Long-term Stored Platelets Coupled to Thrombospondin-1 Detection for Rapid and Accurate Identification of Pathogenic HIT Antibodies

Abstract

Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening disorder characterized by antibodies to Platelet Factor 4 (PF4)-polyanion complexes which cause thrombocytopenia and thrombosis. Currently used technically-simple frontline assays such as the PF4-polyanion enzyme-linked immunosorbent assays (ELISAs) lack specificity, and more accurate functional assays such as the serotonin release assay (SRA) and PF4-dependent P-selectin expression assay (PEA) have long turnaround times due to technical complexity and availability only in the reference laboratory setting. There is a critical need for accurate near-patient functional testing to guide patient management, but a key barrier to attaining this goal is the short-term viability of platelets. Here, we detail a process of platelet cryopreservation that renders them viable for at least one year and show that PF4-treated cryopreserved platelets, when coupled with ELISA-based measurement of thrombospondin-1 release (a platelet α-granule protein), detects pathogenic HIT antibodies with high accuracy. Furthermore, testing of a cohort of non-pathogenic HIT antibodies that were strongly reactive in PF4/polyanion ELISA but negative in functional assays demonstrated negative results in the thrombospondin-1 release assay, confirming high specificity of this technique. These findings have the potential to transform the diagnostic testing paradigm in HIT by making in-hospital functional testing available for rapid and accurate diagnosis.