Coomb’s Test- Direct and Indirect Coomb’s Test

• Coomb’s test is a direct agglutination reaction, more commonly known as antiglobulin test.
• It was discovered by Coombs, Mourant and Race in 1945 originally for the detection of incomplete anti-Rh antibodies.
• In the test, incomplete antibodies do not agglutinate erythrocytes. Incomplete antibody antiglobulin coats the surface of erythrocytes but does not cause any agglutination.
• When such erythrocytes are treated with antiglobulin or Coombs’ serum then the cells are agglutinated.
• Coombs’ Serum or Coomb’s reagent is a special serum from a rabbit or other animal previously immunized with purified human globulin to prepare antibodies directed against IgG and complement (eg. rabbit antiserum against human globulin).
• It is used in the direct and indirect Coomb’s tests and also called antihuman globulin.

Objectives of Coomb’s Test

To detect red blood cells sensitized with IgG alloantibodies, IgG autoantibodies or complement components.

Principle of Coomb’s Test

Under certain conditions, complement proteins or more commonly, incomplete antibodies (IgG) attach to red cell membrane by the Fab portion of the immunoglobulin. These cells are said to be sensitized. Sensitization of red cells can occur in vivo or vitro.  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 as a result of which the sensitized red cells do not agglutinate.

Adding of the Coomb’s reagent (antiglobulin serum) however, completes the reaction. When the serum is added to the sensitized cells, the Fab portion of the anti-human globulin 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.

Thus, if human IgG antibody has already attached to the patient’s red cells in vivo (in the bloodstream), or the patient serum contains incomplete antibodies that can attach to RBCs in-vitro, then the addition of anti-human IgG will cause the cells to agglutinate. This is a positive Coomb’s test.

Types of Coomb’s Test

There are two types of Coombs tests: the direct Coomb’s test and the Indirect Coomb’s test.

Direct Coomb’s Test (Direct Antiglobulin Test)

• The direct test is more common and checks for antibodies that are attached to the surface of red blood cells.
• In this test, the sensitization of red blood cells (RBCs) with incomplete antibodies takes place in vivo.
• The cell-bound antibodies can be detected by this test in which antiserum against human immunoglobulin is used to agglutinate patient’s red cells.

Indirect Coomb’s Test (Indirect Antiglobulin Test)

• The indirect test checks for unattached antibodies that are floating in the bloodstream.
• In this test, the sensitization of RBCs with incomplete antibodies takes place in vitro.
• The patient’s serum is mixed with normal red cells and antiserum to human immunoglobulin is added. Agglutination occurs if antibodies are present in the patient’s serum.

Requirements for Coomb’s Test

Test tubes, centrifuge, Anti-human globulin (AHG) reagent, pre-sensitized red cells (Coombs’ control cells), Saline.

Procedure of Coomb’s Test

The use of antihuman globulin serum to detect sensitization of red cells in vitro is a two stage technique constitute indirect antiglobulin test (IAT). On the other hand, sensitization of red cells in vivo is detected by one stage technique – the direct antiglobulin test (DAT).

Direct Coomb’s Test

1. Red cells suspected of being sensitized is washed 3 to 4 times in large volume of saline.
2. Two drops of anti-human globulin serum is added to the sedimented cells.
3. It is mixed well and centrifuged at 1500 rpm for one minute.
4. Agglutination is examined by holding against a lighted background and tapping the bottom of the tube.
5. If the agglutination is not seen, the tube is left at room temperature for 10 min. then re-centrifuged and read. A weaker reacting antibody may will show delayed reaction and this is considered as positive.
6. If haemagglutination is not seen in step 5, one drop of presensitized red blood cells (5% suspension is saline) is added. This should result in haemagglutination of pre-sensitized cells indicating that the antihuman globulin (AHG) is reactive and the result is valid.

Indirect Coomb’s Test

1. 4% saline suspension of the test cells is prepared.
2. Two drops of cell suspension is added to a small test tube.
3. Two drops of antiserum is added to the cell suspension.
4. It is incubated in a water bath at 37°C for 30 min.
5. The tube from the water bath is removed and washed 3 to 4 times with large volume of saline. It is completely decanted after last washing.
6. Two drops of anti human globulin (AHG) is immediately added and mixed well.
7. It is centrifuged at 1500 rpm for one minute and examined for haemagglutination.
8. In case of negative haemagglutination, pre-sensitized reagent cells is added to test the reactivity of AHG. Agglutination must be seen with the addition of Coombs’ control cells.

Result Interpretation of Coomb’s Test

Positive:  A clumping of the red blood cells (agglutination) during the test.

Agglutination of blood cells during a direct Coomb’s test suggests that antibodies may be present on red blood cells of the patient and that the condition of hemolysis may persist.

Agglutination of blood cells during an indirect Coomb’s test suggest the presence of antibodies circulating in bloodstream that could cause the immune system to react to any red blood cells that are considered foreign to the body — particularly those that may be present during a blood transfusion.

Negative: No clumping or agglutination of red blood cells.

Applications of Coomb’s Test

1. Coomb’s test is one of the blood tests employed to help find out the kind of anemia an anemic patient is suffering from.
2. Indirect test is administered to determine if there was a potential bad reaction to a blood transfusion.
3. Blood banks use the indirect Coombs test to determine whether there is likely to be an adverse reaction to blood to be transfused. 
4. Coombs’ tests are used for detection of anti-Rh antibodies.
5. It is also used to detect incomplete antibodies in brucellosis and other diseases.
6. The indirect Coombs test is used in prenatal testing of pregnant women.
7. The test is done on the newborn’s blood sample, usually in the setting of a newborn with jaundice.
8. It helps in the detection of conditions like hemolytic anemia, chronic lymphocytic leukemia, erythroblastosis fetalis, infectious mononucleosis, mycoplasmal infection, syphilis, systemic lupus erythematosus etc.

Limitations of Coomb’s Test

• Sometimes, especially in older adults, a Coomb’s test will have an abnormal result even without any other disease or risk factors.
• The test can only be rarely used to diagnose a medical condition.

References

1. http://www.shareyouressays.com/knowledge/useful-notes-on-coombs-test/115418
2. Coombs, R. R.; Mourant, A. E.; Race, R. R. (1945). “A new test for the detection of weak and incomplete Rh agglutinins”. British journal of experimental pathology. 26: 255–66. 
3. Lydyard, P.M., Whelan,A.,& Fanger,M.W. (2005).Immunology (2 ed.).London: BIOS Scientific Publishers.
4. Parija S.C. (2012). Textbook of Microbiology & Immunology.(2 ed.). India: Elsevier India.
5. Sastry A.S. & Bhat S.K. (2016). Essentials of Medical Microbiology. New Delhi : Jaypee Brothers Medical Publishers.