What is Indirect Coombs Test ?
Introduction
This test is performed to detect presence of Rh-antibodies or other antibodies in patient’s serum in case of the following:
- To check whether an Rh-negative woman (married to Rh-positive husband) has developed Anti-Rh antibodies.
- Anti-D may be produced in the blood of any Rh- negative person by exposure to D antigen by –
- Transfusion of Rh positive blood.
- Pregnancy, if the infant is Rh-positive (if father is Rh-positive).
- Abortion of Rh-positive fetus.
Requirements
- Test tubes: (10x 75 mm)
- Pasteur pipettes
- Incubator
- Centrifuge
Specimen
Serum (It need not be a fasting sample)
Reagents
- Anti-D serum
- Antihuman serum
Additional Requirements
Coomb’s control cells :- Make pooled ‘O’ Rho (D) positive cells from at least three different O’ positive blood samples. Wash these cells three times in normal saline (The cells should be completely free from serum with no free antibodies). Make 5% saline suspension of these cells.
What is Cross Matching in Blood Bank
Procedure
- Label three test tubes as T’ (test serum,) PC (positive control) and NC (negative control).
- In the tube labeled as ‘T add two drops of test serum.
- In the tube ‘PC’ add one drop of Anti-D serum.
- In the tube ‘NC’ add one drop of saline.
- Add one drop of 5% saline suspension of the pooled ‘O’ Rho (D) positive cells in each tube.
- Incubate all 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).
- Add two drops of Coomb’s 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.
Observation
| Observation | Conclusions | |
| Positive control (PC) | a) Agglutination b) No Agglutination | a) Correctly performed test procedures b) Coombs serum may not be proper, repeat the test. |
| Negative control (NC) | It should show no agglutination, since saline does not contain Anti-D or any other antibodies. | |
| Test (serum) (T) | a) Agglutination (and if PC results are correct) b) No agglutination | a) Patient’s serum contains Anti-D b) Patíent’s serum does not contain Anti-D |