Effect of swab pooling on the Accula point-of-care RT-PCR for SARS-CoV-2 detection

IntroductionSwab pooling may allow for more efficient use of point-of-care assays for SARS-CoV-2 detection in settings where widespread testing is warranted, but the effects of pooling on assay performance are not well described.MethodsWe tested the Thermo-Fisher Accula rapid point-of-care RT-PCR platform with contrived pooled nasal swab specimens.ResultsWe observed a higher limit of detection of 3,750 copies/swab in pooled specimens compared to 2,250 copies/swab in individual specimens. Assay performance appeared worse in a specimen with visible nasal mucous and debris, although performance was improved when using a standard laboratory mechanical pipette compared to the transfer pipette included in the assay kit.ConclusionClinicians and public health officials overseeing mass testing efforts must understand limitations and benefits of swab or sample pooling, including reduced assay performance from pooled specimens. We conclude that the Accula RT-PCR platform remains an attractive candidate assay for pooling strategies owing to the superior analytical sensitivity compared to most home use and point-of-care tests despite the inhibitory effects of pooled specimens we characterized

Human Skeletal Muscle Study Through 2D and 3D Model Design

Through the creation of a 2D and 3D model of human skeletal muscle, structural and functional data can be collected. The initiation mechanisms of Facioscapulohumeral muscular dystrophy’s (FSHD), a genetic dystrophy that causes increasing muscle degeneration, can be identified through the creation of a reproducible model. Here, we share how the details of various facets and properties of the 2D and 3D design affect a human skeletal muscle in vitro. The specifics of how various extracellular matrix play a role in 2D patterning of skeletal muscle is discussed alongside the intricacies of creating a hydrogel based 3D skeletal muscle bundle. While the models have not yet reached optimal design compared to other techniques, this design is close to reaching a state to produce collectible data

Human Skeletal Muscle Study Through 2D and 3D Model Design

Through the creation of a 2D and 3D model of human skeletal muscle, structural and functional data can be collected. The initiation mechanisms of Facioscapulohumeral muscular dystrophy’s (FSHD), a genetic dystrophy that causes increasing muscle degeneration, can be identified through the creation of a reproducible model. Here, we share how the details of various facets and properties of the 2D and 3D design affect a human skeletal muscle in vitro. The specifics of how various extracellular matrix play a role in 2D patterning of skeletal muscle is discussed alongside the intricacies of creating a hydrogel based 3D skeletal muscle bundle. While the models have not yet reached optimal design compared to other techniques, this design is close to reaching a state to produce collectible data

Human Skeletal Muscle Study Through 2D and 3D Model Design

Through the creation of a 2D and 3D model of human skeletal muscle, structural and functional data can be collected. The initiation mechanisms of Facioscapulohumeral muscular dystrophy’s (FSHD), a genetic dystrophy that causes increasing muscle degeneration, can be identified through the creation of a reproducible model. Here, we share how the details of various facets and properties of the 2D and 3D design affect a human skeletal muscle in vitro. The specifics of how various extracellular matrix play a role in 2D patterning of skeletal muscle is discussed alongside the intricacies of creating a hydrogel based 3D skeletal muscle bundle. While the models have not yet reached optimal design compared to other techniques, this design is close to reaching a state to produce collectible data

Striated myocyte structural integrity: Automated analysis of sarcomeric z-discs.

As sarcomeres produce the force necessary for contraction, assessment of sarcomere order is paramount in evaluation of cardiac and skeletal myocytes. The uniaxial force produced by sarcomeres is ideally perpendicular to their z-lines, which couple parallel myofibrils and give cardiac and skeletal myocytes their distinct striated appearance. Accordingly, sarcomere structure is often evaluated by staining for z-line proteins such as α-actinin. However, due to limitations of current analysis methods, which require manual or semi-manual handling of images, the mechanism by which sarcomere and by extension z-line architecture can impact contraction and which characteristics of z-line architecture should be used to assess striated myocytes has not been fully explored. Challenges such as isolating z-lines from regions of off-target staining that occur along immature stress fibers and cell boundaries and choosing metrics to summarize overall z-line architecture have gone largely unaddressed in previous work. While an expert can qualitatively appraise tissues, these challenges leave researchers without robust, repeatable tools to assess z-line architecture across different labs and experiments. Additionally, the criteria used by experts to evaluate sarcomeric architecture have not been well-defined. We address these challenges by providing metrics that summarize different aspects of z-line architecture that correspond to expert tissue quality assessment and demonstrate their efficacy through an examination of engineered tissues and single cells. In doing so, we have elucidated a mechanism by which highly elongated cardiomyocytes become inefficient at producing force. Unlike previous manual or semi-manual methods, characterization of z-line architecture using the metrics discussed and implemented in this work can quantitatively evaluate engineered tissues and contribute to a robust understanding of the development and mechanics of striated muscles

Striated myocyte structural integrity: Automated analysis of sarcomeric z-discs.

