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A manual <jats:styled-content style=\"fixed-case\">ATR<\/jats:styled-content> assay using <jats:italic>Oxyuranus\u00a0scutellatus<\/jats:italic> (Ox) venom as a prothrombin activator was established for detecting antithrombin\u2010resistant prothrombin. However, this assay was limited because of Ox snake venom availability and its throughput capacity. Here, we have improved the <jats:styled-content style=\"fixed-case\">ATR<\/jats:styled-content> assay using bovine factors Xa and Va (<jats:styled-content style=\"fixed-case\">FX<\/jats:styled-content>a\/Va) as prothrombin activators and have optimised assay conditions for an automated instrument (<jats:styled-content style=\"fixed-case\">ACL TOP<\/jats:styled-content> 500).<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Diluted plasma was incubated with a prothrombin activator mix (phospholipids, CaCl<jats:sub>2<\/jats:sub>, and bovine <jats:styled-content style=\"fixed-case\">FX<\/jats:styled-content>a\/Va), followed by inactivation with antithrombin for 10, 20 and 30\u00a0minutes. We added a chromogenic substrate S\u20102238, and assessed changes in absorbance\/min at 405\u00a0nm. We also adapted assay conditions for <jats:styled-content style=\"fixed-case\">ACL TOP<\/jats:styled-content> 500.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Optimum conditions for <jats:styled-content style=\"fixed-case\">FX<\/jats:styled-content>a\/Va treatment were 6.25% phospholipids, 5 <jats:styled-content style=\"fixed-case\">mM<\/jats:styled-content> Ca<jats:styled-content style=\"fixed-case\">CL<\/jats:styled-content><jats:sub>2<\/jats:sub>, 0.01\u00a0\u03bcg\/<jats:styled-content style=\"fixed-case\">mL FX<\/jats:styled-content>a and 0.1 \u03bcg\/<jats:styled-content style=\"fixed-case\">mL FV<\/jats:styled-content>a. <jats:styled-content style=\"fixed-case\">ATR<\/jats:styled-content> assay kinetics with the <jats:styled-content style=\"fixed-case\">FX<\/jats:styled-content>a\/Va activator was comparable with that with the Ox activator in heterozygous reconstituted plasma with the recombinant wild\u2010type or antithrombin\u2010resistant prothrombin. Using <jats:styled-content style=\"fixed-case\">ACL TOP<\/jats:styled-content> 500, optimum conditions for the <jats:styled-content style=\"fixed-case\">FX<\/jats:styled-content>a\/Va treatment were 10.0% phospholipids, 5 <jats:styled-content style=\"fixed-case\">mM<\/jats:styled-content> CaCl<jats:sub>2<\/jats:sub>, 0.02\u00a0\u03bcg\/<jats:styled-content style=\"fixed-case\">mL FX<\/jats:styled-content>a and 0.2 \u03bcg\/<jats:styled-content style=\"fixed-case\">mL FV<\/jats:styled-content>a. The automated <jats:styled-content style=\"fixed-case\">ATR<\/jats:styled-content> assay with the <jats:styled-content style=\"fixed-case\">FX<\/jats:styled-content>a\/Va activator demonstrated good detectability for antithrombin\u2010resistant prothrombin in plasma from a heterozygous carrier with prothrombin Yukuhashi or Belgrade.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>We optimised the <jats:styled-content style=\"fixed-case\">ATR<\/jats:styled-content> assay with the <jats:styled-content style=\"fixed-case\">FX<\/jats:styled-content>a\/Va activator and adapted the assay for <jats:styled-content style=\"fixed-case\">ACL TOP<\/jats:styled-content> 500; the assay showed the ability to clearly detect antithrombin\u2010resistant prothrombin in manual and automated procedures.<\/jats:p><\/jats:sec>","DOI":"10.1111\/ijlh.12786","type":"journal-article","created":{"date-parts":[[2018,2,13]],"date-time":"2018-02-13T11:06:57Z","timestamp":1518520017000},"page":"312-319","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Optimisation of antithrombin resistance assay as a practical clinical laboratory test: Development of prothrombin activator using factors Xa\/Va and automation of assay"],"prefix":"10.1111","volume":"40","author":[{"given":"S.","family":"Tamura","sequence":"first","affiliation":[{"name":"Department of Pathophysiological Laboratory Sciences Nagoya University Graduate School of Medicine  Nagoya Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Y.","family":"Suga","sequence":"additional","affiliation":[{"name":"Department of Pathophysiological Laboratory Sciences Nagoya University Graduate School of Medicine  Nagoya 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