Rapid Determination of Xa Inhibitor Activity in Blood Using a Microfluidic Device that Measures Platelet Deposition and Fibrin Generation Under Flow.

TH open : companion journal to thrombosis and haemostasis Pub Date : 2025-03-25 eCollection Date: 2025-01-01 DOI:10.1055/a-2547-5710
Jason M Rossi, Karen A Panckeri, Soumita Ghosh, Tilo Grosser, Adam Cuker, Scott L Diamond
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Abstract

Background: Patients taking direct oral anticoagulants (DOACs) often present complicated scenarios following major bleeding, stroke, or emergency surgery. Rapid whole blood assays of DOAC levels would aid clinical decisions such as the need for DOAC reversal.

Methods: We developed a single-use, storage-stable, eight-channel microfluidic device to estimate factor Xa (FXa) inhibitor (apixaban or rivaroxaban) levels in venous thromboembolism or atrial fibrillation patients. The assay simultaneously measured whole blood clotting dynamics on collagen/tissue factor (TF; wall shear rate, 200 -1 ) under four ex vivo conditions: no-treatment control, high dose Factor Xa inhibition, low dose or high dose FXa reversal agent (andexanet alfa). Fibrin and platelet deposition dynamics were monitored via two-color epifluorescence microscopy. Plasma samples were also evaluated by LC-MS/MS for DOAC concentrations.

Results: Experiments with healthy volunteer blood spiked with DOAC verified device performance (DOAC IC 50 ∼120 nM) and confirmed that andexanet alfa added to healthy donor blood had no off-target effect on platelet or fibrin signal. Patient whole blood monitored for 15 to 25 minutes (17 minutes mean runtime) allowed calculation of functional DOAC concentrations ranging from 2 to 500 nM that correlated well with LC-MS/MS determination of apixaban or rivaroxaban (R 2  = 0.7 or 0.9, respectively). Platelet dysfunction was not observed in any patient on DOAC. For a threshold of 100 nM DOAC, the area under the curve (AUC) was found to be 0.881 for apixaban and 0.933 for rivaroxaban.

Conclusion: Microfluidic testing of whole blood can provide a rapid estimate of DOAC levels over the on-therapy range.

背景:服用直接口服抗凝剂(DOAC)的患者在大出血、中风或急诊手术后往往会出现复杂的情况。DOAC水平的快速全血检测有助于临床决策,如是否需要逆转DOAC:我们开发了一种一次性使用、储存稳定的八通道微流控装置,用于估算静脉血栓栓塞或心房颤动患者体内的 Xa 因子(FXa)抑制剂(阿哌沙班或利伐沙班)水平。该检测仪在四种体外条件下同时测量了胶原蛋白/组织因子(TF;壁剪切率,200 -1 )上的全血凝结动态:无治疗对照、高剂量 Xa 因子抑制剂、低剂量或高剂量 FXa 逆转剂(andexanet alfa)。通过双色荧光显微镜监测纤维蛋白和血小板的沉积动态。血浆样本也通过 LC-MS/MS 对 DOAC 浓度进行了评估:结果:用添加了 DOAC 的健康志愿者血液进行的实验验证了设备的性能(DOAC IC 50 ∼120 nM),并确认添加到健康献血者血液中的 andexanet alfa 不会对血小板或纤维蛋白信号产生脱靶效应。患者全血监测时间为 15 至 25 分钟(平均运行时间为 17 分钟),可计算出 2 至 500 nM 的功能性 DOAC 浓度,这些浓度与阿哌沙班或利伐沙班的 LC-MS/MS 测定结果有很好的相关性(R 2 分别为 0.7 或 0.9)。服用 DOAC 的患者均未出现血小板功能障碍。对于 100 nM DOAC 临界值,阿哌沙班和利伐沙班的曲线下面积(AUC)分别为 0.881 和 0.933:结论:全血微流控检测可快速估算治疗范围内的 DOAC 水平。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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