Evaluation of functional assays for the diagnosis of heparin induced thrombocytopenia using 5B9, a monoclonal IgG that mimics human antibodies.

Serotonin Release Assay (SRA) is considered as the "gold standard" for detecting pathogenic heparin-induced thrombocytopenia (HIT) antibodies. However, this method is time-consuming, expensive and uses radioelements. Heparin-induced multiple electrode aggregometry (HIMEA), light transmission aggregometry (LTA) with platelet rich plasma (PRP) or washed platelets (WP), ATP release, and flow cytometry (FC) are available alternatives.To evaluate the performance of these assays, comparatively with SRA, for detecting HIT antibodies, using 5B9, a monoclonal IgG fully mimicking human HIT antibodies.Heparin-dependent platelet activation induced by 5B9 (50/20/10 µg/mL) was evaluated by all assays performed on the same day using platelets from 20 healthy donors. The three methods exhibiting the highest sensitivity to 5B9 were then assessed by testing samples from patients with either likely (n=10), or undeterminate/unlikely HIT (n=10).All methods exhibited good sensitivity for detecting 5B9 50 µg/mL, but only SRA and HIMEA were positive with 100% of donors using 5B9 20 µg/mL, followed by FC (83%). SRA detected 5B9 10 μg/mL with 90% of donors, while HIMEA and FC were positive in 45% and 44% of cases, respectively. Whereas SRA was positive with 9/10 samples from likely HIT, HIMEA and FC were positive with 7 and 6 of them, respectively. Neither SRA nor HIMEA was positive with undeterminate/unlikely HIT samples, while FC was positive or doubtful in three cases.SRA likely remains the most sensitive and specific assay for detecting platelet activating HIT antibodies, but HIMEA or FC are potential alternatives, despite being less performant.