Optimized Cell-Based Platform for Platelet-Free Detection of Heparin-Induced Thrombocytopenia Antibodies.

Accurate detection of heparin-induced thrombocytopenia (HIT) antibodies is crucial for diagnosing and managing thrombotic events. Conventional immunoassays, however, often lack specificity and require confirmatory testing with fresh human platelets. To address this limitation, we optimized our previously developed cell-based enzyme-linked immunosorbent assay (ELISA) for improved HIT detection under various experimental conditions. Platelet factor 4 was immobilized on breast cancer cells (MDA-MB-231) to capture monoclonal HIT-like (KKO) and non-HIT (RTO) antibodies, which served as models to evaluate assay performance under different pH levels, ionic strengths (NaCl), and fixation methods (ethanol, paraformaldehyde, glutaraldehyde). To identify the most suitable substrate, additional cancer cell lines (HCT-116, MCF-7, HepG2) were tested under live and fixed conditions, with selected conditions validated using human HIT sera. Optimal detection tested with monoclonal antibodies was achieved using 50 mM NaCl and 4% paraformaldehyde fixation. Notably, live MDA-MB-231 and HCT-116 cells demonstrated superior sensitivity and specificity compared to fixed cells. Furthermore, these cell lines enable the efficient detection of HIT antibodies using flow cytometry, a robust and platelet-free diagnostic method. Our findings establish live MDA-MB-231 and HCT-116 cells as highly promising platforms for clinical applications in HIT antibody detection.