IMMUNOLOGIC PRINCIPLES
Primary Immunologic Components:
Provides basis for blood bank testing and
reaction
o
o
Antigen:
surface
Antibody:
found on RBC
found on plasma/ serum
ANTIGEN
Substances that have the capability to
stimulate antibody production
o
o
o
Allergen
Bacteria
Blood groups
Characteristics of an Antigen
1. Chemical nature
a. Protein
o MW, complex
b. Lipids
o less immunogenic as it is less
stable
c. Polysaccharide
o Carriers
o Haptens/ incomplete
immunogens
d. Nucleic Acid
o least immunogenic
o easily degraded
2. Molecular weight
>10, 000 D
3. Complexity
4. Stability
5. Foreignness
3. Blood Group Antigen
ABO, Rh (D) antigens
ANTIBODY
Also known as immunoglobulin (Ig)
Serum protein formed in response to
immunization
Characteristics of an Antibody
1. Protein
2. Produced in response to antigen stimulation
3. Specific for stimulating antigen
Classification of Blood Group Antigen
1. Alloantibody
Reacts with foreign antigen not
present on patients own RBC
Most produced as a result of immune
stimulation via transfusion or
pregnancy (delivery)
2. Autoantibody
Reacts with the antigen on patients
own cell and with the same antigen on
cells of other individual
Reacts at both warm and cold
temperatures
Autoimmune disease
Classes of Immunoglobulins
1. IgM
General Rule:
MW Complex Foreignness stability =
immunogenic
Important Cellular Antigen
1. Histocompatibility Antigen
Nucleated cells: WBCs are commonly
express cell surface antigen that
stimulates immune response
MHC/ HLA
Gene product identified on WBC
Influences survival/ rejection rate of
organ transplantation
Important in determining Graft vs. Host
Disease
2. Autoantigens
Self antigen
Antigen bearing cells RBC
Structure: pentamer (5 monomer
linked by J-chain)
10% of Immunoglobulin pool
Half life: 5-6 days
First to be produced in immune
response
Most effective agglutinin due to
binding sites
Funtions:
o Best in complement fixation
(requires only 1 molecule of IgM
o Agglutination
o Cytolytic reaction
2. IgG
-
Structure: monomer
70-75% of Immunoglobulin pool
Half life: 23 days
Major immunoglobulin in the serum
Average level is achieved before 16
years old
Functions:
o Immunity to newborn
(transplacental except IgG2)
o Agglutination
o Precipitation
o
o
o
Opsonisation
Complement fixation (except
IgG4)
Neutralization of toxins and
tissue
IgG
Monomer
Divalent
Smallest Ab
150, 000 D
7s
Shortest Ab: 250 A
Y shaped
Can cross the placenta
Placenta has specific
receptors for IgG
Can provide passive
immunity to the
newborn
Warm ab
370C
Protein reacting
Produced after IgM
IgM
Pentamer
Decavalent
Largest Ab
950, 000 D
19s
Longest Ab: 1,000 A
Star or Crab
shaped
Cannot cross the
placenta
Cannot provide
passive immunity to
the newborn
Presence on newborn
may indicate
intrauterine infection
Cold ab
Saline reacting
Produced early/ first
infection
Chronic Ab
Acute Ab
Capable of complement fixation
facilitates IgA transport
4. IgE
Structure: monomer
0.004 % Ig pool
HL: 5-6 days
Reaginic antibody
Affinity to basophils & mast cells
release of histamine & heparin
Attached to eosinophils (allergies &
parasitic infection)
Type I Hypersensitivity
Can cause transfusion reaction but not
HTR
Most heat labile
Structure: monomer
<1 % Ig pool
Extremely susceptible to proteolysis
HL: 2-3 days
Least significant in BB
Immunoregulation
Found on surface of B cells
5. IgD
Conglutinin
-
Causes clumping with AHG (IgG)
Protein reacting
Albumin/ AHG:
visible
clumping
NSS:
no clumping
Warm antibody: reacts at 370C
(clinically significant)
Agglutinin
3. IgA
-
Structure: monomer
15-20 % Immunoglobulin pool
30 % ABO antibody
Anaphylaxis (Type I Hypersensitivity),
selective IgA deficiency cannot be
administered with plasma products
Enhance reaction of IgG in terms of
hemolysis
Major antibody on secretions
Tears
Saliva
Colostrums
Milk
Intestinal fluids
Synthesis of IgA:
a. Synthesized by plasma cells plasma IgA
b. Synthesized by cells of intestinal wall
binds to glycoprotein secretory
component secretory IgA
Secretory IgA:protects IgA from digestion
by
proteolytic enzymes
Substances that causes immediate
clumping (IgM)
NSS reacting
Cold antibody: reacts at refrigeratorroom temperature (4-260C)
IMMUNE RESPONSE
Types of Immune Response
A. Primary Immune Response
- 1st encounter with antigen
- IgM
B. Secondary Immune Response
- Memory response
- Anamnestic response
- IgG
Stages of Immune Response
1. Lag Phase
- No detectable antibody
- Serologic test is not recommended
2. Log Phase
- Increasing concentration of antibody
(10x)
- Use of sensitive serologic test
3. Plateau/ Stable Phase
- Rate of antibody production is equal to
antibody degradation
- Best time to use serologic test
4. Decline Phase
- Antibodies are catabolised
- antibody titer : leads to false
negative serologic test result
- Degree of degradation is more than
the degree of production
