BACKGROUND Heparin-induced thrombocytopenia (HIT) is usually caused by anti-platelet factor 4 (PF4)/heparin antibodies, leading to intravascular platelet activation. These antibodies can be detected by PF4/polyanion antigen assays or platelet activation assays. While antigen assays are very sensitive and recognize immunoglobulin (Ig)G, IgA, and IgM antibodies, the role of IgM and IgA HIT-antibodies is debated. Platelet activation assays recognize IgG and are more specific for clinical HIT. METHODS We analyzed sera from 755 consecutive patients referred for diagnostic testing for HIT using a PF4/heparin enzyme-linked immunosorbent assay (ELISA) for IgG, IgA, and IgM and by the heparin-induced platelet activation (HIPA) test. Clinical information was provided by the treating physicians. RESULTS A total of 108 of 755 (14.3%) patients tested positive, 105 (13.9%) in the PF4/heparin IgG/A/M ELISA [28 (26.7%) only for IgM/A]; 53 (7.0%) sera were positive in the HIPA, of those 50 tested also positive in the ELISA. In 77 patients sufficient clinical information was provided. Available clinical information for 17 of the 28 patients who had only IgM and/or IgA detected showed plausible alternative (non-HIT) explanations in four of seven who had thromboembolic complications and in nine of 10 who had isolated HIT. CONCLUSIONS Detection of IgG, IgM and IgA class antibodies by PF4/heparin ELISA yields a positive test result about twice as often as does a platelet activation assay, with only a minority of the additional patients detected likely having HIT. Thus, there is a potential for considerable over-diagnosis of HIT by laboratories that utilize only an ELISA for diagnostic testing.