1. The Antiglobulin Test ( Direct & Indirect )
Islamic University-Gaza
The Antiglobulin Test ( Direct & Indirect )
Mr. Mohammed A. Jaber

2. The Antiglobulin Test
Antiglobulin serum (Coombs’Serum) was discovered by Coombs etal 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).

3. 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.

4. 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.

5. DIRECT ANTIGLOBULIN TEST (DAT)

6. 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)

7. 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.

8. Procedure of DAT
Take 2-3 drops of blood to be tested in a clean labeled tube.
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.
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.
Mix, Centrifuge at 1000 rpm for 1 minutes immediately.
Gently shake the tube to dislodge the cell button and see for agglutination, use optical aid if needed. Record the result.
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.

9. 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
Compatibility testing.
Screening and detection of unexpected antibodies in serum.
Determination of red cells phenotype K, Lea, Fya Fyb, Jka, Jkb and sub-group of Rh etc by using known sera.

10. Indirect antiglobulin test

11. Procedure:
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.
Add 1 drop of 3-5% suspension of washed O Rh (D) positive red cells to the serum in the tube.
Mix and incubate at 37°C for minutes.
Centrifuge at 1000 rpm for 1 minutes.
Examine for hemolysis and/or agglutination. Use optical aid if necessary. Agglutination at this stage indicates the presence of saline (complete) antibodies.
If no agglutination is seen, wash cells 3-4 times in large volume of saline. Decant supernatant in each wash as completely as possible.

12. Procedure: Add 2 drops of AHG serum to the cells.
Mix and centrifuge at 1000 rpm for 1 minutes immediately.
Gently shake the tube to dislodge the button and examine for agglutination, using optical aid. Record the result.
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.
Auto control should be kept with IAT.

14. BOVINE ALBUMIN(22%)-IAT
One Stage Method - Additive method
Procedure:
Two drops of albumin 22.5% are added in step (2) of saline-IAT
Mix and incubate for minutes at 37°C
Proceed further as in saline-IAT procedure.