Company The Antiglobulin Test

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( Direct & Indirect )
 The Antiglobulin Test

• Antiglobulin serum (Coombs’Serum) was

discovered by Coombs et. al. in 1945.
• The antiglobulin test can be used to detect red
cells sensitized with IgG alloantibodies, IgG
autoantibodies or complement components.
• Sensitization of red cells can occur in vivo or vitro.
The use of AHG serum to detect sensitization of
red cells in vitro is a two stage technique known as
indirect antiglobulin test (IAT). The sensitization of
red cells in vivo is detected by one stage
technique the direct antiglobulin test (DAT).
 PRINCIPLE
• Washed red cells coated with IgG and / or
complement components C3b or C3d will
show agglutination with broad spectrum
AHG serum.
• The coating (sensitization) of red cells can
occur in vivo or in vitro following
incubation at 37°C with serum containing
antibody.
 PRINCIPLE
• The incomplete antibodies (IgG) attach to red cell
membrane by the Fab portion of the immunoglobulin
molecule (IgG).
• The IgG molecules attached to the red cells are unable
to bridge the gap between sensitized red cells which are
separated from each other by the negative charge on
their surface and the sensitized red cells do not
agglutinate.
• When AHG serum is added to the washed sensitized
cells, the Fab portion of the AHG molecule (anti-IgG)
reacts with the Fc portions of two adjacent IgG
molecules attached to red cells thereby bridge the gap
between sensitized red cells and cause agglutination.
DIRECT ANTIGLOBULIN TEST
(DAT)
 DAT

• The direct antiglobulin test (DAT) detects

sensitized red cells with IgG and/or
complement components C3b and C3d in
vivo.
• In vivo coating of red cells with IgG and/or
complement may occur in:
• (1) Hemolytic disease of newborn (HDN)
• (2) Autoimmune hemolytic anemias (AIHA)
• (3) Drug induced hemolytic anemias (AIHA)
• (4) Hemolytic transfusion reaction (HTR)
 Blood Sample

• Blood Sample - It should be as fresh as

possible not more than 24 hours old,
otherwise,
• the sample should be taken in EDTA (1.5
mg EDTA for I ml of blood) to protect the
uptake of complement.
 Procedure of DAT
1. Take 2-3 drops of blood to be tested in a clean labeled tube.
2. Wash the red cells 3-4 times in a large volume of saline to
remove free globulin molecules. Remove all supernatant
after each wash. Completely decant the final supernatant
wash.
3. Add 2 drops of polyspecific AHG serum in 1 drop of
sensitized washed red cells or in 1 drop of 3-5 % suspension
of sensitized cells immediately.
4. Mix, Centrifuge at 1000 rpm for 1 minutes immediately.
5. Gently shake the tube to dislodge the cell button and see for
agglutination, use optical aid if needed. Record the result.
6. Add 1 drop of IgG coated red cells to a negative test. Mix,
centrifuge at 1000 rpm for 1 min. Immediately look for
agglutination. If a negative result (no agglutination) is
obtained the test result is invalid and whole test should be
repeated. If agglutination is obtained, the result is valid.
 IAT
• Indications
• The IAT is done to determine the presence of
sensitization of red cells with IgG and/or
complement in vitro in the following conditions.
• TRANSFUSION MEDICINE Technical Manual
1. Compatibility testing.
2. Screening and detection of unexpected antibodies
in serum.
3. Determination of red cells phenotype K, Lea, Fya
Fyb, Jka, Jkb and sub-group of Rh etc by using
known sera.
Indirect antiglobulin test
 Procedure:
1. Place 2-3 drops of the test serum in a tube. Serum should
be fresh for detecting complement components and
complement binding antibodies, otherwise, fresh AB serum
should be added to it.
2. Add 1 drop of 3-5% suspension of washed O Rh (D) positive
red cells to the serum in the tube.
3. Mix and incubate at 37°C for 30-40 minutes.
4. Centrifuge at 1000 rpm for 1 minutes.
5. Examine for hemolysis and/or agglutination. Use optical aid
if necessary. Agglutination at this stage indicates the
presence of (complete) antibodies.
6. If no agglutination is seen, wash cells 3-4 times in large
volume of saline. Decant supernatant in each wash as
completely as possible.
 Procedure:
7. Add 2 drops of AHG serum to the cells.
8. Mix and centrifuge at 1000 rpm for 1 minutes
immediately.
9. Gently shake the tube to dislodge the button and
examine for agglutination, using optical aid.
Record the result.
10. Add 1 drop of IgG coated red cells to any test
that is negative. Mix and centrifuge at 1000 rpm
for 1 minutes. Look for agglutination. If there is no
agglutination, the test result is invalid and the
whole test is repeated. If agglutination is obtained
the result is valid.
Source of errors in
coombs test
False negative results:-
 Failure to wash red blood cells adequately, since globulins not bound
to RBCs will neutralize the AHG reagent.
– The washing process and the addition of AHG reagent must be
undertaken as quickly as possible to minimize loss of bound
antibodies by elution.
 Improper storage, bacterial contamination and contamination with
human serum will impair the AHG reagent activity.
• Not adding the AHG reagent
• Improper centrifugation
• Number of cells present in the test:
– too many cells give weak reactions
– too few cells will impair the reading of the agglutination
False positive results:

DAT and IAT ;

In specimens containing potent cold-reactive antibodies
agglutination may occur before adding the AHG reagent.
Dirty glassware may cause clumping of cells.
Over centrifugation