BASIC
IMMUNOHAEMATOLOGY
TRAINING
BASIC IMMUNOLOGY:
ANTIBODIES/ANTIGENS
BLOOD GROUPS
THE PRINCIPLE OF THE GEL TECHNOLOGY
COMMON IMMUNOHAEMATOLOGY TESTS
BASIC IMMUNOLOGY
The immune system comprises a series of
mechanisms responsible for distinguishing
‘self’ from ‘non-self’, and for mounting
defence against infectious
agents and other foreign substances.
The Immune response
Historically, the immune system was separated
into two branches: Humoral immunity, for which
the protective function of immunization could be
found in the humor (cell-free bodily fluid or serum)
and Cellular immunity, for which the protective
function of immunization was associated with cells.
T cells provide protection against different
pathogens.
BASIC IMMUNOLOGY
Humoral Component:
– Essentially comprised of antibody proteins
and the complement system.
– Immunohaematology is predominately
concerned with this branch of the immune
system.
BASIC IMMUNOLOGY
Antigen: any substance that
causes the immune system to
mount a response by producing
antibodies against it.
Antibody: a specific protein
produced by the immune system
when it detects a foreign substance
or antigen.
BASIC IMMUNOLOGY
Antibody Sub-classes
BASIC IMMUNOLOGY
It is the IgG and IgM antibodies and their reactions with red blood cell
antigens that form the basis of blood banking.
THE ABO BLOOD GROUP
SYSTEM
The first human blood group antigen system to be discovered and
remains the single most important system in transfusion.
In the transfusion medicine laboratory it is essential to determine an
individual’s blood group in order to ensure a compatible product is
provided.
Almost all normal, healthy people older than three months of age have
naturally occurring antibodies to the ABO antigens they lack.
These antibodies (generally IgM) can readily lead to life-threatening
haemolysis if the incorrect blood group is transfused.
The ABO Blood Group Forward
and Reverse
Blood Anti-A Anti-B A cells B cells
Group Reagent Reagent Reagent Reagent
A + 0 0 +
B 0 + + 0
O 0 0 + +
AB + + 0 0
The Rh System
• Comprises Antigens D, C, c, E, e
• Rh D is the most important as it is highly
immunogenic - 40% of exposures likely to
produce anti-D
• Antigen D is present on the red cell, then
Rh Positive e.g. A Pos
• Antigen D is absent on the red cell, then
Rh Negative e.g. O Neg
Variations of D
• Some individuals have weakened or
variant expressions of D antigen
• Patients with ? D variant or D weak
treated as Rh negative
• Donors with ? D variant or D weak treated
as Rh Positive.
• Anti-D Clones differ for patient (VI-) and
donor (VI+) testing
Other Red Cell Antigen Groups
Gel Technology
In the case of red cell antigens and antibodies, these interactions cause a
phenomenon known as HAEMAGGLUTINATION or clumping of the red
cells. We are able to visualise this reaction between the antibody and the
antigen.
The principle of gel technology
• The gel acts as a sieve or density gradient
• Antibodies added to the gel supernatant agglutinate
the red cells
• Under specified centrifugal conditions:
• Large agglutinates remain on or near the
interface of the gel/supernatant
• Smaller agglutinates pass down the column of gel
depending on size
• Non- agglutinated cells pass to the bottom of the
gel
THE AHG TEST
AHG Test = Anti-Human Globulin test
The AHG test is also known as the Coombs test
Incubation of cells with plasma promotes
sensitization – Ab/Ag attachment.
AHG reagent enhances crosslinking of Antibody
bound to RBCs.
Production of the AHG reagent
THE INDIRECT AHG TEST
THE INDIRECT AHG TEST
The IAHG test can be used to detect either red cell
antigens or red cell antibodies.
The IAHG test is utilised in the laboratory for numerous
tests:
– Antibody detection
– Antibody identification
– Compatibility testing (Crossmatching)
– Antigen typing
– Antibody titration
THE DIRECT AHG TEST
THE DIRECT AHG TEST
The DAT is designed to demonstrate the presence of an
antibody or complement, or both, that has caused in vivo
sensitisation of the red blood cells.
The DAT is useful in investigating the following disorders:
– Autoimmune haemolytic anaemia (AIHA)
– Haemolytic disease of the newborn (HDN)
– Haemolytic transfusion reactions
– Drug-induced haemolytic anaemia
Group & Reverse
Most commonly used grouping
card. Used for pre-transfusion
and antenatal testing.
Forward grouping tests
patient cells against reagent
Anti-A, Anti-B, and
Anti-D.
Reverse grouping tests patient
plasma against reagent A cells
and B cells.
Combined reaction pattern used
for interpretation.
Group Check
Used for first presentation of
patient to confirm ABO/D
typing. Different anti-A, anti-B
and Anti-D to the Group and
Reverse card.
Newborn & Rh/Kell
Babies of Rh(D) Negative Mothers.
Mother of Rh(D) Positive baby will
require Anti-D injection to mop up
Rh(D) Positive cells that may have
crossed into her circulation at
delivery.
Newborn card has two Anti-D
antibodies as even incomplete
D antigen can stimulate a response.
Also includes Direct Coombs to test
for sensitization of babies cells.
HDN – haemolytic disease of the
Newborn.
Beside ABO/Rh(D), most common
antigen test is Rh antigens C,c,E,e
and Kell.
Antibody Screens
Pre-transfusion and
antenatal testing.
Screening cells are Group
O, so will
not react with ABO
antibodies.
Rh(D) Negative mothers
have repeat
screens through
pregnancy.
Antibody Screens
For Positive Antibody Screen tests, reaction pattern from the screening cells can give
some indication of the Antibody specificity, but is not conclusive.
=> Antibody Identification Panel is the next step.
Antibody Identification Panel
Antibody Identification Panel
Antibody specificity is derived from the reaction pattern by a process
of elimination. Sometimes multiple antibodies are present, requiring
different detection techniques and/or alternative panels.
Once antibodies identified from panel results, related antigen test(s)
performed on patient red cells to confirm absence of antigen.
Crossmatch
Directly tests compatibility of
donor
red cells with recipient plasma.
Many laboratories use ‘Computer
Crossmatching’ for most patients
– requires specific LIS criteria.
Full crossmatch always required
if an antibody is detected or there
is a history of antibodies.
Direct Coombs
Tests for the presence of in
vivo sensitisation of red cells.
Can be a polyspecific screen
or monospecific Anti-IgG and
Anti-C3d.
