An extensional strain sensing mechanosome drives adhesion-independent platelet activation at supraphysiological hemodynamic gradients.

BACKGROUND: Supraphysiological hemodynamics are a recognized driver of platelet activation and thrombosis at high-grade stenosis and in blood contacting circulatory support devices. However, whether platelets mechano-sense hemodynamic parameters directly in free flow (in the absence of adhesion receptor engagement), the specific hemodynamic parameters at play, the precise timing of activation, and the signaling mechanism(s) involved remain poorly elucidated. RESULTS: Using a generalized Newtonian computational model in combination with microfluidic models of flow acceleration and quasi-homogenous extensional strain, we demonstrate that platelets directly mechano-sense acute changes in free-flow extensional strain independent of shear strain, platelet amplification loops, von Willebrand factor, and canonical adhesion receptor engagement. We define an extensional strain sensing "mechanosome" in platelets involving cooperative Ca2+ signaling driven by the mechanosensitive channel Piezo1 (as the primary strain sensor) and the fast ATP gated channel P2X1 (as the secondary signal amplifier). We demonstrate that type II PI3 kinase C2α activity (acting as a "clutch") couples extensional strain to the mechanosome. CONCLUSIONS: Our findings suggest that platelets are adapted to rapidly respond to supraphysiological extensional strain dynamics, rather than the peak magnitude of imposed wall shear stress. In the context of overall platelet activation and thrombosis, we posit that "extensional strain sensing" acts as a priming mechanism in response to threshold levels of extensional strain allowing platelets to form downstream adhesive interactions more rapidly under the limiting effects of supraphysiological hemodynamics.

Paper-Based Acoustofluidics for Separating Particles and Cells.

Paper is emerging as a versatile platform for automated fluid handling with a broad range of applications in medical diagnostics and analytical chemistry. However, selectively controlling analyte transport in paper to achieve concentration or selection has been a challenge for functional analysis. Here, by combining paper-based microfluidics with acoustics, we present a rapid and powerful method to size dependently control movement of microparticles and cells in paper using surface acoustic waves (SAW). We demonstrate the unique capability of the paper-based SAW approach to trap and concentrate microparticles in paper and release them when required, achieving collection efficiency of over 98%. Given the correlation between collection efficiency, size, and applied power, the paper-based SAW approach is applied to isolate a mixture of microparticles (1.1, 3.2, and 5 µm in diameter) into different regions and also to trap and concentrate human prostate cancer PC3 cells at a predetermined site. This paper-based SAW approach provides opportunities to develop powerful and low-cost selection and analysis tools, capable of processing complex multicomponent samples, with potential applications in medical diagnostics.

Active Micropump-Mixer for Rapid Antiplatelet Drug Screening in Whole Blood.

There is a need for scalable automated lab-on-chip systems incorporating precise hemodynamic control that can be applied to high-content screening of new more efficacious antiplatelet therapies. This paper reports on the development and characterization of a novel active micropump-mixer microfluidic to address this need. Using a novel reciprocating elastomeric micropump design, we take advantage of the flexible structural and actuation properties of this framework to manage the hemodynamics for on-chip platelet thrombosis assay on type 1 fibrillar collagen, using whole blood. By characterizing and harnessing the complex three-dimensional hemodynamics of the micropump operation in conjunction with a microvalve controlled reagent injection system we demonstrate that this prototype can act as a real-time assay of antiplatelet drug pharmacokinetics. In a proof-of-concept preclinical application, we utilize this system to investigate the way in which rapid dosing of human whole blood with isoform selective inhibitors of phosphatidylinositol 3-kinase dose dependently modulate platelet thrombus dynamics. This modular system exhibits utility as an automated multiplexable assay system with applications to high-content chemical library screening of new antiplatelet therapies.

Bubble oscillations at low frequency ultrasound for biological applications.

Bubbles oscillating in the presence of ultrasound is commonly employed in biomedical applications for drug delivery, ultrasound enhanced thrombolysis, and the transport and manipulation of cells. This is possible because bubbles tend to interact with the ultrasound to undergo periodic shape changes known as shape-mode oscillation, concomitant with the generation of liquid agitation or streaming. This phenomenon is examined both experimentally and theoretically on a single bubble at a frequency of (45 ± 1) kHz. Effects of ultrasonic frequency and power on the flowfield were explored. Experiments revealed different trends in the development of liquid streaming velocities at different acoustic forcing conditions (5.53, 6.80 and 7.02 Vpp), with lowest (0.5 mm/s) and highest (1.1 mm/s) values of time-averaged mean streaming velocity occurring at 6.80 Vpp and 7.02 Vpp, respectively. Simulations captured the simultaneous evolution of bubble-shapes that helped create flow vortices in the liquid surrounding the bubble. These vortices collectively responsible in generating signature patterns in the liquid for a dominant shape-mode of the bubble, and could also generate localised shear stresses for practical application. The velocity and pressure profiles in the liquid around the bubble confirmed the connection of the applied and reflected soundwaves in driving this phenomenon.

