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Optimisation of antithrombin resistance assay as a practical clinical laboratory test: Development of prothrombin activator using factors Xa/Va and automation of assay.
Int J Lab Hematol 2018; 40(3):312-319IJ

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 (FXa/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, CaCl2 , and bovine FXa/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 FXa/Va treatment were 6.25% phospholipids, 5 mM CaCL2 , 0.01 μg/mL FXa and 0.1 μg/mL FVa. ATR assay kinetics with the FXa/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 FXa/Va treatment were 10.0% phospholipids, 5 mM CaCl2 , 0.02 μg/mL FXa and 0.2 μg/mL FVa. The automated ATR assay with the FXa/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 FXa/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.

Authors+Show Affiliations

Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.Department of Transfusion Medicine, Nagoya University Hospital, Nagoya, Japan.Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan. Japan Society for the Promotion of Science, Tokyo, Japan.Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

29436777

Citation

Tamura, S, et al. "Optimisation of Antithrombin Resistance Assay as a Practical Clinical Laboratory Test: Development of Prothrombin Activator Using Factors Xa/Va and Automation of Assay." International Journal of Laboratory Hematology, vol. 40, no. 3, 2018, pp. 312-319.
Tamura S, Suga Y, Tanamura M, et al. Optimisation of antithrombin resistance assay as a practical clinical laboratory test: Development of prothrombin activator using factors Xa/Va and automation of assay. Int J Lab Hematol. 2018;40(3):312-319.
Tamura, S., Suga, Y., Tanamura, M., Murata-Kawakami, M., Takagi, Y., Hottori, Y., ... Kojima, T. (2018). Optimisation of antithrombin resistance assay as a practical clinical laboratory test: Development of prothrombin activator using factors Xa/Va and automation of assay. International Journal of Laboratory Hematology, 40(3), pp. 312-319. doi:10.1111/ijlh.12786.
Tamura S, et al. Optimisation of Antithrombin Resistance Assay as a Practical Clinical Laboratory Test: Development of Prothrombin Activator Using Factors Xa/Va and Automation of Assay. Int J Lab Hematol. 2018;40(3):312-319. PubMed PMID: 29436777.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Optimisation of antithrombin resistance assay as a practical clinical laboratory test: Development of prothrombin activator using factors Xa/Va and automation of assay. AU - Tamura,S, AU - Suga,Y, AU - Tanamura,M, AU - Murata-Kawakami,M, AU - Takagi,Y, AU - Hottori,Y, AU - Kakihara,M, AU - Suzuki,S, AU - Takagi,A, AU - Kojima,T, Y1 - 2018/02/13/ PY - 2017/10/08/received PY - 2018/01/11/accepted PY - 2018/2/14/pubmed PY - 2018/10/13/medline PY - 2018/2/14/entrez KW - ACL TOP 500 KW - FXa/Va activator KW - Ox venom KW - antithrombin resistance KW - prothrombin SP - 312 EP - 319 JF - International journal of laboratory hematology JO - Int J Lab Hematol VL - 40 IS - 3 N2 - 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 (FXa/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, CaCl2 , and bovine FXa/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 FXa/Va treatment were 6.25% phospholipids, 5 mM CaCL2 , 0.01 μg/mL FXa and 0.1 μg/mL FVa. ATR assay kinetics with the FXa/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 FXa/Va treatment were 10.0% phospholipids, 5 mM CaCl2 , 0.02 μg/mL FXa and 0.2 μg/mL FVa. The automated ATR assay with the FXa/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 FXa/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. SN - 1751-553X UR - https://www.unboundmedicine.com/medline/citation/29436777/Optimisation_of_antithrombin_resistance_assay_as_a_practical_clinical_laboratory_test:_Development_of_prothrombin_activator_using_factors_Xa/Va_and_automation_of_assay L2 - https://doi.org/10.1111/ijlh.12786 DB - PRIME DP - Unbound Medicine ER -