Getting a good view: in vitro imaging of platelets under flow

2010

We report a novel device to analyze cell-surface interactions under controlled fluid-shear conditions on wellcharacterised protein surfaces. Its performance is demonstrated by studying platelets interacting with immobilised von Willebrand Factor at arterial vascular shear rates using just 200 μL of whole human blood per assay. The device's parallelplate flow chamber, with 0.1 mm 2 cross sectional area and height-to-width ratio of 1:40, provides uniform, well-defined shear rates along the chip surface with negligible vertical wall effects on the fluid flow profile while minimizing sample volumetric flow. A coating process was demonstrated by ellipsometry, atomic force microscopy, and fluorescent immunostaining to provide reproducible, homogeneous, uniform protein layers over the 0.7 cm 2 cell-surface interaction area. Customized image processing quantifies dynamic cellular surface coverage vs. time throughout the whole-blood-flow assay for a given drug treatment or disease state. This device can track the dose response of anti-platelet drugs, is suitable for point-of-care diagnostics, and is designed for adaptation to mass manufacture.

Journal of visualized experiments : JoVE, 2017

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca(2+)-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca(2+)/Mg(2+) buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked co...

Methods in Molecular Biology, 2013

Fluorescence microscopy techniques have provided important insights into the structural and signalling events occurring during platelet adhesion under both static and blood flow conditions. However, due to limitations in sectioning ability and sensitivity these techniques are restricted in their capacity to precisely image the adhesion footprint of spreading platelets. In particular, investigation of platelet adhesion under hemodynamic shear stress requires an imaging platform with high spatial discrimination and sensitivity and rapid temporal resolution. This chapter describes in detail a multimode imaging approach combining total internal reflection fluorescence microscopy (TIRFM) with high speed epifluorescence and differential interference contrast (DIC) microscopy along with a novel microfluidic perfusion system developed in our laboratory to examine platelet membrane adhesion dynamics under static and flow conditions.

The Analyst, 2011

Platelet aggregation is essential for vascular haemostasis and thrombosis. To improve the therapy of arterial thrombotic disorders and identify novel therapeutic targets it is imperative to study basic mechanisms of platelet thrombus formation. To date most data on biology, physiology and pharmacology of platelet aggregation have been obtained by studying this phenomenon under static or quasi-dynamic conditions at the macroscale level. There is a widespread recognition for the need of new technologies that will help to further elucidate the role of platelets in physiological and pathological thrombus formation and to design more effective and specific antithrombotic drugs. Micro-and nanofluidic devices, capable of reaching nanoscale resolution, can be used for this purpose setting the scene for the development of novel methods for studying platelet function in physiology, pathology and therapeutics.

Annals of Biomedical Engineering, 2008

Microscopic steps and crevices are inevitable features within prosthetic blood-contacting devices. This study aimed to elucidate the thrombogenicity of the associated microscopic flow features by studying the transport of fluorescent platelet-sized particles in a suspension of red blood cells (RBCs) flowing through a 100 µm:200 µm sudden expansion. Micro-flow visualization revealed a strong influence of hematocrit upon the path of RBCs and spatial concentration of particles. At all flow rates studied (Re = 8.3-41.7) and hematocrit 20% and lower, RBC streamlines were found to detach from the microchannel wall creating an RBC-depleted zone inside the step that was much larger than the cells themselves. However, the observed distribution of particles was relatively homogeneous. By contrast, the RBC streamlines of samples with hematocrit equal to or greater than 30% more closely followed the contour of the microchannel, yet exhibited enhanced concentration of particles within the corner. The corresponding size of the cell depletion layer was comparable with the size of the cells. This study implies that local platelet concentration in blood within the physiological range of hematocrit can be elevated within the flow separation region of a sudden expansion and implicates the role of RBCs in causing this effect.

2nd Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine and Biology. Proceedings (Cat. No.02EX578), 2002

Platelet function is suggestive of pathological conditions in heart related diseases. With current insufficient prognostic devices, we see the need for a device to assess complete platelet function. This work presents our preliminary microfluidic device for the analysis of shear-induced platelet activation, which is crucial in studying the pathological role of platelets. In our novel device, the polydimethylsiloxane microchannels were coated using a novel layer-by-layer self-assembly technique to provide controlled nanometer-thick layers of fibrinogen. Anticoagulated platelet rich plasma labeled with a fluorescin isothiocynate-tagged anti-GpIIb/IIIa-antibody was passed through these microchannels and several experimental runs for different shear rates were carried out. Finally, fluorescence assays confirmed the activation of platelets in the microchannels for various shear rates. Control experiments showed that the extent of adhesion on bare PDMS surfaces was less than on the surfaces assembled with fibrinogen. Based on image processing, the extent of platelet adhesion to the fibrinogen substrate was determined for each of the shear rates. The extent of adhesion (ϕ ϕ ϕ ϕ) was modeled as a third order polynomial in shear rate for substrate fibrinogen.

