Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
Thromb Res. 2011 Dec;128(6):e166-70. doi: 10.1016/j.thromres.2011.08.002. Epub 2011 Aug 27.
Fast and accurate monitoring is crucial in the successful regulation of coagulation therapy. For the treatment of venous thromboembolism, both unfractionated heparin (UFH) and low molecular weight heparins (LMWHs) are commonly administered. The chromogenic anti-factor Xa (FXa) assay is currently considered the 'gold standard' assay for monitoring LMWH. However different commercial chromogenic methods often differ when tested with the same samples. Fluorogenic anti-FXa assays have the potential to offer greater benefits over chromogenic assays in terms of greater specificity, sensitivity and they are not so influenced by sample opacity or turbidity.
Commercial plasmas were spiked with pharmacologically relevant concentrations (0-1 U/ml) of UFH, enoxaparin, and tinzaparin. The fluorogenic assay was carried out using previously optimized concentrations of 12 nM FXa and 2.7μM fluorogenic substrate, in addition to 6μl of 100mM CaCl(2) and 44μl of plasma. The Biophen® and Coamatic chromogenic assays were carried out according to the manufacturer's instructions. Reaction rates and endpoint values were analyzed and statistical analysis by means of one-way analysis of variance (ANOVA) was performed.
The fluorogenic anti-FXa assay was found to have the broadest therapeutic range of 0-1 U/ml with CVs of<5% for UFH and tinzaparin and CVs<9% for enoxaparin. Despite their limited measuring range, good assay reproducibility was observed with both chromogenic kits.
This study indicated that the fluorogenic assay is the most sensitive assay with the broadest dynamic range for monitoring LMWH therapy when compared with standard chromogenic assays.
快速准确的监测对于成功调节凝血治疗至关重要。在治疗静脉血栓栓塞症时，通常使用未分级肝素（UFH）和低分子肝素（LMWH）。显色抗因子 Xa（FXa）测定目前被认为是监测 LMWH 的“金标准”测定。然而，当用相同的样本进行测试时，不同的商业显色方法往往存在差异。荧光抗 FXa 测定法在特异性、灵敏度方面具有优于显色测定法的潜在优势，并且受样本不透明度或浊度的影响较小。
将药理学相关浓度（0-1 U/ml）的 UFH、依诺肝素和亭扎肝素加入商业血浆中。使用先前优化的浓度（12 nM FXa 和 2.7μM 荧光底物）进行荧光测定法，此外还使用 6μl 100mM CaCl(2)和 44μl 血浆。按照制造商的说明进行 Biophen®和 Coamatic 显色测定法。分析反应速率和终点值，并通过单向方差分析（ANOVA）进行统计分析。
发现荧光抗 FXa 测定法具有最宽的治疗范围（0-1 U/ml），UFH 和亭扎肝素的 CV<5%，依诺肝素的 CV<9%。尽管其测量范围有限，但两种显色试剂盒均表现出良好的测定重现性。
与标准显色测定法相比，本研究表明荧光测定法是监测 LMWH 治疗时最敏感、动态范围最广的测定法。
