM has 5 such pairs joined together by the J chain.
▪ IgM antibody agglutinate the cells bearing
corresponding antigen.
Properties of human immunoglobulins
Properties IgG IgM IgA

H chain, Class Gamma Mu Alpha


Serum conc (mg/dl) 1000 – 1500 85 - 205 200 - 350

Molecular weight 150,000 900,000 180,000

Complement fixation Occasionally Yes No

Structure Monomer Pentamer Mono / diamer


Crosses placenta Yes No No
Secretions No No Yes
IgM Antibodies (Complete)

• Agglutinate in saline phase


• Pentavalent
• Usually naturally occurring
• Do not cross placenta
• React at temperature
varying from 4 – 20oC
• Example: ABO antibodies
IgG Antibodies (Incomplete)
• Agglutinate in IAT phase
• However, may cause
agglutination in saline phase
using albumin / enzymes
• Monovalent
• Usually immune in nature
• Can cross placenta
• React at 37oC
• Example: Rh antibodies
14 nm 35 nm

IgG IgM

24 nm

Intracellular distance
Complete and Incomplete antibody

Complete antibody -IgM

Incomplete antibody -IgG


Antibody (Immune) Response

IgG

IgM
Naturally occurring Vs Immune
Antibodies

Feature Naturally occurring Immune

Antigen stimulus Obscure, possibly Human red cell


from microbial origin antigens
Type of Immunoglobulin IgM IgG

Optimum temperature < 22o C at 37o C

Clinical significance Acute HTR HDN, DHTR

Examples ABO antibodies Rh, Kell, Kidd,


Duffy antibodies
Clinical Significance of Antibody

Clinically significant Clinically insignificant

• ABO • Lewis
• Rh • M,N
• Kell • P1
• Duffy • Lutheran
• Kidd • A1
• Ss
Types of Antigen-Antibody Reactions

❖ Agglutination
❖ Sensitization
❖ Haemolysis
❖ Neutralization (inhibition)
❖ Precipitation
❖ Immunofluorescence
❖ Complement fixation
❖ Radio Immunoassay
Antigen-Antibody Reaction
Stages of ag-ab reaction
Stage of sensitization Stage of agglutination
• Only coating of red cells • Characterized by
with IgG antibody without formation of bridges
causing agglutination between sensitized red
• Bond holding ag-ab cells resulting in visible
complex may dissociate aggregate of red cells
& re-associate till the
state of equilibrium is
reached
Factors affecting stage of sensitization
• Antigen – Antibody ratio
– Two volume of serum and one volume of 5% red cells
– Sensitivity of test depends upon number of antibody
molecules bound per red cells
• pH
– Most antibodies detected at neutral pH
• Temperature
– IgM antibodies react optimally at cold temp while IgG at 37oC
• Incubation time
– Time needed to reach ag-ab reaction at equilibrium
– Too short incubation – weaker reaction
– Prolonged incubation results in dissociation of antibody
Factors affecting stage of agglutination
• Size & Class of antibody
– IgM antibody, being a pentamer can bind antigen sites
up to 35 nm apart
– IgG antibody, being a monomer can bind antigen sites up
to 14 nm apart
• Antigenic sites
– Antigens located on surface of red cell membrane (ABO)
result in strong agglutination
– Antigens embedded in membrane (Rh antiges) result in
weaker agglutination
• Zeta potential
– Electrostatic repulsive force between red ells
Electron Cloud Around Each Cell

zeta potential
Effect of adding LISS

▪ Reduction in zeta
potential using LISS
▪ RBCs come closer
together
▪ Strong agglutination
▪ Reduction in
incubation time
Red Cell Serological Techniques

➢ ABO and Rh (D) typing


➢ Weak ‘D’ or Du testing
➢ Antibody screening & identification
➢ Pre-transfusion testing
➢ Indirect antiglobulin test
➢ Direct antiglobulin test
Genetics: Basics
• Gene
– A biological unit of inheritance
– Dominant gene – always expressed as antigen regardless of
whether it is in homozygous or heterozygous state
– Recessive gene – produce antigen only when in homozygous state
• Chromosome
– Linear arrangement of genes
• Locus
– Hypothetical seat of gene on a chromosome
• Allele
– Al alternative form of gene at particular locus
Genetics: Basics

• Genotype
– Sum total of genes present on chromosome
regardless of whether or not they produce detectable
products.
– Determined through testing of genes & family study
• Phenotype
– Detectable products (antigens) demonstrated
through direct testing only
Genotype Vs Phenotype
Phenotype Genotype
A1 A1A1
A1A2
A1O

A2 A2A2
A2O

B BB
BO

O OO
Genetics: Basics
• Homozygous:
– presence of identical pair of allelic genes on both
chromosomes
– double dose of antigens
– stronger reaction with corresponding antibody
– example: KK
• Heterzygous:
– different alleles of genes on pair of chromosome
– weaker reaction with corresponding antibody
– example: Kk

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