PLoS ONE, 2011

To activate clot formation and maintain hemostasis, platelets adhere and spread onto sites of vascular injury. Although this process is well-characterized biochemically, how the physical and spatial cues in the microenvironment affect platelet adhesion and spreading remain unclear. In this study, we applied deep UV photolithography and protein micro/nanostamping to quantitatively investigate and characterize the spatial guidance of platelet spreading at the single cell level and with nanoscale resolution. Platelets adhered to and spread only onto micropatterned collagen or fibrinogen surfaces and followed the microenvironmental geometry with high fidelity and with single micron precision. Using micropatterned lines of different widths, we determined that platelets are able to conform to micropatterned stripes as thin as 0.6 mm and adopt a maximum aspect ratio of 19 on those protein patterns. Interestingly, platelets were also able to span and spread over non-patterned regions of up to 5 mm, a length consistent with that of maximally extended filopodia. This process appears to be mediated by platelet filopodia that are sensitive to spatial cues. Finally, we observed that microenvironmental geometry directly affects platelet biology, such as the spatial organization and distribution of the platelet actin cytoskeleton. Our data demonstrate that platelet spreading is a finely-tuned and spatially-guided process in which spatial cues directly influence the biological aspects of how clot formation is regulated.

Methods in Molecular Biology, 2011

The platelet is a specialized adhesive cell that plays a key role in thrombus formation under both physiological and pathological blood fl ow conditions. Platelet adhesion and activation are dynamic processes associated with rapid morphological and functional changes, with the earliest signaling events occurring over a subsecond timescale. The relatively small size of platelets combined with the dynamic nature of platelet adhesion under blood fl ow means that the investigation of platelet signaling events requires techniques with both high spatial discrimination and rapid temporal resolution. Unraveling the complex signaling processes governing platelet adhesive function under conditions of hemodynamic shear stress has been a longstanding goal in platelet research and has been greatly infl uenced by the development and application of microimaging-based techniques. Advances in the area of epi-fl uorescence and confocal-based platelet calcium (Ca 2+) imaging have facilitated the in vitro and in vivo elucidation of the early signaling events regulating platelet adhesion and activation. These studies have identifi ed distinct Ca 2+ signaling mechanisms that serve to dynamically regulate activation of the major platelet integrin α IIb β 3 and associated adhesion and aggregation processes under fl ow. This chapter describes in detail a ratiometric calcium imaging protocol and associated troubleshooting procedures developed in our laboratory to examine live platelet Ca 2+ signaling dynamics. This technique provides a method for high-resolution imaging of the Ca 2+ dynamics underpinning platelet adhesion and thrombus formation under conditions of pathophysiological shear stress.

2010

Background: Platelet aggregation is essential for vascular haemostasis and thrombosis. Inhibition of platelet aggregation underpins the pharmacological and clinical effects of antiplatelet drugs. These effects are commonly quantified using methods that assess platelet aggregation under no-flow conditions in macroscale. These devices neither mimic the conditions found in human microvasculature nor detect microaggregates. Aim: The aim of our study was to develop a new method of flow-induced platelet microaggregation using a commercially available (Q-Sense TM E 4 system) nanoscale resolution device, Quartz Crystal Microbalance with Dissipation (QCM-D), that measures mass deposition on sensor crystals as changes in their frequency of vibrations f and energy dissipation D. Material and methods: Human platelets were perfused through QCM-D and f and D were recorded in real time. Phase-contrast, confocal imaging, atomic force microscopy and flow cytometry were also used to study flow-induced platelet microaggregate formation on the surface of fibrinogen-coated crystals. Results: Microaggregates were detected by the device by changes in f and D in a platelet concentration-, flow-and shear stress-dependent manner, confined to the sensor surface and imaged by phase-contrast, confocal and atomic force microscopy. Conclusions: QCM-D is a sensitive device capable of measuring flow-induced platelet microaggregation.

The International Journal of Artificial Organs, 2005

A method for quantitative analysis of platelet deposition under flow is discussed here. The model system is based upon perfusion of blood platelets over an adhesive substrate immobilized on a glass coverslip acting as the lower surface of a rectangular flow chamber. The perfusion apparatus is mounted onto an inverted microscope equipped with epifluorescent illumination and intensified CCD video camera. Characterization is based on information obtained from a specific image analysis method applied to continuous sequences of microscopical images. Platelet recognition across the sequence of images is based on a time-dependent, bidimensional, gaussian-like pdf. Once a platelet is located, the variation of its position and shape as a function of time (i.e., the platelet history) can be determined. Analyzing the history we can establish if the platelet is moving on the surface, the frequency of this movement and the distance traveled before its resumes the velocity of a non-interacting ce...