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Conventional immunoassays, however, often lack specificity and require confirmatory testing with fresh human platelets. To address this limitation, we optimized our previously developed cell\u2010based enzyme\u2010linked immunosorbent assay (ELISA) for improved HIT detection under various experimental conditions. Platelet factor 4 was immobilized on breast cancer cells (MDA\u2010MB\u2010231) to capture monoclonal HIT\u2010like (KKO) and non\u2010HIT (RTO) antibodies, which served as models to evaluate assay performance\u00a0under different pH levels, ionic strengths (NaCl), and fixation methods (ethanol, paraformaldehyde, glutaraldehyde). To identify the most suitable substrate, additional cancer cell lines (HCT\u2010116, MCF\u20107, 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\u00a0mM NaCl and 4% paraformaldehyde fixation. Notably, live MDA\u2010MB\u2010231 and HCT\u2010116 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\u2010free diagnostic method. Our findings establish live MDA\u2010MB\u2010231 and HCT\u2010116 cells as highly promising platforms for clinical applications in HIT antibody detection.<\/jats:p>","DOI":"10.1002\/adbi.202500079","type":"journal-article","created":{"date-parts":[[2025,8,28]],"date-time":"2025-08-28T06:19:19Z","timestamp":1756361959000},"source":"Crossref","is-referenced-by-count":1,"title":["Optimized Cell\u2010Based Platform for Platelet\u2010Free Detection of Heparin\u2010Induced Thrombocytopenia Antibodies"],"prefix":"10.1002","volume":"9","author":[{"given":"Li\u2010Yu","family":"Chen","sequence":"first","affiliation":[{"name":"Institute for Bioprocessing and Analytical Measurement Techniques  37308 Heilbad Heiligenstadt Germany"},{"name":"Department of Infection Biology Leibniz Institute for Natural Product Research and Infection Biology  07745 Jena Germany"},{"name":"Institute of Microbiology Friedrich\u2010Schiller\u2010University  07743 Jena Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Uwe","family":"Schirmer","sequence":"additional","affiliation":[{"name":"Institute for Bioprocessing and Analytical Measurement Techniques  37308 Heilbad Heiligenstadt Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter F.","family":"Zipfel","sequence":"additional","affiliation":[{"name":"Department of Infection Biology Leibniz Institute for Natural Product Research and Infection Biology  07745 Jena Germany"},{"name":"Institute of Microbiology Friedrich\u2010Schiller\u2010University  07743 Jena Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Doris","family":"Heinrich","sequence":"additional","affiliation":[{"name":"Institute for Bioprocessing and Analytical Measurement Techniques  37308 Heilbad Heiligenstadt Germany"},{"name":"Faculty of Mathematics and Natural Sciences Technische Universit\u00e4t Ilmenau  98694 Ilmenau Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9237-3482","authenticated-orcid":false,"given":"Thi\u2010Huong","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Institute for Bioprocessing and Analytical Measurement Techniques  37308 Heilbad Heiligenstadt Germany"},{"name":"Faculty of Mathematics and Natural Sciences Technische Universit\u00e4t Ilmenau  98694 Ilmenau Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,8,27]]},"reference":[{"key":"e_1_2_10_1_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41420-019-0188-0"},{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1055\/s-0038-1656329"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood.V96.1.182"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1172\/JCI90958"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2013-11-539262"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood.V95.5.1533.005k01_1533_1540"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/clc.23061"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2104840"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2105385"},{"key":"e_1_2_10_10_1","first-page":"328","volume":"18","author":"Dragonetti D.","year":"2020","journal-title":"Blood Transfus."},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood.2020008762"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1111\/jth.15650"},{"key":"e_1_2_10_13_1","first-page":"470","volume":"22","author":"Pelzl L.","year":"2024","journal-title":"JTH"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.3324\/haematol.2021.279000"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tmrv.2006.05.001"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1538-7836.2010.03974.x"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1002\/ajh.24025"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.1111\/jth.15362"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.1053\/j.seminhematol.2022.03.003"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1182\/bloodadvances.2022007766"},{"key":"e_1_2_10_21_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1537-2995.1994.34594249047.x"},{"key":"e_1_2_10_22_1","doi-asserted-by":"publisher","DOI":"10.1055\/s-0037-1614536"},{"key":"e_1_2_10_23_1","doi-asserted-by":"publisher","DOI":"10.1111\/ijlh.12236"},{"key":"e_1_2_10_24_1","doi-asserted-by":"crossref","unstructured":"On Behalf Of The Gfht\u2010Hit Study Group B.Tardy\u2010Poncet A.Montmartin M.Piot M.Alhenc\u2010Gelas P.Nguyen I.Elalamy A.Greinacher E. 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