Off-the-shelf cryopreserved platelets for the detection of HIT and VITT antibodies

Heparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically-simple Platelet Factor-4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but non-specific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay endpoints, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to significantly extend platelet viability by optimizing storage conditions and developed a simple functional assay endpoint by measuring the release of platelet α granule protein, Thrombospondin-1 (TSP1) in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several month-old cryopreserved platelets were treated with PF4 or heparin and evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets (Heparin-TRA) relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated but not heparin-treated cryopreserved platelets consistent with recent data suggesting the requirement for PF4-treated platelets for consistent detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically-simple functional testing available for rapid and accurate in-hospital diagnosis.