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Coombs test
Coombs test is also known as anti-globulin test. The Coombs test tests for
antibodies that may stick to the red blood cells and cause red blood cells to
die too early. It was discovered by Coombs, Mourant and Race in 1945.
Coombs reagent is antihuman globulin. It is made by injecting human
globulin into animals, which produce polyclonal antibodies specific for
human immunoglobulins and human complement system factors.
Principle of Coombs test
Red cells coated with complement or IgG antibodies do not agglutinate
directly when centrifuged. These cells are said to be sensitized with IgG or
complement. In order for agglutination to occur an additional antibody,
which reacts with the Fc portion of the IgG antibody, or with the C3b or C3d
component of complement, must be added to the system. This will form a
“bridge” between the antibodies or complement coating the red cells,
causing agglutination.
Types of Coombs test
1-Direct Coombs Test (Direct Anti-globulin Test- DAT)
The direct Coombs test is used to detect antibodies (IgG or C3) that are
stuck to the surface of red blood cells. Many diseases and drugs can cause
this. These antibodies sometimes destroy red blood cells and cause anemia.
Clinical conditions that can result in vivo coating of red blood cells with
antibody or complement or both are:
1. Hemolytic disease of the new born (HDN).
2. Hemolytic transfusion reaction (HTR)
3. Auto-immune and drug-induced hemolytic anemia.
4. Rh (-) ve pregnant women with Rh (+) ve fetus.
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Procedure of Direct Coombs Test
1. Prepare a 5 % suspension in isotonic saline of the red blood cells to be
tested.
2. With clean pipette add one drop of the prepared cell suspension to a
small tube.
3. Wash three times with normal saline to remove all the traces of serum.
4. Decant completely after the last washing.
5. Add two drops of Anti-human serum.
6. Mix well and centrifuge for one minute at 1500 RPM.
7. Re-suspend the cells by gentle agitation and examine macroscopically
and microscopically for agglutination.
2-Indirect Coombs Test (Indirect Anti-globulin Test- IAT)
The indirect Coombs test looks for free-flowing antibodies against certain
red blood cells. It is most often done to determine if you may have a reaction
to a blood transfusion.
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This is the test that is done on the mother’s blood sample as part of her
prenatal labs. Frequently referred to as the “antibody screen”, this test
identifies a long list of minor antigens that could either cause problems in
the newborns or cause problems in the mother if transfusion is necessary.
Approximately 5% of patients have a positive IAT due to IgG antibodies,
IgM antibodies, or both.
Indirect-Coombs-Test
Procedure of Indirect Coombs Test
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 1 drop of anti D
serum.
4. In the test tube labeled as NC (Negative control), take 1 drop of
normal saline.
5. Add one drop of 5 % suspension of the pooled Rho (D) positive cells
in each tube.
6. Incubate all the three tubes for one hour at 37°C.
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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
Keep for 5 minutes and then centrifuge at 1,500 RPM for one minute.
9. Re-suspend the cells and examine macroscopically as well as
microscopically. Result Interpretation of Coombs Test
Negative Result:
No clumping of cells (no agglutination). This means you have no antibodies
to red blood cells.
Positive Result:
Clumping (agglutination) of the blood cells during a direct Coombs test
means that you have antibodies on the red blood cells and that you may have
a condition that causes the destruction of red blood cells by your immune
system (hemolysis). This may be due to
Hemolytic anemia.
Chronic lymphocytic leukemia or similar disorder.
Erythroblastosis fetalis (hemolytic disease of the newborn).
Infectious mononucleosis.
Mycoplasma infection.
Syphilis.
Systemic lupus erythematosus.
Transfusion reaction, such as one due to improperly matched units of
blood.
