{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"medRxiv"}],"indexed":{"date-parts":[[2026,7,3]],"date-time":"2026-07-03T00:33:24Z","timestamp":1783038804174,"version":"3.54.6"},"posted":{"date-parts":[[2025,11,6]]},"group-title":"Hematology","reference-count":28,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,11,6]],"date-time":"2025-11-06T00:00:00Z","timestamp":1762387200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,4,14]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:sec>\n                  <jats:title>BACKGROUND<\/jats:title>\n                  <jats:p>Platelet factor 4-polyanion enzyme-linked immunosorbent assays (ELISAs) are considered highly sensitive for diagnosing heparin-induced thrombocytopenia (HIT), such that current practice guidelines recommend use of ELISA-negative results to exclude HIT. Once HIT is ruled out, alternative, non-heparin-based anticoagulant treatments are ceased, and heparin reintroduction frequently occurs.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>METHODS<\/jats:title>\n                  <jats:p>Antigen-based and PF4-dependent functional testing were used to study PF4\/polyvinyl sulfonate ELISA-negative platelet-activating antibodies in HIT-suspected patients and mice immunized with PF4\/heparin.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>RESULTS<\/jats:title>\n                  <jats:p>Three patients with clinical presentations consistent with HIT tested negative in an ELISA using PF4-polyvinylsulfonate (PF4\/PVS), an antigenic target very commonly used for HIT antibody detection. All three patients demonstrated PF4-dependent platelet activation in functional testing that was sensitive to blockade of platelet Fc\u03b3RIIa receptors and inhibited by high concentrations of heparin, consistent with pathogenic HIT antibodies. Functional testing-based screening of 500 ELISA-negative patients identified three patients whose sera activated platelets in a PF4- and Fc\u03b3RIIa-dependent manner, and had clinical histories consistent with HIT. Five of the six ELISA-negative HIT patients were re-exposed to heparin, which precipitated a decrease in platelet counts in all re-exposed patients, and one patient developed a new thrombus. To advance the study of ELISA-negative HIT antibodies, mice were immunized with PF4\/heparin, and functional and antigenic assays were simultaneously used to successfully identify an ELISA-negative, PF4-dependent platelet-activating murine monoclonal antibody that recapitulated the serological characteristics of ELISA-negative HIT patients.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>CONCLUSIONS<\/jats:title>\n                  <jats:p>Recognition of ELISA-negative HIT is critical to avoid harm due to the cessation of alternative anticoagulation therapy and re-exposure of these patients to heparin.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.10.29.25338884","type":"posted-content","created":{"date-parts":[[2025,11,6]],"date-time":"2025-11-06T17:30:19Z","timestamp":1762450219000},"source":"Crossref","is-referenced-by-count":1,"title":["Pathogenic PF4\/Polyvinylsulfonate ELISA-negative Antibodies in HIT"],"prefix":"10.64898","author":[{"given":"Adam J.","family":"Kanack","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noah P.","family":"Splinter","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emily E.","family":"Mauch","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leben","family":"Tefera","sequence":"additional","affiliation":[{"name":"Department of Cardiovascular Medicine, Cleveland Clinic, Cleveland, OH"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Morayma","family":"Reyes Gil","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, Cleveland Clinic, Cleveland, OH"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sakshi","family":"Jasra","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Vermont, Burlington, VT"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew","family":"Goodwin","sequence":"additional","affiliation":[{"name":"Department of Pathology, University of Vermont, Burlington, VT"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristi J","family":"Smock","sequence":"additional","affiliation":[{"name":"Department of Pathology, Spencer Fox Eccles School of Medicine at the University of Utah, Salt Lake City, UT"},{"name":"ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hadiyah","family":"Ahmad","sequence":"additional","affiliation":[{"name":"Department of Medicine, Mayo Clinic, Rochester, MN"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aneel","family":"Ashrani","sequence":"additional","affiliation":[{"name":"Department of Medicine, Mayo Clinic, Rochester, MN"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathaniel L.","family":"Robinson","sequence":"additional","affiliation":[{"name":"Department of Surgery, Mayo Clinic, Rochester, MN"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana I.","family":"Casanegra","sequence":"additional","affiliation":[{"name":"Department of Cardiovascular Medicine, Mayo Clinic, Rochester, MN"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Curtis G.","family":"Jones","sequence":"additional","affiliation":[{"name":"Retham Technologies, Wauwatosa, WI"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shannon M.","family":"Pechauer","sequence":"additional","affiliation":[{"name":"Versiti Blood Research Institute, Milwaukee, WI"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erin","family":"Yttre","sequence":"additional","affiliation":[{"name":"Versiti Blood Research Institute, Milwaukee, WI"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard H.","family":"Aster","sequence":"additional","affiliation":[{"name":"Versiti Blood Research Institute, Milwaukee, WI"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mindy C.","family":"Kohlhagen","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rachel R.","family":"Leger","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David L.","family":"Murray","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine and 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