Indirect antiglobulin test / antibody screen

Principle

• Detects unexpected IgG red cell antibodies in the patient's plasma (in vitro sensitisation).
• Plasma is incubated at 37 °C with group O reagent red cells of known phenotype; bound antibody is detected after washing by anti-human globulin.

Specimen

• EDTA plasma or serum. If transfused or pregnant within the previous 3 months, the sample must be collected within 3 days of planned transfusion.

Reagents and equipment

• 2 or 3 group O screening cells covering clinically important antigens: D, C, E, c, e, K, k, Fya, Fyb, Jka, Jkb, S, s, M, N, P1, Lea, Leb. Ideally homozygous expression for antigens showing dosage (e.g. Jka, Jkb, Fya, Fyb, S, s).
• Enhancement medium (LISS, PEG or albumin), polyspecific or anti-IgG AHG, IgG check cells.

Procedure

• Add 2 drops plasma to 1 drop of each screening cell (LISS may be added per manufacturer); an immediate spin reading is optional.
• Incubate at 37 °C: about 10–15 min with LISS, about 15 min with PEG (do not centrifuge PEG tubes before washing), 30–60 min in saline.
• Wash 3–4 times with saline, add AHG, centrifuge, read and grade.
• Add check cells to all negative tubes.

Results

• Any positive reaction indicates an antibody (allo- or auto-) and requires antibody identification.
• Include an autocontrol or DAT when screen is positive.

Quality control and pitfalls

• Test weak IgG (e.g. anti-D) positive control and negative control daily.
• Inadequate washing neutralises AHG; check cells detect this.
• Rouleaux, cold antibodies and drug antibodies (e.g. daratumumab, anti-CD38, reacting with all cells) cause interference.
• Screens do not detect antibodies to low-frequency antigens absent from screening cells.

Clinical use

• Pre-transfusion testing, antenatal screening for antibodies causing HDFN, and investigating transfusion reactions.