Module 1.

2
Antigen, Antibody, Complement System and Basic Genetics
Basic Immunology
• Transfusion medicine is based on the knowledge of Antigen (Ag) &
Antibody (Ab).
• Ag is defined as a foreign or non-self substance.
• Ab is a protein produced if a foreign Ag is introduced into an
immunocompetent individual.
Characteristics of Blood Group Antibodies:
1. Polyclonal or Serum Ab = produced in response to a single
Ag with more than 1 epitope.
2. Monoclonal Ab = produced in response to an Ag with a
single epitope. They are preferred in testing because they
are highly specific, well characterized, & uniformly reactive.
3. Naturally-occurring Ab = found in the serum of individuals
who have never been previously exposed to RBC Ag by
transfusion, injection, or pregnancy. They are usually IgM
cold Ab that react best at RT or lower and are hemolytic
when active at 370C.
4. Immune Ab = found in serum of individuals who have been
transfused or pregnant. They are usually IgG warm Ab that
react best at 370C & require the use of AHG or Coomb’s sera
for detection.
Naturally-occurring Ab:
• React to Ag of the ABH, Hh, Ii, MN, & P blood group systems.

Immune Ab:
➢React to Ag of Rh, Kell, Duffy, Kidd, & Ss blood group systems.
Alloantibodies
• Produced after exposure to genetically different, or
nonself, Ag of the same species, such as a different
RBC Ag after transfusion.
• Transfused components may elicit the formation of
alloantibodies against Ag (red cell, white cell &
platelet) not present in the recipient.

6. Autoantibodies
➢Produced in response to self Ag.
➢They can cause reactions in the recipient if they have
a specificity that is common to the transfused blood.
 Factors that influence agglutination
reactions:
1. Centrifugation
2. Ag-Ab ratio
Prozone effect = excess of Ab.
Postzone effect = excess of Ag.
3. Effect of pH = between 6.5 to 7.5 similar to normal
plasma or serum.
4. Temperature
IgM = cold Ab; react best at RT or below.
IgG = warm Ab; react best at 370C.
5. Ab type
6. Enhancement media = used to discover presence of
IgG Ab.
Examples of Enhancement Media:
1. Saline = 0.85% NaCl
2. Protein Media = AHG, 22% Albumin
3. LISS(low ionic strength saline)/Low Salt Media = 0.2% NaCl
4. PEG = polyethylene glycol
5. Proteolytic Enzymes = Ficin, Papain, Trypsin, Bromelain
Antigen and Antibody in blood banking
• Antibody production is a result of blood group
antigen (foreign-transfusion or pregnancy), or can be
naturally occurring.
• Blood group antigens are integral part of the RBC
membrane; Antigens are found on the surface of the
RBC
• B Cells produce antibody molecules that are specific
for a target antigen (part of the surface of RBC) = Anti-
A
• Antigenic determinants on RBC = elicit the production
of different antibodies
• Complement System: set or group of serum proteins that
generates membrane attack that causes cell destruction.
(Hemolysis)

Classical pathway
• Antibody-Antigen complex activates Complement = CLASSICAL
PATHWAY
IgG = 2 molecules; IgM= 1 molecule
Alternative pathway
• Activated by presence of bacterial polysaccharide or binding of IgA

Complement activation
1. Anaphylatoxins = induce inflammation
2. Vasoactive amines
3. Chemotaxins = promotes attraction of phagocytes to the area
4. Opsonins = enhances phagocytosis
5. Receptors
• Antigens-have different levels of immunogenicity
for them to cause immune response.
Factors affecting immunogenicity
1. Chemical composition and complexity (protein is
considered the most immunogenic)
2. Degree of foreignness
3. Size
4. Dosage
5. Route of administration
Antibodies – are proteins (immunoglobulin-Ig)
• 5 Classification:
1. IgG- Clinically significant in BB = crosses the
placenta
2. IgM- Can be Clinically significant in BB
3. IgA – found in secretions as a dimer, in tears,
sweats, etc; found in blood as a monomer.
4. IgE – allergic reactions
5. IgD
IgM antibodies:
• Produced initially in response to foreign antigen
• Large pentamer structure
• Contains 10 potential antigenic sites
• In BB – reaction in saline procedures
• 5-10 % of Ig class
• Short half-life 5-6 days
• Activates Complement with great efficiency.
IgG antibodies
• Monomer
• Accounts for 80% of Ig
• Found in extravascular fluid
• 2 antigen binding sites
• In BB- reaction less apparent in saline procedures, need
additive to show reaction.
• Half-life 23 days
• Cross placenta
• Activates complement
• Subclasses:
• IgG1 = major subclass, most efficient in placental transfer
• IgG2 = cannot cross the placenta
• IgG3 = most efficient in complement fixation
• IgG4 = cannot fix complement
• Immune response (Primary vs. Secondary)
• Primary = LONGER lag phase; normal increase Ab; IgM
• Secondary = SHORTER lag phase; 10x increase Ab; IgG
• Lag phase = phase of Ab synthesis where no Ab is produced.

