China
Africa
Explore chapters and articles related to this topic
Methods in exercise immunology
Published in R. C. Richard Davison, Paul M. Smith, James Hopker, Michael J. Price, Florentina Hettinga, Garry Tew, Lindsay Bottoms, Sport and Exercise Physiology Testing Guidelines: Volume II – Exercise and Clinical Testing, 2022
Nicolette C. Bishop, Neil P. Walsh
More detailed assessment of immune cell types and their receptors can be determined using flow cytometry. This is a method of single-cell analysis, whereby the cell population is suspended in a clear saline solution and funnelled through a nozzle to create a stream of single cells. The cells flow past a set of laser sources and as the light hits each cell it is scattered in different directions; this gives an indication of the cell morphology. Light scattered in a forward direction (‘forward scatter’) gives an indication of cell size; bigger cells will cause more scattering. Light scattered sideways (‘side scatter’) gives an indication of volume of cytoplasmic granules inside the cell. The degree of scattering is detected, converted into an electrical signal, and visualised by the cytometer software; hence, populations of cells with similar properties are clustered together. Because the three main circulating immune cell types (neutrophils, monocytes and lymphocytes) differ in size and granularity, plotting forward scatter against side scatter allows the identification of these different cell populations (Figure 4.4.1). Specific cell subpopulations within these populations can be subsequently identified by ‘tagging’ receptors on the surface of the cells using fluorescent emitting chemicals (fluorochromes) joined to antibodies which then bind to cell surface receptors. Once excited by the laser, each fluorochrome emits light at a specific wavelength and is gathered by the flow cytometer’s detectors.
Laboratory techniques to study the cellular and molecular processes of disorders
Published in Louis-Philippe Boulet, Applied Respiratory Pathophysiology, 2017
Quantitative flow cytometry was first invented in the 1950s and it is commonly used to detect microorganisms, organelles, and cell types in a sample [19,20]. Samples are first suspended in a biological fluid at high speed and subjected to a constant flux. The criteria for target selection may be a general parameter such as size and granularity or molecule-specific. Fluorophore or antibody conjugated with a fluorophore is added into the solution depending on the selection criteria. A single particle or cell is forced through an opening and illuminated by a light source, generally a laser. As each particle passes the light source, the incoming light is scattered and the intensity of the scatter is detected and transformed into a voltage pulse. Scatters can be described as either forward or side. The intensity of the forward scatter is proportional to the size of the particle or cell, and the side scatter is proportional to the granularity or complexity within the cell.
Flow Cytometry
Published in Wojciech Gorczyca, Atlas of Differential Diagnosis in Neoplastic Hematopathology, 2014
In FC analysis, cells are tagged with fluorochrome-conjugated monoclonal antibodies directed toward specific surface, cytoplasmic, or nuclear antigens. Intrinsic physical properties of the cells, especially their size and cytoplasmic granularity, are measured simultaneously with fluorescence emission as the fluorochrome-tagged cells pass through laser light. The side scatter (SSC; right-angle/orthogonal light scatter) corresponds to the granularity of the cytoplasm (y-axis in Figure 3.1). The cells with agranular cytoplasm (i.e., lymphocytes) have low SSC, whereas the cells with granular cytoplasm (i.e., neutrophils, eosinophils, hypergranular promyelocytes) have high SSC. Forward scatter (FSC; forward angle light scatter) corresponds to the cell size (Figures 3.2 and 3.3). Large cells have higher FSC than smaller cells: Note the difference between myeloblasts and monoblasts in Figure 3.2 and the difference among small lymphocytes (red dots), large lymphocytes (blue dots), and cancer cells (orange dots) in Figure 3.3.
The utility of flow cytometric platelet forward scatter as an alternative to mean platelet volume
Published in Platelets, 2022
David Connor, David Rabbolini, Marie-Christine Morel-Kopp, Kate Fixter, Dea Donikian, Mayuku Kondo, Onki Chan, Susan Jarvis, Walter Chen, Timothy Brighton, Vivien Chen, Christopher Ward, Joanne Joseph
There are potential limitations to the use of FSC measurements to classify platelet size. There are few reference controls to measure day-to-day performance of individual flow cytometers. Standardized beads may be used, however, conventional latex beads (or similar) have a refractive index that may not necessarily match the refractive index of platelets. Forward scatter settings and results will also vary widely between laboratories and models of flow cytometer. The reference ranges described in this study are applicable for this study and for this individual analyzer only. Each laboratory is required to establish their own reference ranges for each individual analyzer and develop protocols for monitoring of day-to-day variability in forward scatter parameter settings. A method of normalization would also be required to compare data between machines.
Guidelines for panel design, optimization, and performance of whole blood multi-color flow cytometry of platelet surface markers
Published in Platelets, 2020
Xavier Busuttil-Crellin, Conor McCafferty, Suelyn Van Den Helm, Hui Ping Yaw, Paul Monagle, Matthew Linden, Vera Ignjatovic
Parameters such as forward-scatter (FSC) to determine size, and side-scatter (SSC) to determine granularity can be used to discriminate between populations of cells. The unique characteristics of platelets makes flow cytometry perfectly suited for their identification. The use of fluorescently labeled antibodies attached to antigens of interest provides detailed information of antigenic abundance and alludes to the functional physiology of platelets. For example, mean fluorescence intensity (MFI), the brightness intensity of an event, can be used to compare antigen presentation levels. Additionally, percentage positive provides a measure of total events that present an antigen of interest. With these factors in mind, multi-colored flow cytometry is an attractive technique for the characterization of platelets and their receptor phenotype.
Y-RNA subtype ratios in plasma extracellular vesicles are cell type- specific and are candidate biomarkers for inflammatory diseases
Published in Journal of Extracellular Vesicles, 2020
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, Esther N.M. Nolte-‘T Hoen
High-resolution flow cytometric analysis of PKH67-labelled EV was performed on a BD Influx flow cytometer (BD Biosciences, San Jose, CA) with an optimized configuration for small particle analysis as previously described [50,51]. We applied fluorescence threshold triggering to discriminate PKH67 labelled EV from non-fluorescent noise signals. Forward scatter (FSC) was detected at a 15–25 degree collection angle. Fluorescent polystyrene 100 and 200 nm beads (FluoSpheres, Invitrogen, Carlsbad, CA) were used to calibrate the fluorescence and reduced width-FSC settings before each measurement. Sucrose gradient fractions were diluted 10–20 times in PBS and vortexed just before measurement. Samples were measured at maximally 10,000 events per second, which is far below the electronic pulse processing limit of the BD Influx [52]. Serial dilutions of peak fractions were included to control for potential “invisible swarm” effects [53].