Cd4 T-Lynphocyte count associated with patient infected with Typei &Typeii HIV attending Malali clinic and maternity, Kaduna Kaduna - Nigeria

Background: More than 25 million people in developing countries are living with HIV infection. An enormous global effort is now underway to bring antiretroviral treatment to at least 2.5 million of those infected. While drug prices have dropped considerably, the cost and technical complexity of laboratory tests essential for the management of HIV disease, such as CD4 cell counts, remain prohibitive. New, simple, and affordable methods for measuring CD4 cells that can be implemented in resource-scarce settings are urgently needed [5, 6]. Methods and Findings: Here we describe the development of a prototype for a simple, rapid, and affordable method for counting CD4 lymphocytes. Microliter volumes of blood without further sample preparation are stained with fluorescent antibodies, captured on a membrane within a minimized flow cell and imaged through microscope optics with the type of charge-coupled device developed for digital camera technology [10]. An associated computer algorithm converts the raw digital image into absolute CD4 counts and CD4 percentages in real time. The accuracy of this prototype system was validated through testing in the United States and Botswana, and showed close agreement with standard flow cytometry (r = 0.95) over a range of absolute CD4 counts, and the ability to discriminate clinically relevant CD4 count thresholds with high sensitivity and specificity [1,4]

Detection of SARS-CoV-2 Specific Memory B cells to Delineate Long-Term COVID-19 Immunity

Background: The ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, represents a serious worldwide health concern. A deeper understanding of the immune response to SARS-CoV-2 will be required to refine vaccine development and efficacy as well as to evaluate long-term immunity in convalescent patients. With this in mind, we investigated the formation of SARS-CoV-2 specific BMEMORY cells from patient blood samples. Methods: A standard flow cytometry-based protocol for the detection of SARS-CoV-2 specific B cells was applied using fluorochrome-coupled SARS-CoV-2 spike (S) full-length protein. Cohorts of 26 central European convalescent mild/moderate COVID-19 patients and 14 healthy donors were assessed for the levels of SARS-CoV-2 S- specific BMEMORY cells. Results: Overall B cell composition was not affected by SARS-CoV-2 infection in convalescent patients. Our analysis of SARS-CoV-2 specific BMEMORY cells in samples collected at different time points revealed that S-protein specific B cells remain in peripheral blood at least up to 6 months after COVID-19 diagnosis. Conclusions: Detection of SARS-CoV-2 specific BMEMORY cells may improve our understanding of the long-term adaptive immunity in response to SARS-CoV-2, allowing for an improved public health response and vaccine development during the COVID-19 pandemic. Further validation of the study in larger and more diverse populations and a more extended observation period will be required.

De novo identification and visualization of important cell populations for classic Hodgkin lymphoma using flow cytometry and machine learning

ObjectivesAutomated classification of flow cytometry data has the potential to reduce errors and accelerate flow cytometry interpretation. We desired a machine learning approach that is accurate, intuitively easy to understand, and highlights the cells that are most important in the algorithm’s prediction for a given case.MethodsWe developed an ensemble of convolutional neural networks (CNNs) for classification and visualization of impactful cell populations in detecting classic Hodgkin lymphoma, using two-dimensional (2D) histograms. Data from 977 and 245 clinical flow cytometry cases were used for training and testing, respectively. 78 non-gated 2D histograms were created per flow cytometry file. SHAP values were calculated to determine the most impactful 2D histograms and regions within the histograms. The SHAP values from all 78 histograms were then projected back to the original cells data for gating and visualization using standard flow cytometry software.ResultsThe algorithm achieved 67.7% recall (sensitivity), 82.4 % precision, and 0.92 AUROC. Visualization of the important cell populations in making individual predictions demonstrated correlations with known biology.ConclusionsThe method presented enables model explainability while highlighting important cell populations in individual flow cytometry specimens, with potential applications in both diagnosis and discovery of previously overlooked key cell populations.

Visual and fluorescence characterisation of particulate aerosols in ice cores with imaging flow cytometry

<p>Current efforts to examine and quantify so-called ‘biomarkers’ present in polar ice samples offer exciting potential as biological and biochemical proxies for past climate and ocean dynamics. Here we present a new rapid and easily replicable method to provide measurements of the microscopic particulate content of ice samples from polar environments. Using an Amnis® Imagestream® Imaging Flow Cytometer, melted snow and ice samples from Patriot Hills in the Ellsworth Mountains, Antarctica were analysed for their particulate (biological and non-biological) content. Selective use of a nucleic acid stain pre-treatment allows for a straightforward gating strategy that resolves both autofluorescent and non-autofluorescent biological material in sample replicates. In the Patriot Hills samples this method clearly identifies marine picoplankton, along with non-biological particulates such as tephra and minerogenic material. Crucially, the 60x Brightfield images provided by the Imagestream offer a significant additional capability above standard flow cytometry systems; each object identified by the machine can be visually differentiated (automatically or manually) from particulates with similar fluorescence properties. Back-trajectory analysis with the NOAA Hybrid Single-Particle Lagrangian Integrated Trajectory (HySPLIT) model indicates that these ice-bound marine organisms originate from the Weddell and Amundsen-Bellingshausen Seas. This technique, when paired with established chemical and biochemical methods, shows considerable potential in providing valuable information about the nature and origin of aerosols and biomarker signals trapped in past ice layers.</p>

Cultured pluripotent planarian stem cells retain potency and express proteins from exogenously introduced mRNAs

Planarians possess naturally occurring pluripotent adult somatic stem cells (neoblasts) required for homeostasis and whole–body regeneration. However, methods for culturing neoblasts are currently unavailable, hindering both mechanistic studies of potency and the development of transgenic tools. We report the first robust methodologies for culturing and delivering exogenous mRNA into neoblasts. We identified culture media for maintaining neoblasts in vitro, and showed via transplantation that the cultured stem cells retained pluripotency. By modifying standard flow cytometry methods, we developed a new procedure that significantly improved yield and purity of neoblasts. These methods facilitated the successful introduction and expression of exogenous mRNAs in neoblasts, overcoming a key hurdle impeding the application of transgenics in planarians. The tissue culture advances reported here create new opportunities to advance detailed mechanistic studies of adult stem cell pluripotency in planarians, and provide a systematic methodological framework to develop cell culture techniques for other emerging research organisms.

Analysis of serous body fluids using the CELL-DYN Sapphire hematology analyzer

Correct cell enumeration and differential analysis of body fluids are important in the diagnosis and management of several diseases. Currently, microscopic analysis is still considered the “gold standard”. The aim of the present study was to evaluate the analytical performance of the CELL-DYN Sapphire hematology analyzer for automated differentiation of cells in serous fluids and to explore whether manual analysis of the raw data files could improve the differential count compared with reference microscopy.A total of 105 serous fluids (39 peritoneal and 66 pleural effusions) were analyzed by the CELL-DYN Sapphire using standard whole-blood algorithm. Additionally, we performed optimized manual gating of the Sapphire raw data file using standard flow cytometry software.The standard Sapphire algorithm showed substantial deviations from the reference microscopic differentiation: polymorphonuclear cell counts were too high because they contained some monocytic cells. However, when optimized manual gating strategy is used, a good correlation and negligible bias were found.We have demonstrated that with a modified algorithm, CELL-DYN Sapphire will provide reliable identification and enumeration of blood cells in peritoneal and pleural fluids