Direct antiglobulin test (DAT)

Principle

• Detects IgG and/or complement (C3d) coating red cells in vivo.
• Anti-human globulin (AHG) bridges antibody or complement molecules on adjacent cells, causing agglutination.

Specimen

• EDTA blood is preferred: EDTA chelates calcium and prevents in vitro complement binding that can occur in clotted samples stored cold.
• Cord blood for suspected HDFN (wash well to remove Wharton's jelly).

Procedure

• Prepare a 2–5% suspension of patient red cells.
• Wash 3–4 times with large volumes of saline, decanting completely, to remove unbound globulins that would neutralise AHG.
• Add polyspecific AHG (anti-IgG + anti-C3d), centrifuge immediately and read, grading 0 to 4+.
• If positive, repeat with monospecific anti-IgG and anti-C3d; test a saline control (should be negative).
• Add IgG-sensitised check (Coombs control) cells to negative tubes containing anti-IgG; they must agglutinate, otherwise the test is invalid and must be repeated.

Interpretation

• IgG positive: warm autoimmune haemolytic anaemia, haemolytic transfusion reaction, HDFN, drug-induced haemolysis.
• C3d only: cold agglutinin disease, paroxysmal cold haemoglobinuria, some drug-induced and some warm AIHA cases.
• A positive DAT also occurs in a small proportion of healthy donors and more often in hospital patients (e.g. after IVIG or with hypergammaglobulinaemia); always interpret with clinical and haemolysis findings.
• If recently transfused and DAT is IgG positive, prepare an eluate to identify alloantibody.

Quality control and pitfalls

• False negatives: inadequate washing, delay after washing (antibody elutes), inactive AHG, under-centrifugation, too many cells.
• False positives: clotted or refrigerated samples (in vitro C3 binding), over-centrifugation, dirty glassware, contaminated saline, Wharton's jelly.
• Run positive and negative controls each day.

Clinical use

• Investigation of haemolysis, suspected transfusion reactions, HDFN and drug-induced immune haemolysis.