Immune response influenced by:

• Age
• Route of exposure
• Genetic make-up
• Health
Antigen-Antibody interaction:
• Formation of antigen –antibody complex causes an
immune reaction.
• Amount is determined by:
1. Goodness to fit
2. Size
3. Shape
4. Charge
Antigen-Antibody reaction in Vivo vs. Vitro
• Primary concern in Blood Bank- In Vivo
• Reduce the potential of exposure to foreign antigens which
would result in antibody production.
• Identify antigens present on the RBC
• Identify any antibodies produced – found in the
serum/plasma
Identify antigen-antibody complex by:
• Hemolysis (Complement reaction associated with
complex)
• Agglutination (antigen/antibody complex), like
clotting of the blood

2 stages of agglutination:
1. Sensitization- antibody binds to an antigen. Is
influenced by amount of antigen and antibody
present.
• Sensitization enhanced by:
1. Serum to cell ratio
2. Temperature
3. Incubation
4. pH
5. Ionic strength
2. Lattice Formation stage (cell interaction = visible
agglutination, linkage between antigen and
antibody)
• Factors that influence Lattice formation
1. Zeta potential = degree of negative charge on the surface of a red blood cell
2. Serum to cell ratio (zone of equivalence)
3. Prozone effect
4. Centrifugation

Grading agglutination (lattice formation)

0(neg), 1+, 2+, 3+, 4+
Indicators of antibody-antigen complex
• Hemolysis
• Agglutination
Hemolysis is an indication of antigen-antibody complex
that results in cell destruction. Identified usually in
the Antiglobulin Test (presence of agglutination with the addition
of AHG indicates antibody binding to a specific red cell antigen)
COMPLEMENT Fixation
• The complement fixation test (CFT) was extensively
used in syphilis serology after being introduced by
Wasserman in 1909.
• Complement is a protein (globulin) present in normal
serum.
• Whole complement system is made up of nine
components: C1 to C9
• Complement proteins are heat labile and are destroyed
by heating at 56°C for 20 – 30 minutes.
• Complement binds to Ag-Ab complex
• When the Ag is in an RBC it causes lysis of RBC’s.
 Principle
• Complement takes part in many of the immunological reactions.
It gets absorbed during the combination of antigens and
antibody.

• This property of antigen–antibody complex to fix the

complement is used in complement fixation test for the
identification of specific antibodies.

• The haemolytic system containing sheep erythrocytes (RBC) and

its corresponding antibody (amboceptor) is used as an indicator
which shows the utilization or availability of the complement.
• Sheep RBCs coated with hemolysin
• If the complement is fixed then there will be no lysis of sheep
erythrocytes, thus denoting a positive test.

• If the complement is available then there will be haemolysis

which is a property of complement, denoting a negative test.
 Components of CFT
Test System
• Antigen: It may be soluble or particulate.
• Antibody: Human serum (May or may not contain Antibody
towards specific Antigen)
• Complement: It is pooled serum obtained from 4 to 5 guinea pigs.
It should be fresh or specially preserved as the complement
activity is heat labile (stored at -30 °C in small fractions). The
complement activity should be initially standardized before using
in the test.
Indicator System (Haemolytic system)
• Erythrocytes: Sheep RBC
• Amboceptor (Hemolysin): Rabbit antibody to sheep red cells
prepared by inoculating sheep erythrocytes into rabbit under
standard immunization protocol.
Positive Test
• Step 1:
At 37°C
Antigen + Antibody + Complement Complement gets fixed
(from serum) 1 Hour

• Step 2:
At 37°C
Fixed Complement complex + Haemolytic system No Haemolysis
1 Hour (Test Positive)
 Negative Test
◼ Step 1:
At 37°C
Antigen + Antibody absent + Complement Complement not fixed
1 Hour

◼ Step 2:
At 37°C
Free Complement + Haemolytic system Haemolysis
1 Hour (Test Negative)
 Results and Interpretations:
• No haemolysis is considered as a positive test.
• haemolysis of erythrocytes indicative of a negative test.
1 2 3 4

• Microtiter plate showing Haemolysis (Well A2, A3 and B4)

and No Haemolysis (Well A1, A2, A3)
Basic Genetics
Genes:
➢The Ag present on an individual’s RBC
membrane are inherited.
➢Each Ag is controlled by a gene, which is
the unit of inheritance.
➢If the gene for a particular Ag is present,
that Ag would be expected to be
expressed.
Chromosomes:
• Each cell, except for mature RBCs, consists of cytoplasm & a
nucleus.
• If the nucleus is observed under the microscope at
approximately the time of cell division, several long, threadlike
structures will be visible.
• These structures are referred to as chromosomes.
• Each species has a specific number of chromosomes, & the
chromosomes occur in pairs.
• Humans have 46 chromosomes (23 chromosome pairs)
• The paired chromosomes are similar in size & shape & have
their own distinct functions.
• A complete set of 23 chromosomes is inherited from each
parent.
Complete set of chromosomes:
Gene Location or Linkage:
• Because the gene is the unit of inheritance, it must also be
located within the nucleus.
• Genes are exceedingly small particles that, when associated in
linear form, make up the chromosome.
• They are too small to see under the normal brightfield
microscope but together are visible as the chromosome.
• Genes are made up of DNA.
• Each trait that is inherited is controlled by the presence of a
specific gene.
• The genes responsible for a particular trait always occur at
exactly the same point or position on a particular
chromosome.
• The position is referred to as the Locus (plural: Loci) of the
gene.
Genetic Terms:
Syntenic genes = genes for different inherited traits
are known to be carried on the same
chromosome. The genes on a single chromosome
are too far apart to display absolute linkage in
inheritance.

