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

ABSTRACT: 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 ?L 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 ?m 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 ?m was necessary to support platelet adhesion under flow, suggesting a threshold injury size is necessary for stable platelet adhesion. Integrating 50 ?m 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.

SUBMITTER: Hansen RR

PROVIDER: S-EPMC3544986 | biostudies-other | 2013 Feb

REPOSITORIES: biostudies-other

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

Hansen Ryan R RR Wufsus Adam R AR Barton Steven T ST Onasoga Abimbola A AA Johnson-Paben Rebecca M RM Neeves Keith B KB

Annals of biomedical engineering 20120922 2

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 ...[more]

PMID: 23001359

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Project description:Nonsurgical bleeding is the most frequent complication of left ventricular assist device (LVAD) support. Supraphysiologic shear rates generated in LVAD causes impaired platelet aggregation, which increases the risk of bleeding. The effect of shear rate on the formation size of platelet aggregates has never been reported experimentally, although platelet aggregation size can be considered to be directly relevant to bleeding complications. Therefore, this study investigated the impact of shear rate and exposure time on the formation size of platelet aggregates, which is vital in predicting bleeding in patients with an LVAD. Human platelet-poor plasma (containing von Willebrand factor, vWF) and fluorochrome-labeled platelets were subjected to a range of shear rates (0-10 000 s-1 ) for 0, 5, 10, and 15 minutes using a custom-built blood-shearing device. Formed sizes of platelet aggregates under a range of shear-controlled environment were visualized and measured using microscopy. The loss of high molecular weight (HMW) vWF multimers was quantified using gel electrophoresis and immunoblotting. An inhibition study was also performed to investigate the reduction in platelet aggregation size and HMW vWF multimers caused by either mechanical shear or enzymatic (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13-ADAMTS13, the von Willebrand factor protease) mechanism under low and high shear conditions (360 and 10 000 s-1 ). We found that the average size of platelet aggregates formed under physiological shear rates of 360-3000 s-1 (200-300 μm2 ) was significantly larger compared to those sheared at >6000 s-1 (50-100 μm2 ). Furthermore, HMW vWF multimers were reduced with increased shear rates. The inhibition study revealed that the reduction in platelet aggregation size and HWM vWF multimers were mainly associated with ADAMTS13. In conclusion, the threshold of shear rate must not exceed >6000 s-1 in order to maintain the optimal size of platelet aggregates to "plug off" the injury site and stop bleeding.

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

Publications

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

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