Coomb's test

Coomb's test: Introduction, principle, application, clinical significance, requirements, procedure, interpretation and result.

1. COOMB’s TEST By: Nityanand Upadhyay Associate Professor Department of MLT Integral University, Lucknow

2. This is also known as Anti-Globulin Test. Antiglobulin serum ( Coombs’Serum ) was discovered by Coombs etal in 1945. Coomb’s test is an Investigation done for identification of anti-D antibody or Rh antibody. The Rh - antibody is not naturally occurring antibody Introduction

3. The antiglobulin test can be used to detect red cells sensitized with IgG alloantibodies , IgG autoantibodies or components.

4. 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. PRINCIPLE

5. 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.

6. Two Types 1. Direct Antiglobulin Test/ Direct Coomb’s (DCT). 2. Indirect Antiglobulin Test/ Indirect Coomb’s Test (ICT). Types

7. This test is performed to detect anti-D antibody attached with RBC,s to get sensitized . The anti-D attached with RBC’s are found in certain clinical conditions. The DCT is performed in - DCT

8. 1.HDN/ Erythroblastosis . 2. Transfusion Reaction. 3. Drugs Induced sensitized RBC. 4. Autoimmune Haemolytic Anaemia

9. DCT

10. Test Tubes (10x75mm) Centrifuge NS Micropipettes Tips Incubator REQUIRMENTS

11. Specimen: 2 Ml. Blood drawn into EDTA is preferred but oxalated , or clotted, citrated whole blood may be used (specimen need n.ot be fasting sample). Reagent: AHG (Anti Human Globulin)/ Coomb’s .

12. 1. Prepare a 5 % suspension in isotonic saline of the red blood cells to be tested. 2. With clean Pasture 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. PROCEDURE

13. 4. Decant completely after the last washing 5. Add two drops of Antihuman serum (AHG). 6. Incubate at 37 Centigrade for 5-10 min. 6. Mix well and centrifuge for one minute at 1500 RPM. 7. Resuspend the cells by gentle agitation and examine macroscopically and microscopically for agglutination.

14. Looking for Agglutination Reaction INTERPRETATION

15. 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 ).  RESULT

16. Hemolytic anemia, Chronic lymphocytic leukemia or similar disorder, Erythroblastosis fetalis (hemolytic disease of the newborn), Infectious mononucleosis, Mycoplasmal infection, Syphilis, Systemic lupus erythematosus and Transfusion reaction, such as one due to improperly matched units of blood. POSTIVE CASES IN…

17. The ICT is used to detect presence of Rh -antibody in patient serum. e.g. 1. The Rh-ve women married with Rh+ve husband. 2. The Rh-ve person received Rh+ve Blood. ICT(INDIRECT COOMB’s TEST

18. 3. Rh-ve mother carry Rh+ve featus . 4. Abortion of Rh+ve featus .

19. Test Tubes (10x75mm) Centrifuge NS Micropipettes Tips Incubator REQUIRMENTS

20. Specimen: Serum (need not be fasting) Reagents: 1. Antihuman serum 2. Anti-D serum Additional Requirements: Coombs control cells A. Make a pooled ‘O’ Rho (D) positive cells from at least three different ‘O’ positive blood samples. B. Wash these cells three times in normal saline (these cells should be completely free from serum with no free antibodies).

21. Label three test tubes as T (test serum) PC (Positive control) and NC (negative control). In the tube labeled as T (Test), take 2 drops of test serum. In the test tube labeled as PC (Positive control), take 1 drop of anti D serum. In the test tube labeled as NC (Negative control), take 1 drop of normal saline. PROCEDURE

22. Add one drop of 5 % saline suspension of the pooled ‘O’ Rho (D) positive cells in each tube. Incubate all the three tubes for one hour at 37°C. 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).

23. 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. Resuspend the cells and examine macroscopically as well as microscopically.

24. Look for agglutination reaction 1. PC Tube- Agglutination ( Test correctly performed ). No agglutination( Coombs serum may not be proper. Repeat the test again.) INTERPRETATION

25. 2. Negative control (NC)- It should show no agglutination, since saline does not contain Anti-D or any other antibodies. 3. Test (Serum) (T). a. Agglutination (and if PC results are correct)- Patients serum contains AntiD . b. No Agglutination- Negative.

26. Thanks…………..