COOMBS TEST
Antiglobulin serum (Coombs’ Serum) was discovered by
Coombs 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-vivo or in-vitro can be:
One stage technique , the direct Antiglobulin test
(DAT).
Two stage technique , the indirect Antiglobulin test
(IAT).
PRINCIPLE
Normal human red blood cells, in presence of
antibody directed towards the antigen they
possess, may fail to agglutinate when centrifuged
and become sensitized.
This may be due to the particular nature of the
antigen and antibody involved.
This will form a "bridge" between the antibodies or
complement coating the red cells, causing
agglutination.
The coating (sensitization) of red cells can occur in
vivo or in vitro following incubation at 37°C with
serum containing antibody.
COOMBS FORMATION
May be made by injecting rabbits , goats or sheep
with purified human IgG or C3, then harvesting the
antibodies produced by the rabbit.
Monoclonal technology may be used to make
monoclonal antiglobulin reagent.
Types of AHG reagent
Polyspecific Anti-human Globulin: Anti-IgG and
Anti-C3b, -C3d
Monospecific reagents: Anti-IgG alone or Anti-
C3b,-C3d alone
APPLICATION OF DAT
This test is performed to detect anti- D Ab or other
Ab attached to red cell surface within the blood
stream.
This occurs in in following conditions
APPLICATION OF 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.
BLOOD SAMPLE
Whole Blood Sample - It should be as
fresh as possible not more than 24
hours old,
otherwise, the sample should be taken
in EDTA.
Clotted sample for Serum
DIRECT ANTIGLOBULIN TEST (DAT)
PROCEDURE OF DAT
1. Prepare a 5 % suspension.
2. Add one drop of the prepared 5% cell suspension into test tube
3. Wash three times with normal saline to remove all the traces of serum.
4. Decant completely to obtain dry button of RBCs
5. Add two drops of Anti-human serum/ coombs serum
6. Mix well and centrifuge for 20 seconds at 3400 RPM
7. Resuspend the cells by gentle agitation and examine macroscopically
and microscopically for agglutination, use optical aid if needed,
Record the result.
8. Add 1 drop of check cells to a negative test. Mix, centrifuge at 3400
rpm for 15 seconds. 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.
PROCEDURE OF IAT
PROCEDURE OF IAT
1. Label three test tubes as T (test serum) PC (Positive control) and NC (negative control).
2. In the tube labeled as T (Test), take 2 drops of test serum.
3. In the test tube labeled as PC (Positive control), take 2 drops of test serum
4. In the test tube labeled as NC (Negative control), take 2 drop of normal saline.
5. Add one drop of 5 % saline suspension of the ‘O’ Rho (D) positive cells in each tube.
6. Incubate all the three tubes for one 30 minutes at 37°C.
7. Wash the cells three times in normal saline to remove excess serum with no free antibodies, (in the
case of inadequate washings of the red cells, negative results may be obtained).
8. Add two drops of Coombs serum (anti human serum) to each tube.
9. Centrifuge at 3400 RPM for 20 seconds.
10. In the test tube labeled as PC (Positive control), add one drop of check cells.
11. Resuspend the cells and examine macroscopically as well as microscopically.
RESULT INTERPRETATION
• Positive Result:
• Agglutination indicates a Positive Coombs Test.
• Negative Result;
• No agglutination indicates a negative Coombs Test.
• Agglutination with Grading
• 4+ = Single large clump, No free cells, Clear back ground.
• 3+ = Several large clumps, no or occasional free red cells, clear back ground.
• 2+ = Medium size clumps, many free red cells, turbid back ground.
• 1+ = Small size clumps, too many free red cells, turbid back ground.
• Weak Positive = Few tiny clumps, almost all free cells, turbid background.
SOURCES OF ERROR IN AHG TESTS
•False negative results: General DAT & IAT
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
FALSE POSITIVE RESULTS:
DAT
In specimens containing potent cold-reactive
antibodies agglutination may occur before adding the
AHG reagent.
Dirty glassware may cause clumping of cells.
Over centrifugation
• .
Rh D ANTIBODY TITRATION
Antibody titration is a semi-quantitative means of assessing the
amount of antibody in the serum.
This is usually done in Rh-incompatible mothers.
It should be done after detection of the antibody by IAT
PROCEDURE OF RH D ANTIBODY
1. Set up 12 test tubes in a rack. Label them as 1/1, 1/2, 1/4,1/8, 1/16, 1/32, 1/64, 1/128,
1/256, 1/512 and 1/1024.
2. Add 2 drops of saline in each starting from the second (1/2) tube.
3. Add 2 drops of the patient's serum in the first and second tubes.
4. Mix well and transfer 2 drops from the 2nd test tube to the third. Mix well and transfer 2
drops to the next tube and so on until the last tube is reached.
5. Discard 2 drops from the last tube.
6. Add one drop of 3-5% known O Rh D positive cell suspension in each tube and centrifuge
for 15 seconds at the rate of 3400 RPM. Look for agglutination.
7. Incubate the test tubes at 37°C for 30 min.
8. Wash the cells three times with normal saline.
9. After the last wash, add 2 drops of Coomb‘s serum in all of the test tubes.
10. Centrifuge for 20 seconds at 3400 rpm and read the results and observe for
agglutination
CONTINUED….
• Interpretations:
The highest dilution showing agglutination indicates the titer of the
antibody in the serum.
While reporting the titer, the dilution prior to the highest one
showing agglutination is reported.
