Premium
Optimisation of antithrombin resistance assay as a practical clinical laboratory test: Development of prothrombin activator using factors Xa/Va and automation of assay
Author(s) -
Tamura S.,
Suga Y.,
Tanamura M.,
MurataKawakami M.,
Takagi Y.,
Hottori Y.,
Kakihara M.,
Suzuki S.,
Takagi A.,
Kojima T.
Publication year - 2018
Publication title -
international journal of laboratory hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.705
H-Index - 55
eISSN - 1751-553X
pISSN - 1751-5521
DOI - 10.1111/ijlh.12786
Subject(s) - antithrombin , prothrombinase , chemistry , activator (genetics) , thrombin , plasminogen activator , chromatography , chromogenic , prothrombin time , microbiology and biotechnology , biochemistry , immunology , medicine , biology , heparin , platelet , gene
Abstract Introduction Antithrombin resistance ( ATR ) is a novel thrombotic risk in abnormal prothrombins. A manual ATR assay using Oxyuranus scutellatus (Ox) venom as a prothrombin activator was established for detecting antithrombin‐resistant prothrombin. However, this assay was limited because of Ox snake venom availability and its throughput capacity. Here, we have improved the ATR assay using bovine factors Xa and Va ( FX a/Va) as prothrombin activators and have optimised assay conditions for an automated instrument ( ACL TOP 500). Methods Diluted plasma was incubated with a prothrombin activator mix (phospholipids, CaCl 2 , and bovine FX a/Va), followed by inactivation with antithrombin for 10, 20 and 30 minutes. We added a chromogenic substrate S‐2238, and assessed changes in absorbance/min at 405 nm. We also adapted assay conditions for ACL TOP 500. Results Optimum conditions for FX a/Va treatment were 6.25% phospholipids, 5 mM Ca CL 2 , 0.01 μg/ mL FX a and 0.1 μg/ mL FV a. ATR assay kinetics with the FX a/Va activator was comparable with that with the Ox activator in heterozygous reconstituted plasma with the recombinant wild‐type or antithrombin‐resistant prothrombin. Using ACL TOP 500, optimum conditions for the FX a/Va treatment were 10.0% phospholipids, 5 mM CaCl 2 , 0.02 μg/ mL FX a and 0.2 μg/ mL FV a. The automated ATR assay with the FX a/Va activator demonstrated good detectability for antithrombin‐resistant prothrombin in plasma from a heterozygous carrier with prothrombin Yukuhashi or Belgrade. Conclusion We optimised the ATR assay with the FX a/Va activator and adapted the assay for ACL TOP 500; the assay showed the ability to clearly detect antithrombin‐resistant prothrombin in manual and automated procedures.