As sarcomeres produce the force necessary for contraction, assessment of sarcomere order is paramount in evaluation of cardiac and skeletal myocytes. The uniaxial force produced by sarcomeres is ideally perpendicular to their z-lines, which couple parallel myofibrils and give cardiac and skeletal myocytes their distinct striated appearance. Accordingly, sarcomere structure is often evaluated by staining for z-line proteins such as α-actinin. However, due to limitations of current analysis methods, which require manual or semi-manual handling of images, the mechanism by which sarcomere and by extension z-line architecture can impact contraction and which characteristics of z-line architecture should be used to assess striated myocytes has not been fully explored. Challenges such as isolating z-lines from regions of off-target staining that occur along immature stress fibers and cell boundaries and choosing metrics to summarize overall z-line architecture have gone largely unaddressed in previous work. While an expert can qualitatively appraise tissues, these challenges leave researchers without robust, repeatable tools to assess z-line architecture across different labs and experiments. Additionally, the criteria used by experts to evaluate sarcomeric architecture have not been well-defined. We address these challenges by providing metrics that summarize different aspects of z-line architecture that correspond to expert tissue quality assessment and demonstrate their efficacy through an examination of engineered tissues and single cells. In doing so, we have elucidated a mechanism by which highly elongated cardiomyocytes become inefficient at producing force. Unlike previous manual or semi-manual methods, characterization of z-line architecture using the metrics discussed and implemented in this work can quantitatively evaluate engineered tissues and contribute to a robust understanding of the development and mechanics of striated muscles

Striated myocyte structural integrity: Automated analysis of sarcomeric z-discs

As sarcomeres produce the force necessary for contraction, assessment of sarcomere order is paramount in evaluation of cardiac and skeletal myocytes. The uniaxial force produced by sarcomeres is ideally perpendicular to their z-lines, which couple parallel myofibrils and give cardiac and skeletal myocytes their distinct striated appearance. Accordingly, sarcomere structure is often evaluated by staining for z-line proteins such as α-actinin. However, due to limitations of current analysis methods, which require manual or semi-manual handling of images, the mechanism by which sarcomere and by extension z-line architecture can impact contraction and which characteristics of z-line architecture should be used to assess striated myocytes has not been fully explored. Challenges such as isolating z-lines from regions of off-target staining that occur along immature stress fibers and cell boundaries and choosing metrics to summarize overall z-line architecture have gone largely unaddressed in previous work. While an expert can qualitatively appraise tissues, these challenges leave researchers without robust, repeatable tools to assess z-line architecture across different labs and experiments. Additionally, the criteria used by experts to evaluate sarcomeric architecture have not been well-defined. We address these challenges by providing metrics that summarize different aspects of z-line architecture that correspond to expert tissue quality assessment and demonstrate their efficacy through an examination of engineered tissues and single cells. In doing so, we have elucidated a mechanism by which highly elongated cardiomyocytes become inefficient at producing force. Unlike previous manual or semi-manual methods, characterization of z-line architecture using the metrics discussed and implemented in this work can quantitatively evaluate engineered tissues and contribute to a robust understanding of the development and mechanics of striated muscles

iCLOTS: open-source, artificial intelligence-enabled software for analyses of blood cells in microfluidic and microscopy-based assays

Abstract While microscopy-based cellular assays, including microfluidics, have significantly advanced over the last several decades, there has not been concurrent development of widely-accessible techniques to analyze time-dependent microscopy data incorporating phenomena such as fluid flow and dynamic cell adhesion. As such, experimentalists typically rely on error-prone and time-consuming manual analysis, resulting in lost resolution and missed opportunities for innovative metrics. We present a user-adaptable toolkit packaged into the open-source, standalone Interactive Cellular assay Labeled Observation and Tracking Software (iCLOTS). We benchmark cell adhesion, single-cell tracking, velocity profile, and multiscale microfluidic-centric applications with blood samples, the prototypical biofluid specimen. Moreover, machine learning algorithms characterize previously imperceptible data groupings from numerical outputs. Free to download/use, iCLOTS addresses a need for a field stymied by a lack of analytical tools for innovative, physiologically-relevant assays of any design, democratizing use of well-validated algorithms for all end-user biomedical researchers who would benefit from advanced computational methods

Multiplatform analyses reveal distinct drivers of systemic pathogenesis in adult versus pediatric severe acute COVID-19

In this work, authors take a multiomics and microfluidics-based approach to elucidate the mechanism of endothelial damage in critical illness associated with SARS-CoV-2