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
- Monoclonal or polyclonal anti-A.
- Monoclonal or polyclonal anti-B.
- Anti-A, B.
- A1 and B red cells.
Procedures
For red cell testing (also may be referred to as forward grouping or direct grouping).
- Label tubes with patient initials, MRN and reagent identification (antisera).
- Dispense 1 drop of anti-A into a clean, labelled test tube.
- Dispense 1 drop of anti-B into a clean, labelled test tube.
- Dispense 1 drop of anti-A, B into a clean, labelled test tube.
- Add 1 drop of the 2-5% cell suspension of patient red cells to each tube.
- Mix the contents of the tubes thoroughly but gently.
- Centrifuge tubes for 20 seconds.
- Gently re-suspend the cell button and examine macroscopically.
- Grade and record results immediately while holding tube in your hand.
- Confirm results with reverse grouping.
For serum/plasma testing (also may be referred to as reverse grouping). This test is omitted in neonates.
- Label tubes with patient initials, MRN and reagent (A1 or B cells).
- Add 2 drops of patient serum/plasma to two clean, labelled test tubes.
- Add 1 drop of A1 cells to the tube labelled A1.
- Add 1 drop of B cells to the tube labelled B.
- Mix the contents of the tubes thoroughly but gently.
- Centrifuge tubes for 20 seconds.
- Gently re-suspend the cell button and examine macroscopically.
- Grade and record results immediately while holding tube in your hand.
- Verify results are expected/ align with the forward red cell testing.
Interpretation
- 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.
- 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.
- Any discrepancy (anomaly) between cell and serum/plasma testing should be investigated and resolved.
Notes
- 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)
- Positive reactions usually are 3+ to 4+ agglutination with reagent antibodies.
- Reactions between patient plasma and reagent red cells (reverse grouping) are generally weaker than cell typing (forward grouping) agglutination.
- Weak reactions (weak or 1+) in the serum testing may be enhanced by:
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- 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).