The use of microfluidics in hemostasis: clinical diagnostics and biomimetic models of vascular injury.

PURPOSE OF REVIEW: This article reviews the application of microfluidic technologies in hemostasis. The emphasis is on promising developments in devices for clinical applications and novel approaches to modeling complex hemodynamics. RECENT FINDINGS: Microfluidics combined with micropatterning of prothrombotic substrates provides devices for measuring platelet function and coagulation with low blood volumes (∼100 µl) over a wide range of shear stresses. This technology has been applied to the diagnosis of bleeding and thrombotic disorders, as well as to dosing and monitoring of anticoagulation and antiplatelet agents. Microfluidic devices that mimic vascular geometries such as bifurcations, stenosis, and complex interconnected networks yield complex flow fields that have revealed new mechanisms of platelet adhesion and aggregation. Applying techniques from tissue engineering by endothelializing these networks is beginning to close the gap between in-vitro and in-vivo models of vascular injury. SUMMARY: Microfluidic technology enables researchers to create in-vitro models of vascular disease with unprecedented control of the biochemical and biophysical conditions. Two promising directions are flow-dependent clinical assays and biomimetic vascular networks. These approaches are particularly well suited for modeling the microvasculature. However, caution should be used when extrapolating results from microfluidic channels to the pathophysiology of thrombosis in large arteries and veins.

Sources of variability in platelet accumulation on type 1 fibrillar collagen in microfluidic flow assays.

Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s⁻¹ through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (Lag(T)), the rate of platelet accumulation (V(PLT)), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to V(PLT) and SC at all wall shear rates. A longer Lag(T) for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s⁻¹ and 300 s⁻¹. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay.

High content evaluation of shear dependent platelet function in a microfluidic flow assay.

The high blood volume requirements and low throughput of conventional flow assays for measuring platelet function are unsuitable for drug screening and clinical applications. In this study, we describe a microfluidic flow assay that uses 50 µL of whole blood to measure platelet function on ~300 micropatterned spots of collagen over a range of physiologic shear rates (50-920 s(-1)). Patterning of collagen thin films (CTF) was achieved using a novel hydrated microcontact stamping method. CTF spots of 20, 50, and 100 µm were defined on glass substrates and consisted of a dense mat of nanoscale collagen fibers (3.74 ± 0.75 nm). We found that a spot size of greater than 20 µm was necessary to support platelet adhesion under flow, suggesting a threshold injury size is necessary for stable platelet adhesion. Integrating 50 µm CTF microspots into a multishear microfluidic device yielded a high content assay from which we extracted platelet accumulation metrics (lag time, growth rate, total accumulation) on the spots using Hoffman modulation contrast microscopy. This method has potential broad application in identifying platelet function defects and screening, monitoring, and dosing antiplatelet agents.