Lehmann Marcus, Wallbank Alison M, Dennis Kimberly A, Wufsus Adam R, Davis Kara M, Rana Kuldeepsinh, Neeves Keith B
Chemical and Biological Engineering Department, Colorado School of Mines , Golden, Colorado 80401, USA.
Biomicrofluidics. 2015 Nov 18;9(6):064106. doi: 10.1063/1.4935863. eCollection 2015 Nov.
In vitro assays of platelet function and coagulation are typically performed in the presence of an anticoagulant. The divalent cation chelator sodium citrate is among the most common because its effect on coagulation is reversible upon reintroduction of divalent cations. Adding divalent cations into citrated blood by batch mixing leads to platelet activation and initiation of coagulation after several minutes, thus limiting the time blood can be used before spontaneously clotting. In this work, we describe a herringbone microfluidic mixer to continuously introduce divalent cations into citrated blood. The mixing ratio, defined as the ratio of the volumetric flow rates of citrated blood and recalcification buffer, can be adjusted by changing the relative inlet pressures of these two solutions. This feature is useful in whole blood assays in order to account for differences in hematocrit, and thus viscosity. The recalcification process in the herringbone mixer does not activate platelets. The advantage of this continuous mixing approach is demonstrated in microfluidic vascular injury model in which platelets and fibrin accumulate on a collagen-tissue factor surface under flow. Continuous recalcification with the herringbone mixer allowed for flow assay times of up to 30 min, more than three times longer than the time achieved by batch recalcification. This continuous mixer allows for measurements of thrombus formation, remodeling, and fibrinolysis in vitro over time scales that are relevant to these physiological processes.
血小板功能和凝血的体外测定通常在抗凝剂存在的情况下进行。二价阳离子螯合剂柠檬酸钠是最常用的抗凝剂之一，因为重新引入二价阳离子后，其对凝血的影响是可逆的。通过批量混合向枸橼酸化血液中添加二价阳离子会导致血小板活化，并在几分钟后引发凝血，从而限制了血液在自发凝血前可使用的时间。在这项工作中，我们描述了一种人字形微流控混合器，用于将二价阳离子连续引入枸橼酸化血液中。混合比定义为枸橼酸化血液和重新钙化缓冲液的体积流速之比，可以通过改变这两种溶液的相对入口压力来调节。这一特性在全血检测中很有用，以便考虑血细胞比容的差异，进而考虑粘度的差异。人字形混合器中的重新钙化过程不会激活血小板。这种连续混合方法的优势在微流控血管损伤模型中得到了证明，在该模型中，血小板和纤维蛋白在流动状态下积聚在胶原-组织因子表面。使用人字形混合器进行连续重新钙化，使得流动检测时间长达30分钟，比批量重新钙化所达到的时间长三倍多。这种连续混合器能够在与这些生理过程相关的时间尺度上体外测量血栓形成、重塑和纤维蛋白溶解。
