HEPARIN-INDUCED THROMBOCYTOPENIA - PREVALENCE IN A LARGE COHORT OF PATIENTS AND CONFIRMED ROLE OF PF4 HEPARIN COMPLEX AS THE MAIN ANTIGEN FOR ANTIBODIES/

We performed a retrospective study on the prevalence of heparin-induce d thrombocytopenia CHIT) in 233 patients receiving hog mucosa heparin therapy. Of these, 82 patients received s.c. calcium heparin, 130 pati ent received unfractionated (UF) i.v. heparin, and 21 patients receive d low molecular weight heparins (LMWH). An additional four patients, r eferred to our consultation and diagnosed by us as having clinically a ctive type II HIT (HIT-Il) were also studied. The mean platelet count of the 233 patients receiving heparin showed a significant decrease af ter 2 days of heparin treatment and a following significant increase 6 days later (basal: 257 +/- 147 x 10(9) platelets/L; day 2: 239 +/- 12 2, p < 0.0002; day 6: 286 +/- 119, p < 0.004). Of the 212 patients rec eiving UF heparin, 13 (6%) fulfilled the criteria for HIT-IT: seven of these had received i.v. heparin (mean daily dose 26,600 +/- 4,082 IU +/- SD) and six had received s.c. heparin (mean daily dose 21,428 +/- 6,900 IU). Their mean basal platelet count was 226 +/- 100 SD x 10(9) platelets/L and the nadir during heparin treatment was 78 +/- 39 x 10( 9) platelets/L. Thrombotic complications occurred in four (30.7%) of t he 13 patients with HIT-II. Since the immunological mechanism has been demonstrated for HIT-II and since platelet factor 4 (PF4) was identif ied as the co-factor for the binding of heparin-related antibodies, we set up our own enzyme-linked immunosorbent assay (ELISA) for testing antibodies against PF4/heparin complex bound through electrostatic bri dges to the solid phase. The highest binding capacity of HIT-related I gG to the multimolecular complex was obtained at 20 mu g/ml for PF4 an d 3 mu g/ml for heparin, corresponding to 250 ng of PF4 and 42 ng of h eparin in each microtiter well. Such binding was inhibited in a dose-d ependent manner by increasing amounts of heparin, protamine hydrochlor ide, and a monoclonal antibody anti-human PF4 clone 10B2. We observed that HIT-related antibodies bound also to PF4/LMWH complexes but the o ptimal PF4/glycosaminoglycan ratio appeared more critical for LMWH (en oxaparin, fraxiparin, and parnaparin) than for UF heparin. Sera from e ight patients with HIT-II were tested by PF4/heparin ELISA; six of the se had IgG against the complex PF4/heparin and three also had IgM. The persistence of HIT-related antibodies was investigated in three patie nts: in one such antibodies were still detectable 3 years after the ac ute episode, while in the other two, they disappeared after 6 months a nd 1 year, respectively.