- Label a small (e.g. 75×12 mm) clean glass tube with the number of the donor blood and write this number also on the patient’s blood transfusion request form.
- Pipette 3 volumes of patient’s serum the tube.
- Add 1 volume of donor’s washed 3% red cell suspension and mix.
- Centrifuge at slow speed e.g. at 150 g for 1 minute or 500 g for 10 seconds.
- Tilting the tube back and forth, examine for haemolysis or agglutination. Haemolysis or agglutination means that the donor blood is ABO incompatible. The blood MUST NOT BE GIVEN TO THE PATIENT. When there is haemolysis or agglutination, recheck the ABO group of the patient and donor blood and also check that the correct patient’s blood sample has been tested.
- When there is no agglutination, mix the contents of the tube and incubate at 37 C for 20–30 minutes.
- Centrifuge at slow speed. Tilting the tube back and forth, examine for haemolysis or agglutination. Haemolysis or agglutination indicate that the blood is incompatible and must not be given to the patient.
- When there is no haemolysis or agglutination, perform an indirect AHG test. Fill the tube with saline, centrifuge (high speed), and remove the supernatant fluid. Wash the cells a further 3 times. At the end of the final wash remove all the supernatant fluid. Careful washing of the cells is essential. Traces of globulin left in the tube will neutralize the AHG reagent.
- Resuspend the cells by tapping the bottom of the tube. Add 2 drops of AHG reagent and mix.
- Centrifuge at slow speed, e.g. at 150 g for 1 minute or at 500 g for 10–15 seconds.
- Tilting the tube back and forth, look for agglutination. When no agglutination is seen, transfer a few of the cells to a slide and check for agglutination microscopically using the 10 objective. When there is no agglutination, check that the AHG has not been neutralized by adding 1 drop of AHG control sensitized cells to the tube. Repeat steps 10–11. The control cells will show agglutination, providing the AHG is active and the test has been performed correctly. When there is agglutination after adding AHG reagent, this means that the patient’s serum contains an immune IgG antibody reactive against the donor’s cells which may cause a transfusion reaction.
- Enter the test results in the Blood Transfusion Records book.
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