Tag Archives: ABO

1. 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.
2. Pipette 3 volumes of patient’s serum  the tube.
3. Add 1 volume of donor’s washed 3% red cell suspension and mix.
4. Centrifuge at slow speed e.g. at 150 g for 1 minute or 500 g for 10 seconds.
5. 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.
6. When there is no agglutination, mix the contents of the tube and incubate at 37 C for 20–30 minutes.
7. 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.
8. 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.
9. Resuspend the cells by tapping the bottom of the tube. Add 2 drops of AHG reagent and mix.
10. Centrifuge at slow speed, e.g. at 150 g for 1 minute or at 500 g for 10–15 seconds.
11. 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.
12. Enter the test results in the Blood Transfusion Records book.

1. Deterioration of reagents

Occasionally difficulties in ABO grouping are caused by using expired or contaminated reagents or incorrectly prepared or heavily contaminated
physiological saline.

Correction: Appropriate controls should be used to check the reactions of antisera and test cells.
Prepare fresh saline if contamination is suspected (check a sample microscopically).

2. Rouleaux

Rouleaux causes red cells to stack together (like piles of coins), giving the appearance of agglutination when there is no true agglutination. It can occur when
a patient has a protein abnormality, e.g. myelomatosis or when dextran, PVP, or similar product has been given intravenously. In cord blood samples, rouleaux can be caused by contamination
of the sample with gel substances such as Wharton’s jelly when applied to the cord of a newborn (wash the infant’s red cells with saline).

Marked rouleaux can cause discrepancies in serum grouping (particularly when tile grouping) and occasionally in cell grouping when using whole blood, unwashed or insufficient washed red cells.
Rouleaux can usually be distinguished from true agglutination by examining the red cells microscopically.

Correction: Whenever suspected, add a drop of saline to the cells. Rouleaux usually disperses after 1–2 minutes following the addition of saline. Serum grouping should be repeated using serum diluted 1 in 2 in saline (mix 1 drop of saline with 1 drop of serum).
This will cause rouleaux to disperse

3. Autoagglutinins

Occasionally a patient’s serum may contain autoagglutinins which are antibodies that cause the agglutination of a person’s own and other red cells (autoagglutination). They are known to occur in lymphoma, leukaemia, virus pneumonia, systemic
lupus erythematosus and other autoimmune diseases, and occasionally in severe falciparum malaria. Autoagglutination may also occur following
treatment with some herbal preparations. Cold agglutinins active at room temperature are a frequent cause of autoagglutination.
When autoagglutinins are present, a patient’s red cells appear agglutinated before commencing grouping. This can be confirmed by setting up an autocontrol.

Correction: When autoagglutination due to cold agglutinins is suspected, the cell grouping should be repeated after washing the patient’s red cells in warm saline and the serum group and autocontrol
read after incubation at 37°C. The autocontrol should then be negative.

4. Faulty technique

When it is possible that a technical error in blood grouping has occurred, repeat the cell and serum grouping (using a spun tube technique).