PROJECT SUMMARY/ABSTRACT This phase II SBIR proposal is aimed at developing a highly accurate functional in vitro diagnostic (IVD) assay for the detection of pathogenic antibodies in Heparin-induced thrombocytopenia (HIT), an adverse reaction to heparin treatment. This assay is based on recent findings that Platelet Factor 4 (PF4)-treated platelets can be used for the sensitive and specific detection of clinically-significant HIT antibodies. HIT kills more than 5 patients every day in the US and is frequently suspected in heparin-treated hospitalized patients who may have a number of potential causes for thrombocytopenia. To assist with diagnosis and management, physicians rely on two families of HIT assays. The first, the PF4/Polyanion ELISA- based in vitro diagnostic (IVD) assays are sensitive but are highly non-specific such that the positive predictive value of these assays is poor at only 30-50%. The second, more accurate platelet activation-based (functional) assays such as the Serotonin release assay (SRA) are technically complex and are performed only at a few reference laboratories which can lead to a long turnaround times. As a result, frontline ELISAs are used to manage most HIT-suspected patients and many patients with false-positive ELISAs are inappropriately treated with alternative anticoagulants which are expensive and have a significantly worse bleeding profile. Retham’s goal is to revolutionize HIT diagnosis by replacing both the inaccurate ELISA and technically complex SRA with HITDx, a simple yet accurate functional IVD that can be performed in the hospital laboratory. HITDx is based on groundbreaking research that suggests that pathogenic platelet-activating HIT antibodies can bind and activate PF4-treated platelets in a heparin-independent manner. Clinical studies including a 409-patient, prospective, multicenter, blinded study demonstrate that this technology can be leveraged to provide accurate HIT diagnosis. During phase I, Retham Technologies demonstrated that PF4 treated, long term (12-months) stabilized platelets can be coupled to an ELISA-based endpoint for detection of a novel analyte, demonstrating that this technology can be adapted to an IVD assay. The foundational patents covering this technology have issued, and multiple additional patents are pending in various jurisdictions. In this SBIR Phase II proposal, Retham will develop, verify and assess the performance of a self-contained HIT IVD prototype using PF4- treated long-term stored platelets. The utility of HITDx for detection of antibodies that cause vaccine-induced thrombotic thrombocytopenia, a newly recognized complication of COVID-19 vaccines will be assessed in future studies. The project will be led by Curtis Jones who is inventor of this technology and spearheaded the development of novel methods to stabilize platelets. He will be supported by Dr. Padmanabhan, a leading HIT expert, QA/RA and biostatistics consultants, and expert Retham advisory board ...