Direct Antiglobulin Test

The direct antiglobulin test (Coombs test) detects immunoglobulin or complement bound to the surface of red cells in vivo. Washed patient red cells are mixed with antihuman globulin, and agglutination indicates bound antibody or complement. Polyspecific reagent is usually used first, followed by monospecific anti-IgG and anti-C3d to define the pattern.

It is the central test in autoimmune haemolytic anaemia, haemolytic disease of the newborn, delayed haemolytic transfusion reaction and drug induced haemolysis. A positive result alone does not prove haemolysis.

Indications. Haemolysis with spherocytes or reticulocytosis, newborn jaundice with suspected maternal antibodies, suspected transfusion reaction, unexplained anaemia in lymphoproliferative disease or autoimmune disease, drug associated anaemia, and evaluation of a positive antibody screen.

Specimen. EDTA blood. Clotted samples stored in the cold can bind complement in vitro and give false positive results.

Method. Tube or gel column technique with washing to remove unbound plasma proteins. Results are graded 1+ to 4+ in tube and gel methods. Weak reactions are significant only when interpreted with clinical data.

Pattern reporting.

• IgG alone.
• IgG with complement.
• Complement (C3d) alone.
• Negative.

IgG alone or IgG with C3d. Warm autoimmune haemolytic anaemia (idiopathic, or secondary to lymphoproliferative disease, SLE, HIV, immunodeficiency), drug induced haemolysis, haemolytic disease of the newborn (Rh), delayed haemolytic transfusion reaction.

C3d alone. Cold agglutinin disease (primary or secondary to infection or lymphoma), paroxysmal cold haemoglobinuria (Donath-Landsteiner antibody), some warm autoimmune haemolytic anaemia, and drug induced haemolysis.

Positive without haemolysis. Intravenous immunoglobulin, antithymocyte globulin, hypergammaglobulinaemia, lymphoproliferative disease (chronic lymphocytic leukaemia), SLE, HIV, drugs (cephalosporins, piperacillin, methyldopa, fludarabine), recent transfusion with alloantibodies, passenger lymphocyte syndrome after transplant, and about 1 in several thousand healthy donors.

Negative with haemolysis. About 10% of warm autoimmune haemolytic anaemia is DAT-negative from IgA, IgM or low affinity or low density IgG. More sensitive methods (gel, flow cytometry, cold saline washing) and an eluate are needed.

Neonates. ABO incompatibility (group O mother, group A or B infant) gives a weak or negative result despite haemolysis. Rh disease gives a strongly positive result. A positive test in cord blood needs correlation with bilirubin and haemoglobin.

Critical findings needing immediate action. A positive result with acute haemolysis during or after transfusion needs urgent transfusion laboratory review and haematology discussion, and the unit should not be continued until review.

Interferences and pitfalls.

• False positives: cold agglutinins at room temperature, clotted samples stored cold, bacterial contamination, polyagglutination, Wharton jelly in cord samples, high titre rouleaux.
• False negatives: low antibody density, IgA-only binding, inadequate washing, and delayed testing with antibody elution.
• Warm antibody with a negative DAT may need a more sensitive method.
• A positive DAT with a negative haemolysis screen is common and needs no treatment on its own.
• Concurrent immune thrombocytopenia with autoimmune haemolysis (Evans syndrome) should be considered when platelets are low.

Special groups. Cord blood DAT is weakly positive in many group O mothers' infants and needs bilirubin and clinical context. In pregnancy, a positive antibody screen in an Rh negative woman requires antibody identification and titre.

Follow up tests. Reticulocyte count, haemolysis screen, blood film, indirect antiglobulin test with antibody identification, red cell eluate, cold agglutinin titre, Donath-Landsteiner test, immunoglobulin levels, flow cytometry for lymphoproliferative disease, antinuclear antibodies, HIV testing.