21 Tube Tests for ABO Typing

Principle

In red cell (forward) grouping, the presence or absence of ABO antigens is determined by testing the patient cells with anti-A, anti-B, anti- A,B and observing for agglutination. Absence of agglutination is a negative test result, which indicates that the corresponding antigen is not demonstrable. Agglutination of red blood cells with a given reagent is a positive test result, which indicates the presence of the corresponding antigen on the red blood cells. Serum (reverse) grouping demonstrates the presence ( agglutination )or absence(lack of agglutination) of the expected ABO blood group antibodies (anti-A and/or anti-B) by testing the serum or plasma with  A1 and B cells

Specimen

Generally, clotted or anti-coagulated samples may be used. Patient red cells can be suspended in the patients’ serum or plasma, or saline. Washed cells suspended in saline can also be used. Prepare a 2-5% red cell suspension.

Reagents

1. Monoclonal or polyclonal anti-A.
2. Monoclonal or polyclonal anti-B.
3. Anti-A, B.
4. A1 and B red cells.

Procedures

For red cell testing (also may be referred to as forward grouping or direct grouping).

1. Label tubes with patient initials, MRN and reagent identification (antisera).
2. Dispense 1 drop of anti-A into a clean, labelled test tube.
3. Dispense 1 drop of anti-B into a clean, labelled test tube.
4. Dispense 1 drop of anti-A, B into a clean, labelled test tube.
5. Add 1 drop of the 2-5% cell suspension of patient red cells to each tube.
6. Mix the contents of the tubes thoroughly but gently.
7. Centrifuge tubes for 20 seconds.
8. Gently re-suspend the cell button and examine macroscopically.
9. Grade and record results immediately while holding tube in your hand.
10. Confirm results with reverse grouping.

For serum/plasma testing (also may be referred to as reverse grouping). This test is omitted in neonates.

1. Label tubes with patient initials, MRN  and reagent  (A1 or B cells).
2. Add 2 drops of patient serum/plasma to two clean, labelled test tubes.
3. Add 1 drop of A1 cells to the tube labelled A1.
4. Add 1 drop of B cells to the tube labelled B.
5. Mix the contents of the tubes thoroughly but gently.
6. Centrifuge tubes for 20 seconds.
7. Gently re-suspend the cell button and examine macroscopically.
8. Grade and record results immediately while holding tube in your hand.
9. Verify results are expected/ align with the forward red cell testing.

Interpretation

1. Agglutination of tested red cells and agglutination in tests on serum/plasma constitutes positive results and indicates the presence of the corresponding antibody or antigen.
2. A smooth cell suspension after re-suspension of the cell button is a negative result and indicates that the presence of the corresponding antigen or antibody is not demonstrable.
3. Any discrepancy (anomaly) between cell and serum/plasma testing should be investigated and resolved.

Notes

1. Anti-sera is ALWAYS added to tubes first as part of a quality routine (makes it easier to verify the addition especially of colorless anti-sera)
2. Positive reactions usually are 3+ to 4+ agglutination with reagent antibodies.
3. Reactions between patient plasma and reagent red cells (reverse grouping) are generally weaker than cell typing (forward grouping) agglutination.
4. Weak reactions (weak or 1+) in the serum testing may be enhanced by:

• • Room temperature incubation for 5-15 minutes.
  • Cold enhancement techniques- place the tubes at 4 degrees Celsius (include an auto tube and group O cells as controls for cold allo/auto antibodies).