Protocol for inspecting blood cell dynamics with a custom ektacytometer-rheometer apparatus.

Investigating flowing red blood cell (RBC) morphology and orientation is important for elucidating physiology and disease; existing commercially available products are limited to observing cell populations or single cells. In this protocol, we create a custom apparatus that combines coaxial brightfield microscopy with laser diffractometry to inspect near-real-time deformability, morphology, and orientation of flowing RBCs. There are difficulties associated with building optical systems for biological inspection; however, this protocol provides a suitable framework for developing an "ektacytoscope" for studying blood cells. For complete details on the use and execution of this protocol, please refer to McNamee et al. (2020).

Picomolar detection of carbohydrate-lectin interactions on piezoelectrically printed microcantilever array.

Recent advances in micro-electromechanical systems (MEMS) has allowed unprecedent perspectives for label-free detection (LFD) of biological and chemical analytes. Additionally, these LFD technologies offer the potential to design high resolution and high throughput sensing platforms, with the promise of further miniaturization. However, the immobilization of biomolecules onto inorganic surfaces without impacting their sensing abilities is crucial for designing these LFD technologies. Currently, covalent functionalization of self-assembled monolayers (SAMs) present promising pathways for improving assay sensitivity, reproducibility, surface stability and proximity of binding sites to the sensor surface. Herein, we investigate the use of chemical vapor deposition of 3-(glycidyloxypropyl)-trimethoxysilane (GOPTS) as a versatile SAM for the covalent functionalization of a SiO2 microcantilever array (MCA) for carbohydrate-lectin interactions with picogram sensitivity. Additionally, we demonstrate glycan immobilization to MCA is feasible using traditional piezoelectric microarray printer technology. Given the complexity of the glycome, the ability to spot samples in a high-throughput manner establishes our MCA as robust, label-free, and scalable means to analyze carbohydrate-protein interactions These findings demonstrate that GOPTS SAMs provide a suitable biofunctionalization route for MEMS and provides the proof of principle that can be extended to various LFD technologies toward a truly high-throughput and high-resolution platform.

Sublethal Supraphysiological Shear Stress Alters Erythrocyte Dynamics in Subsequent Low-Shear Flows.

Blood is a non-Newtonian, shear-thinning fluid owing to the physical properties and behaviors of red blood cells (RBCs). Under increased shear flow, pre-existing clusters of cells disaggregate, orientate with flow, and deform. These essential processes enhance fluidity of blood, although accumulating evidence suggests that sublethal blood trauma-induced by supraphysiological shear exposure-paradoxically increases the deformability of RBCs when examined under low-shear conditions, despite obvious decrement of cellular deformation at moderate-to-higher shear stresses. Some propose that rather than actual enhancement of cell mechanics, these observations are "pseudoimprovements" and possibly reflect altered flow and/or cell orientation, leading to methodological artifacts, although direct evidence is lacking. This study thus sought to explore RBC mechanical responses in shear flow using purpose-built laser diffractometry in tandem with direct optical visualization to address this problem. Freshly collected RBCs were exposed to a mechanical stimulus known to drastically alter cell deformability (i.e., prior shear exposure (PSE) to 100 Pa × 300 s). Samples were subsequently transferred to a custom-built slit-flow chamber that combined laser diffractometry with direct cell visualization. Cell suspensions were sheared in a stepwise manner (between 0.3 and 5.0 Pa), with each step being maintained for 15 s. Deformability and cell orientation indices were recorded for small-scatter Fraunhofer diffraction patterns and also visualized RBCs. PSE RBCs had significantly decreased visualized and laser-derived deformability at any given shear stress ≥1 Pa. Novel, to our knowledge, observations demonstrated that PSE RBCs had increased heterogeneity of direct visualized orientation with flow vector at any shear, which may be due to greater vorticity and thus instability in 5-Pa flow compared with unsheared control. These findings indicate that shear exposure and stress-strain history can alter subsequent RBC behavior in physiologically relevant low-shear flows. These findings may yield insight into microvascular disorders in recipients of mechanical circulatory support and individuals with hematological diseases that alter physical properties of blood.

Hospital preparedness and the terrorism alert system.

In an attempt to encourage some uniformity of preparation and response, the Department of Home Security issued its Terrorist Advisory Alert color codes. The authors have taken the Department's check-off form and amplified what can be done by healthcare facilities at each alert stage.