**Specific serologic test are used
mainly to prevent cases of cross
reacting reactions
antibody are treated in such a
manner as to disrupt the bonds
between the antigen & antibody
Disassociation of Antigen Antibody Complex
All bonds must be broken simultaneously
-
2-mercaptoethanol
DTT (Dithiothreitol)
breaks down J chain
converts IgM to trimonomers
Detection of Antigen Antibody Reaction
In vitro tests for antigen antibody reaction
Secondary IR in Comparison with Primary IR
Shorter lag phase
Greater antibody production
Longer plateau phase
Gradual decline
ANTIGEN ANTIBODY INTERACTION
Allotype:
are present in
Antigenic determinants that
allelic forms
Immunogenicity:
Capability of a substance to
induce immune
response
Epitope:
Antigen binding site
Paratope:
Antibody binding site
Specificity:
Ability of antibody to bind to a
particular antigen
Cross reactivity:
Antigen determinants of one
type of antigen are similar to
another unrelated type
(homologous protein content of
bacterial cell wall)
Antibody Affinity:
Initial force of attraction
between single Fab & single
epitope
Avidity:
binding strength of antibody with
antigen
Multivalent Ag:
Can combine with more
than one antibody; strength
Immune
Combination of antigen with
specific ab
Complex
Soluble IC
Small IC
Precipitating IC
Large IC
Prozone:
antibody excess
Postzone:
antigen excess
Zone of
ab=ag; visible endpoint: ppt
equivalence
Dosage effect:
weak expression of ag on
RBC
Homozygous> heterozygous
Elution
Process whereby cells that are
coated with
1. Agglutination
- Particulate ag + specific ab
aggregates
2. Precipitation
- Soluble ag + specific ab visible
insoluble
complexes
3. Hemolysis
- Ag + Ab lysis (done w/ cellular
indicator: RBC)
4. Hemagglutination Inhibition
- Determination of secretor status
- (+) no lysis
5. ELISA
- Measurement of immune complex in
an in vitro system
ANTIHUMAN GLOBULIN TEST/ COOMBS TEST
History
-
1908
1945
Moneschi
Coombs, Mourant & Race
Introduced AHG test into
clinical medicine
AHG/ Coombs Serum
-
Green bottle
Reagent is prepared by injecting
rabbits/ goat with whole human serum
or purified serum globulin
Storage: 2-80C
Globulin Foreign Antigen (Rabbit/ Goat)
-
Stimulate the animals to respond
immunologically by synthesizing
antibody that reacts specifically with
globulin of human origin
Principles
1. Antibody molecules and complement
components are globulin
2. Injecting an animal with human globulin
stimulates the animal to produce antibody
to foreign protein
3. AHG reacts with human globulin either
bound or free in the patients serum
4. Washed red blood cells coated with human
globulin are agglutinated by AHG
Chloronoquine
EDTA glycine
Murine monoclonal antibody
II.
Indirect Antihuman Globulin Test (IAT)
-
Types of AHG Reagent
1. Polyspecific AHG Reagent
- Designed to determine if RBCs are
coated with antibody or complement
or both
- Anti- IgG, Anti-C3d, Anti-C3b
2. Monospecific AHG Reagent
- Contains only one antibody
Forms of AHG Test
I.
Direct Antihuman Globulin Test (DAT)
- Used to demonstrate RBC that have
been coated (sensitized) with antibody
in vitro
- DAT Panel: Polyspecific reagent (+)
monospecific reagent
- Use 2 drops of serum and incubate for
15 to 30 mins
Application of DAT
a. HDN
- Maternal IgG crosses the placenta &
binds to infant RBC
- 4+
b. HTR
- Recipient antibody coats donor RBC
- 1+
c. AIHA
- Autoantibody coats own RBC
d. Diagnosis of red cell sensitization reaction
caused by drugs
Diseases that may cause positive reaction to DAT
Viral pneumonia
Infectious hepatitis
Infectious
Mononucleosis
Megaloblastic Anemia
Extreme Reticulocytosis
Acute Intermittent
Purpura
Acute Hemolytic
Anemia
Lead Poisoning
Drugs that may cause positive reaction to DAT
methyldopa
Penicillin
Phenyl hydrazine
Cephalothin
Quinine
Reagents that disassociates Ag-Ab Complex
Used to demonstrate the presence of
free antibody in the patients serum
Achieved by incubation of serum with
red cells of various antigenic make up
at 370C allowing sensitization of red
cells to occur in vitro
Application of IAT
Compatibility testing
HDN
AIHA
Antibody identification
Factors affecting IAT
1. Sensitization Phase
a. Time of Incubation (15-30 mins)
b. Temperature (370C)
Serum cell mixture must be kept
at the incubated temperature until
the start of the washing step to
prevent elution of antibody
Washing of RBC: minimum of 3
times, done through centrifugation
1000 (RCFs) in 20 seconds to
remove unbound or free ab
2. Reaction Medium
a. 22% Albumin
i. Decrease zeta potential by
buffering
ii. Allows ab-coated cells to
come closer together
b. LISS (Low Ionic Strength Solution)
i. Decrease zeta potential
ii. Uses increased amount of
serum with caution
iii. 5-15 mins incubation time
c. PEG
i. Removes water
concentrating antibody
ii. Use monospecific AHG
reagent with anti IgG or it
would lead to a false
negative result
iii. Do not read microscopically
(false +)
