Optimal conditions for the use of 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) in the inactivation of IgM red cell antibodies were investigated. 0.2 m 2-ME was better than 0.01m DTT; higher concentrations of these two substances could not conveniently be used because of gel formation. Incubation at 37 °C led to far more rapid inactivation than incubation at 22 °C; with 2-ME a period of 15 min at 37 °C was sufficient except with very potent antibodies, which were not inactivated even after 3 h incubation. When indirect antiglobulin tests were carried out on 2-ME-treated serum which had not been dialysed, false-positive results were obtained, although only with the mixture of equal volumes of undiluted serum and of 2-ME; results were negative in tests on dilutions of the mixture in saline. False-positive results could also be avoided by incubating the serum for 3 h with iodoacetamide, but this method also involves dilution of the serum. The only method whereby 2-ME-treated serum can be tested without diluting it and without obtaining false-positive reactions is to dialyse it overnight against saline. Since this method also gets rid of the noxious smell of 2-ME it is to be preferred for most purposes.

A patient with autoimmune haemolytic anaemia due to an autoagglutinin of anti-I^T specificity is reported. The antibody was of the IgM class and bound complement; unexpectedly, it reacted optimally at 37 °C. A prozone was noted when testing serial dilutions of the patient’s serum against appropriate red cells in saline, but was not found when testing an eluate prepared from the patient’s red cells. Investigations suggested that the prozone was due to a blocking antibody of high molecular weight, which appeared to be specific for I and I^T.

Two patients with autoimmune haemolytic anaemia of no obvious aetiology are presented. Both cases had IgM autoantibodies which were cold agglutinins with anti-I specificity and were complement binding. Both cases also had IgG autoantibodies which were incomplete, of wide thermal range and also had anti-I specificity, but were not complement binding. The red cells of both patients were coated with C4d/C3d and IgG.