Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland.
Anal Biochem. 2010 Oct 15;405(2):174-83. doi: 10.1016/j.ab.2010.05.030. Epub 2010 Jun 1.
We report an integrated platelet translocation analysis system that measures complex dynamic platelet-protein surface interactions in microliter volumes of unmodified anticoagulated whole blood under controlled fluid shear conditions. The integrated system combines customized platelet-tracking image analysis with a custom-designed microfluidic parallel plate flow chamber and defined von Willebrand factor surfaces to assess platelet trajectories. Using a position-based probability function that accounts for image noise and preference for downstream movement, outputs include instantaneous and mean platelet velocities, periods of motion and stasis, and bond dissociation kinetics. Whole blood flow data from healthy donors at an arterial shear rate (1500 s(-1)) show mean platelet velocities from 8.9+/-1.0 to 12+/-4 microm s(-1). Platelets in blood treated with the antiplatelet agent c7E-Fab fragment spend more than twice as much time in motion as platelets from untreated control blood; the bond dissociation rate constant (k(off)) increases 1.3-fold, whereas mean translocation velocities do not differ. Blood from healthy unmedicated donors was used to assess flow assay reproducibility, donor variability, and the effects of antiplatelet treatment. This integrated system enables reliable, rapid populational quantification of platelet translocation under pathophysiological vascular fluid shear using as little as 150 microl of blood.
我们报告了一个整合的血小板转位分析系统，该系统可在微升体积的未经修饰的抗凝血全血中，在受控的流体剪切条件下测量复杂的动态血小板-蛋白表面相互作用。该整合系统将定制的血小板跟踪图像分析与定制设计的微流控平行板流动室和定义的血管性血友病因子表面相结合，以评估血小板轨迹。使用基于位置的概率函数，该函数考虑了图像噪声和对下游运动的偏好，输出包括瞬时和平均血小板速度、运动和静止期以及键解离动力学。在动脉剪切率（1500 s(-1))下，来自健康供体的全血流数据显示平均血小板速度从 8.9+/-1.0 到 12+/-4 μm s(-1)。在用抗血小板剂 c7E-Fab 片段处理的血液中的血小板在运动中的时间超过未处理对照血液中的血小板的两倍；键解离速率常数（k(off))增加 1.3 倍，而平均转位速度没有差异。来自健康未用药供体的血液用于评估流动分析的重现性、供体变异性以及抗血小板治疗的效果。该整合系统可使用少至 150 微升的血液，可靠、快速地对生理病理血管流体剪切下的血小板转位进行群体定量。
