Computational Tracking of Shear-Mediated Platelet Interactions with von Willebrand Factor.

The imaging of shear-mediated dynamic platelet behavior interacting with surface-immobilized von Willebrand factor (vWF) has tremendous potential in characterizing changes in platelet function for clinical diagnostics purposes. However, the imaging output, a series of images representing platelets adhering and rolling on the surface, poses unique, non-trivial challenges for software algorithms that reconstruct the positional trajectories of platelets. We report on an algorithm that tracks platelets using the output of such flow run experiments, taking into account common artifacts encountered by previously-published methods, and we derive seven key metrics of platelet dynamics that can be used to characterize platelet function. Extensive testing of our method using simulated platelet flow run data was carried out to validate our tracking method and derived metrics in capturing key platelet-vWF interaction-dynamics properties. Our results show that while the number of platelets present on the imaged area is the leading cause of errors, flow run data from two experiments using whole blood samples showed that our method and metrics can detect platelet property changes/differences that are concordant with the expected biological outcome, such as inhibiting key platelet receptors such as P2Y1, glycoprotein (GP)Ib and GPIIb/IIIa. These findings support the use of our methodologies to characterize platelet function among a wide range of healthy and disease cohorts.

Self-Powered Microfluidic Device for Rapid Assay of Antiplatelet Drugs.

We report the development of a microfluidic device for the rapid assay in whole blood of interfacial platelet-protein interactions indicative of the efficacy of antiplatelet drugs, for example, aspirin and Plavix, two of the world's most widely used drugs, in patients with cardiovascular disease (CVD). Because platelet adhesion to surface-confined protein matrices is an interfacial phenomenon modulated by fluid shear rates at the blood/protein interface, and because such binding is a better indicator of platelet function than platelet self-aggregation, we designed, fabricated, and characterized the performance of a family of disposable, self-powered microfluidic chips with well-defined flow and interfacial shear rates suitable for small blood volumes (≤200 µL). This work demonstrates that accurate quantification of cell adhesion to protein matrices, an important interfacial biological phenomenon, can be used as a powerful diagnostic tool in those with CVD, the world's leading cause of death. To enable such measurements, we developed a simple technique to fabricate single-use self-powered chips incorporating shear control (SpearChips). These parallel-plate flow devices integrate on-chip vacuum-driven blood flow, using a predegassed elastomer component to obviate active pumping, with microcontact-printed arrays of 6-µm-diameter fluorescently labeled fibrinogen dots on a cyclic olefin polymer base plate as a means to quantitatively count platelet-protein binding events. The use of SpearChips to assess in whole blood samples the effects of GPIIb/IIIa and P2Y12 inhibitors, two important classes of "antiplatelet" drugs, is reported.

High efficiency ring-lens supercritical angle fluorescence (SAF) detection for optimum bioassay performance.

We present a polymer biochip with embedded optics which allows the detection of supercritical angle fluorescence (SAF) without losses due to total internal reflection within the substrate. The chip design comprises structured spherical and aspherical optical elements on the bottom, while the top is chemically functionalized for direct binding of biomolecules. Furthermore, this design facilitates integration in lab-on-a-chip systems with appropriate microfluidics. In the confocal optical setup an ellipsoidal mirror is used for collection of SAF light above the critical angle of the water-polymer interface, which is detected by a photon-counting detector. The work presented here represents a proof of concept for performing sensitive and rapid point-of-care testing, using this low-cost, robust and disposable optical biochip platform. The performance of the platform was validated using direct binding DNA and human IgG assays which yielded low limits of detection 10 pM for DNA and 10 pg/ml for human IgG.

Individual platelet adhesion assay: measuring platelet function and antiplatelet therapies in whole blood via digital quantification of cell adhesion.

Widespread monitoring of platelet function and the effect of antiplatelet drugs will improve outcomes in cardiovascular patients, but platelet function testing is not routine in clinical practice. We report a rapid, accurate methodology to quantify platelet-protein interactions: a microarray of contact-printed 6-µm fibrinogen dots on a transparent substrate binds platelets from whole blood, one platelet per dot. The fractional occupancy of an array of fibrinogen dots after a predefined incubation time quantitatively assays platelet adhesion to the protein matrix. We demonstrate this technique by measurement of platelet adhesion to fibrinogen as a means to quantify the effect of the P2Y12 and αIIbß3 receptor inhibitors cangrelor and abciximab, respectively, both in vitro--by incubating the drug with a freshly drawn blood sample--and in blood from patients treated with antiplatelet agents. The effects of single- and dual-antiplatelet therapy are also assessed. Results from this platelet-binding assay are well correlated with standard techniques including flow cytometry and light transmission aggregometry. This assay technology, readily integrated with microfluidic platforms, is generally applicable to the assay of cell-protein interactions and promises more effective, rapid assay of drug effects in cardiovascular disease patients.

At-line bioprocess monitoring by immunoassay with rotationally controlled serial siphoning and integrated supercritical angle fluorescence optics.

In this paper we report a centrifugal microfluidic "lab-on-a-disc" system for at-line monitoring of human immunoglobulin G (hIgG) in a typical bioprocess environment. The novelty of this device is the combination of a heterogeneous sandwich immunoassay on a serial siphon-enabled microfluidic disc with automated sequential reagent delivery and surface-confined supercritical angle fluorescence (SAF)-based detection. The device, which is compact, easy-to-use and inexpensive, enables rapid detection of hIgG from a bioprocess sample. This was achieved with, an injection moulded SAF lens that was functionalized with aminopropyltriethoxysilane (APTES) using plasma enhanced chemical vapour deposition (PECVD) for the immobilization of protein A, and a hybrid integration with a microfluidic disc substrate. Advanced flow control, including the time-sequenced release of on-board liquid reagents, was implemented by serial siphoning with ancillary capillary stops. The concentration of surfactant in each assay reagent was optimized to ensure proper functioning of the siphon-based flow control. The entire automated microfluidic assay process is completed in less than 30 min. The developed prototype system was used to accurately measure industrial bioprocess samples that contained 10 mg mL(-1) of hIgG.

Assaying the efficacy of dual-antiplatelet therapy: use of a controlled-shear-rate microfluidic device with a well-defined collagen surface to track dynamic platelet adhesion.

We report the development and demonstration of an assay that distinguishes the pharmacological effects of two widely used antiplatelet therapies, aspirin (COX-1 inhibitor) and clopidogrel (P2Y12 inhibitor). Whole blood is perfused through a low-volume microfluidic device in contact with a well-characterized (ellipsometry, atomic force microscopy) acid-soluble type I collagen surface. Whole human blood treated in vitro with a P2Y12 inhibitor 2-methylthioadenosine 5'-monophosphate triethylammonium salt (2-MeSAMP) extended the time to the start of platelet recruitment, i.e., platelet binding to the collagen surface. Treatment with 2-MeSAMP also slowed the rate of aggregate buildup, with an overall reduced average platelet aggregate area after 8 min of constant blood flow. A far smaller effect was observed for in vitro treatment with aspirin, for which the rate of change of surface coverage is indistinguishable from controls. In whole blood obtained from patients under treatment with dual-antiplatelet therapy (aspirin and clopidogrel), a significant extension of time to platelet recruitment was observed along with a slowed rate of aggregate buildup and an average aggregate size approximately half that of control measurements. Differentiation of the pharmacological effects of these two well-targeted antiplatelet pathways suggests a role for this assay in determining the antiplatelet effects of these and related new therapeutics in clinical settings.