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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 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.

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Acknowledgments

This work was supported by a Scientist Development Grant (K.B.N.) and a Postdoctoral Fellowship (R.R.H) from the American Heart Association, the National Heart, Lung, and Blood Institute (HL100333), the Colorado Office of Economic Development and International Trade, and the Boettcher Foundation’s Webb-Waring Biomedical Research Award.

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Correspondence to Keith B. Neeves.

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Associate Editor Michael R. King oversaw the review of this article.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material includes figures of the μCP apparatus, image processing routine, immunofluorescent images of micropatterned CTF, and the layout and characterization of the msMFA. Supplementary Movies 1, 2, and 3 show platelet accumulation on 20, 50, and 100 μm CTF spots, respectively, using HMC microscopy. The colored circles represent the location of the CTF spots during whole blood flow assay. Supplementary Movie 4 shows the relative fluid velocity via fluorescence polystyrene beads in the six channels of the msMFA. Supplementary Movies 5, 6, and 7 show platelet accumulation in the msMFA on 50 μm CTP spots for donors 2, 3 and 4, respectively.

Supplementary material 1 (MOV 16484 kb)

Supplementary material 2 (MOV 13577 kb)

Supplementary material 3 (MOV 16761 kb)

Supplementary material 4 (MOV 4199 kb)

Supplementary material 5 (MOV 3362 kb)

Supplementary material 6 (MOV 3499 kb)

Supplementary material 7 (MOV 5191 kb)

Supplementary material 8 (PDF 649 kb)

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Hansen, R.R., Wufsus, A.R., Barton, S.T. et al. High Content Evaluation of Shear Dependent Platelet Function in a Microfluidic Flow Assay. Ann Biomed Eng 41, 250–262 (2013). https://doi.org/10.1007/s10439-012-0658-5

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  • DOI: https://doi.org/10.1007/s10439-012-0658-5

Keywords

  • Platelet adhesion
  • Platelet aggregation
  • Flow assays
  • Microfluidics
  • Micropatterning