Coombs Test

2. ANTIGLOBULIN TEST (COOMB‟S TEST)
In some cases, a small antibody molecule such as
IgG can sensitize red blood cells but cannot
produce agglutination.
The small size of the antibody‘s molecules makes
them unable to overcome the forces that cause
red blood cells to repel one another and hence
fail to form cross-linked bridges that connect
cells.

3. In 1945, Coomb et al described a test for detecting
these non-agglutinating, coating (sensitising)
antibodies.
Later, the same test was used to demonstrate the
coating of red blood cells with complement
components as well. This test is known as the
Antiglobulin Test or Coomb‘s Test. The antiglobulin
test is performed in two ways:
The Direct Antiglobulin Test (DAT) and indirect
Antiglobulin Test (IAT).

4. THE DIRECT ANTIGLOBULIN TEST
(DAT)
The Direct Antiglobulin Test initiates the
agglutination of human red blood cells that have
already been sensitized in vivo by antibodies or
complement components.
Coomb‘s serum, containing both anti-human
globulin and anticomplement antibodies, can
detect both of these sensitized cells by inducing
visible agglutination.

5. Indications
It is indicated for the determination of
antibody coated red cells in the
haemolytic disease of newborns,
auto-immune haemolytic anaemia and
following haemolytic transfusion reactions

6. Procedure:
1. Wash the patient‘s red cells three times with normal
saline.
2. Add a volume (drop) of 3% washed red cell suspension in
a test tube.
3. Add 2 drops of Coomb‘s Reagent.
4. Mix and centrifuge for 15 seconds.
5. Re-mix the cells gently and observe for agglutination.
6. Confirm microscopically, for the presence of agglutinates
or otherwise.

7. Interpretations:
Agglutination indicates a positive test which
means that the red cells have been sensitized
in vivo either with an antibody alone or with
complement components.
A valid negative test indicates a lack of in vivo
sensitization or insufficient globulin or
complement molecules on the red cell surface
to allow detection

8. INDIRECT ANTIGLOBULIN TEST
The Indirect Coomb‘s Test is used to
demonstrate circulating antibodies in the serum,
which do not agglutinate cells suspended in
saline. This depends on the combination in vitro
of an antibody with its specific antigen. In the
indirect test, normal O+ Group red cells are
exposed to a serum suspected of containing an
antibody and are subsequently tested after
washing to see whether they have been
sensitised or otherwise.

9. Two steps are required. The first step
involves the incubation of the serum
with known O Group red cells to
allow them to become coated with
the antibody (if it is present in the
serum). The second step involves
testing for sensitised cells as in a
Direct Coomb‘s Test.

10. Indications:
1. Compatibility testing (cross match).
2. Detection and identification of irregular
antibodies.
3. Detection of antibodies, e.g. Kell, Duffy and Kidd,
etc.
4. Investigation of Immune Haemolytic Anaemias.

11. Procedure:
1. Two volumes (drops) of serum are placed in a test tube.
2. One volume (drop) of 3% red cell suspension is added to
it.
3. Mix thoroughly & Incubate at 37°C for 50 minutes.
4. Wash these cells three times with normal saline.
5. After the removal of the saline of the last wash, add 2
drops of Coomb‘s Reagent.
6. Mix and centrifuge for 15 seconds.
7. Re-mix the cells gently and observe for agglutination.
Confirm microscopically.
8. If the test is negative, add 1 drop of check cells to confirm
the validity of the Coomb‘s serum.
9. Reduce the incubation time to 10 minutes if an equal
volume of LISS is added to the patient‘s serum.

12. Interpretations:
The presence of agglutination
indicates the presence of antibodies
in the test serum that are capable of
reacting with the test cells. If a
known antiserum is used, the test
will indicate the presence of the
corresponding antigen.

13. Quality Control:
The antiglobulin serum should be checked
against known, sensitized cells.
The sensitized red cells may either be
commercially purchased or prepared in the
laboratory

14. Preparing Check Cells:
Take 1 ml serum from a D-negative patient who has already
been sensitized by exposure to D-positive fetal cells during
pregnancy/delivery.
The titre of anti-D antibodies should be at least 1/16.
Mix this serum (containing anti-D IgG) with I ml washed O
positive cells and incubate at 37°C for 30 minutes.
Wash the cells and make a 3% suspension with saline.
These IgG-coated check cells may be used to check the
validity of the Coombs Test.

15. SOURCES OF ERROR IN ANTIGLOBULIN
TESTS
False „Negative‟ Tests:
1. The test tubes or pipettes may be dirty.
2. The red cells may have been inadequately washed.
3. Proteins on the fingertips may neutralise AHG and thus a false negative
result may be obtained.
4. The incubation time was too short /too long.
5. The incubation was at a temperature that did not activate the antibody.
6. There was a delay in reading the test or in performing the test, thus
allowing the antibody to be eluted off the red cells
7. The test cells were stored improperly, causing them to loose activity.
8. The antiglobulin serum is inactive (improper storage) or it was not added at
all.
9. A change in the optimal ratio of antibody to antigen.
10. If plasma, rather than serum, was used.
11. Under-centrifugation

16. False „Positive‟ Tests
1. A presence of heavy metal ions can cause
nonspecific agglutination.
2. Bacterial contamination of the test cells due to
improper storage.
3. Refrigerated, clotted blood results in a
nonspecific binding of C4, which can react with
the anti-complement of the antiglobulin serum.
4. Over centrifugation will result in a false positive
test