Characterisation of hydrodynamic trapping in microfluidic cross-slot devices for high strain rate applications.

Hydrodynamic trapping of a particle or cluster of particles based on contact and non-contact approaches has brought prominent insights to micro-nano scale applications. Of the non-contact methods, image-based real-time control in cross-slot microfluidic devices is one of the most promising potential platform for single cellular assays. Here, we report results from experiments conducted in two cross-slot microfluidic channels of different widths, with varying real-time delay of the control algorithm and different magnification. Sustained trapping of 5 µm diameter particles was achieved with high strain rates, of order 102 s-1, higher than in any previous studies. Our experiments show that the maximum attainable strain rate is a function of the real-time delay of the control algorithm and the particle resolution (pixel/µm). Therefore, we anticipate that with further reduced time delays and enhanced particle resolution, considerably higher strain rates can be attained, opening the platform to single cellular assay studies where very high strain rates are required.

A microfluidic method to investigate platelet mechanotransduction under extensional strain.

Background: Blood platelets have evolved a complex mechanotransduction machinery to rapidly respond to hemodynamic conditions. A variety of microfluidic flow-based approaches have been developed to explore platelet mechanotransduction; however, these experimental models primarily focus on the effects of increased wall shear stress on platelet adhesion events and do not consider the critical effects of extensional strain on platelet activation in free flow. Objectives: We report the development and application of a hyperbolic microfluidic assay that allows for investigation of platelet mechanotransduction under quasi-homogenous extensional strain rates in the absence of surface adhesions. Methods: Using a combined computational fluid dynamic and experimental microfluidic approach, we explore 5 extensional strain regimes (geometries) and their effect on platelet calcium signal transduction. Results: We demonstrate that in the absence of canonical adhesion, receptor engagement platelets are highly sensitive to both initial increase and subsequent decrease in extensional strain rates within the range of 747 to 3319/s. Furthermore, we demonstrate that platelets rapidly respond to the rate of change in extensional strain and define a threshold of ≥7.33 × 106/s/m, with an optimal range of 9.21 × 107 to 1.32 × 108/s/m. In addition, we demonstrate a key role of both the actin-based cytoskeleton and annular microtubules in the modulation of extensional strain-mediated platelet mechanotransduction. Conclusion: This method opens a window onto a novel platelet signal transduction mechanism and may have potential diagnostic utility in the identification of patients who are prone to thromboembolic complications associated with high-grade arterial stenosis or are on mechanical circulatory support systems, for which the extensional strain rate is a predominant hemodynamic driver.

Are surgical masks manufactured from sterilisation wrap safe?

BACKGROUND: Due to regional shortages some health services have proposed using surgical masks manufactured from sterilisation wrap. However, there has been little assessment of the safety of this practice. Therefore, we developed our own prototypes and evaluated whether they met regulatory standards. METHODS: Surgical mask prototypes were manufactured from two thickness grades of commercial sterilisation wrap. Safety was assessed in the context of regulatory standards. As it was not previously reported, we developed and performed differential pressure and synthetic blood penetration resistance experiments in accordance with official methodology. RESULTS: Bacterial filtration efficiency was comparable between sterilisation wrap and commercial surgical masks. Both prototypes met regulatory standards for synthetic blood resistance, whilst only our thinner mask fulfilled acceptable differential pressure ('breathability') thresholds. CONCLUSION: Acceptable barrier and breathability properties can be achieved with surgical masks produced from sterilisation wrap. Therefore, this may be a reasonable method to supplement stock if required. Unless there are shortages mandating alternatives, health-care workers should always use approved personal protective equipment.

Elastomeric microvalve geometry affects haemocompatibility.

This paper reports on the parameters that determine the haemocompatibility of elastomeric microvalves for blood handling in microfluidic systems. Using a comprehensive investigation of blood function, we describe a hierarchy of haemocompatibility as a function of microvalve geometry and identify a "normally-closed" v-gate pneumatic microvalve design that minimally affects blood plasma fibrinogen and von Willebrand factor composition, minimises effects on erythrocyte structure and function, and limits effects on platelet activation and aggregation, while facilitating rapid switching control for blood sample delivery. We propose that the haemodynamic profile of valve gate geometries is a significant determinant of platelet-dependent biofouling and haemocompatibility. Overall our findings suggest that modification of microvalve gate geometry and consequently haemodynamic profile can improve haemocompatibility, while minimising the requirement for chemical or protein modification of microfluidic surfaces. This biological insight and approach may be harnessed to inform future haemocompatible microfluidic valve and component design, and is an advance towards lab-on-chip automation for blood based diagnostic systems.