Linked genes = genes are located on the same

chromosome & are normally inherited together.

* The closer the loci of the genes, the closer is the

Linkage.
Alleles:
• Inherited traits are somewhat variable within a
species.
• For example: eye color varies, & it is known to be
inherited.
• Therefore, each possible eye color must be the result
of a gene of that color.
• Alleles are variants of a gene for a particular trait.
• Because we only have 2 genes (1 pair) for any given
trait, our cells will have only 2 alleles.
• However, the number of possible alleles for a trait
varies.
Homozygous Allele:
• A person who has identical alleles for a trait.
• For example: a person with blue eyes carries 2 blue-eye genes & is
homozygous for blue eyes.

Heterozygous Allele:
➢A person who has 2 different alleles for a trait.
➢For example: having a blue-eye gene in addition to a brown-eye gene.
Dominant Allele:
• Alleles that may be stronger than, or may mask the presence of
other alleles.
• For example: brown-eye gene masks the presence of blue-eye gene
and is said to be “dominant” over the blue-eye gene.
Recessive Allele:
➢Gene is weaker than the dominant gene.
➢For example: blue-eye gene is less expressed and is weaker than the
brown-eye gene.
• Individuals who have 1 brown-eye gene & 1 blue-eye gene have eyes
that appear brown.
• One must have 2 blue-eye genes to have blue eyes.
Codominant Allele:
➢As used in transfusion medicine, the various alleles for a particular
blood group system are equally dominant.
➢If the gene is present and there is a suitable testing solution
available, it will be detected.
Phenotype:
• Is seen by tests made directly on the RBCs even
though other Ag may be present.
• Can be detected in the laboratory.
• For example: ABO typing determines if the A or B
antigens are present with the use of typing sera.

Genotype:
➢Refers to the actual total genetic makeup of an
individual.
➢Not usually determined in the laboratory.
➢Usually requires additional studies.
➢For example: family studies
Genetics
• Blood type is determined by genetic inherited patterns.
• Phenotype: observable trait
• Genotype: actual genetic make-up
• Predict genotype, if you know phenotype and can predict phenotype,
if you know genotype.
• Blood type is determined by the antigen present on the RBC.
• Punnet Square
ABO INHERITANCE

Dad = A/O Mom

and
B O
Mom = B/O
A A/B A/O
Dad
O O/B O/O
 ABO groups of the offspring from the various
possible ABO mating

Phenotypes Genotype offspring

AxA AAxAA A (AA)
AAxAO A (AA or AO)
AOxAO A (AA or AO),O(OO)

BxB BBxBO B (BB or BO)

AxAB AAxAB AB (AB) or A (AA)

AOxAB AB (AB), A (AA, AO), B(BO)
Examples:
• AO x BO
B O Phenotype
A AB AO A = 25% AB = 25%
O BO OO B = 25% O = 25%

• BO x BB
B B Phenotype
B BB BB B = 100%
O BO BO

• AB x OO
O O Phenotype

A AO AO A = 50%
B = 50%
B BO BO
• Genes: unit of inheritance on a chromosome. They
are located on specific areas of the cells called genetic
loci.
• Alleles: Form or different forms of a gene of a given
loci
• Ex: A, B and O alleles on the ABO gene locus.
• Polymorphic: having two or more alleles at a given
locus. (Rh system)
• Inheritance Pattern:
1. Co-dominant (equal expression of a gene on an
individual)
2. Recessive or dominant ( only one alleles is
expressed on the cell)
3. Amorphic expression: gene present, but does not
express detectable product.
Mandelian Principle: Independent segregation of traits
4. Mutations
Homozygous vs Heterozygous inheritance
• Homozygous: 2 alleles for a given trait are the same-
genotype are identical genes
• Heterozygous: 2 alleles are inherited are different-
genotype are different.
• Agglutination reactions will be effected by inheritance
pattern.
• Homozygous pattern- stronger reactions
• Heterozygous pattern- weaker reactions
• Inheritance pattern with Cis and Trans position.
(related to the Rh system and how it expresses itself)
• Cis : gene expression is from the same chromosome
• Trans: gene expression is from different or opposite
Chromosome.
• Can help determine agglutination reaction.
• Silent Genes- “Amorph”, present and cause problems,
but do not produce detectable antigen.
• Result in unusual phenotype
• Example is a “Null” type individual- blood type is not
apparent or predictable - see with the Rh system.
Paternity Testing
• Direct exclusion: child has a trait present that neither
parent posses.
• Indirect exclusion: child lacks a gene that should be
inherited from the parent in question.
• Inclusion: when the child has the predictable traits
that are expected form the parent in question.