ABO forward and reverse grouping (tube method)

Principle

• Forward (cell) grouping detects A and B antigens on the patient's red cells using known anti-A and anti-B reagents.
• Reverse (serum/plasma) grouping detects the naturally occurring (mainly IgM) anti-A and anti-B in the patient's plasma using known A1 and B reagent red cells.
• The two results must agree before the group is reported.

Specimen

• EDTA blood (plasma) or clotted blood (serum), correctly labelled.

Procedure

• Prepare a 2–5% saline suspension of patient red cells.
• Forward: 1 drop anti-A, anti-B (and anti-A,B if used) in labelled tubes; add 1 drop cell suspension.
• Reverse: 2–3 drops patient plasma in tubes labelled A1 and B; add 1 drop of each reagent cell.
• Centrifuge immediately per the calibrated setting (e.g. about 15–30 s in a serofuge); gently resuspend and grade agglutination 0 to 4+; note haemolysis (a positive result in reverse grouping).

Results

• Group A: cells react with anti-A; plasma agglutinates B cells.
• Group B: cells react with anti-B; plasma agglutinates A1 cells.
• Group AB: cells react with both; plasma reacts with neither.
• Group O: cells react with neither; plasma agglutinates both A1 and B cells.
• Expected strength: forward usually 3+–4+, reverse usually 2+–4+.

Quality control and pitfalls

• Test reagent antisera and reagent cells daily with known positive and negative cells.
• Discrepancies: weak or missing antibodies (neonates under about 4–6 months, elderly, hypogammaglobulinaemia), weak A/B subgroups (e.g. A2 with anti-A1 in plasma), acquired B antigen, rouleaux, cold autoantibodies or alloantibodies (anti-M, anti-P1, anti-Lea), recent transfusion or stem cell transplant (mixed field), polyagglutinable cells.
• Resolve discrepancies before issuing group-specific components; in an emergency give group O red cells and group AB plasma.
• Neonates: forward grouping only is routinely used.

Clinical use

• Mandatory before transfusion, for donors, in antenatal testing and in transplant work.