Flow cytometry: Principles and Applications

Flow cytometry: Principles and Applications

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  Flow Cytometry PRINCIPLES ANDAPPLICATIONS
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  What is FlowCytometry? ‘Flow Cytometry’ as the name suggests is a technique for cell counting and measurement of different properties of the cell (‘cyto’= cell; ‘metry’=count/measurement). It is a laser based technology that measures and analyses different physical and chemical properties of the cells/particles flowing in a stream of fluid through a beam of light. References: http://www.d.umn.edu/~biomed/flowcytometry/introflowcytometry.pdf
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  A Flow Cytometer Placeyour sample here
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  Historical Perspective: Evolutionof Flow Cytometry 17th Century 1934 1947 to 1949 1953 19651879 1968 1970s onwards… Development of light microscope by Leeuwenhoek. Principles of Droplet formation by Lord Rayleigh. Counting of RBCs by Moldavan by forcing a suspension of cells through capillary tube. Development of Coulter Principle by Wallace Coulter and counting of RBCs using the first Coulter Counter. Optical counting of RBCs by Crosland- Taylor by use of laminar flow principles Development of electrostatic inkjet droplet deflection by Richard Sweet Application of Sweet’s principle and Coulter principle to develop the first cell sorter by M. Fulwyler Development of fluorescence based cell sorter by Wolfgang Gohde Development of FACS and other advances.
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  Principles of workingof Flow Cytometer Coulter Principle Principle s of Laminar Flow Electro statics Optics & Light Scatteri ng Flow Cytome try
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  Components of aFlow Cytometer A flow cytometer is made up of three main systems: fluidics, optics, and electronics.  The fluidics system transports particles in a stream to the laser beam for interrogation.  The optics system consists of lasers to illuminate the particles in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors.  The electronics system converts the detected light signals into electronic signals that can be processed by the computer. For some instruments equipped with a sorting feature, the electronics system is also capable of initiating sorting decisions to charge and deflect particles. References: http://www.d.umn.edu/~biomed/flowcytometry/introflowcytometry.pdf
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  Working of aFlow Cytometer  In the flow cytometer, particles are carried to the laser intercept in a fluid stream. Any suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis.  The portion of the fluid stream where particles are located is called the sample core.  When particles pass through the laser intercept, they scatter laser light. Any fluorescent molecules present on the particle fluoresce.  The scattered and fluorescent light is collected by appropriately positioned lenses.  A combination of beam splitters and filters steers the scattered and fluorescent light to the appropriate detectors.  The detectors produce electronic signals proportional to the optical signals striking them. References: http://www.d.umn.edu/~biomed/flowcytometry/introflowcytometry.pdf
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  Applications of Flow Cytometry Flowcytometry is the sine qua non (without which, nothing)of the modern researcher’s toolbox. Flow cytometry measures multiple characteristics of individual particles flowing in single file in a stream of fluid. Light scattering at different angles can distinguish differences in size and internal complexity, whereas light emitted from fluorescently labeled antibodies can identify a wide array of cell surface and cytoplasmic antigens. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. References: Kuby Immunology, 7th Edition http://www.clinchem.org/content/46/8/1221.full
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  Applications Immunophenotyping Cell subsets aremeasured by labeling population-specific proteins with a fluorescent tag on the cell surface. In clinical labs, immunophenotyping is useful in diagnosing hematological malignancies such as lymphomas and leukemia. Cell Sorting The cell sorter is a specialized flow cytometer with the ability to physically isolate cells of interest into separate collection tubes. The sorter uses sophisticated electronics and fluidics to identify and "kick" the cells of interest out of the fluidic stream into a test tube. DNA Content Analysis The measurement of cellular DNA content by flow cytometry uses fluorescent dyes, such as propidium iodide, that intercalate into the DNA helical structure. The fluorescent signal is directly proportional to the amount of DNA in the nucleus and can identify gross gains or losses in DNA. Cell Cycle Analysis Flow cytometry can analyze replication states using fluorescent dyes to measure the four distinct phases of the cell cycle. Along with determining cell cycle replication states, the assay can measure cell aneuploidy associated with chromosomal abnormalities. Apoptosis The two distinct types of cell death, apoptosis and necrosis, can be distinguished by flow cytometry on the basis of differences in morphological, biochemical and molecular changes occurring in the dying cells. Cell Proliferation Assays The flow cytometer can measure proliferation by labeling resting cells with a cell membrane fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE). When the cells are activated, they begin to proliferate and undergo mitosis. As the cells divide, half of the original dye is passed on to each daughter cell. By measuring the reduction of the fluorescence signal, researchers can calculate cellular activation and proliferation. References: http://www.clinchem.org/content/46/8/1221.full http://www.seattlechildrens.org/research/cores/flow-cytometry/applications-of-flow-cytometry/
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  Fluorescence Activated Cell Sorting(FACS)  Consider a group of lymphocytes from a mouse that have been stained with green fluorescent antibodies specific for CD4 (e.g., fluorescein isothiocyanate, or FITC anti-CD4) and red fluorescent antibodies specific for CD8 (e.g., phycoerythrin, or PE anti-CD8).  Both the labeled cells generate SSC and FSC as they pass through the laser beam creating voltage pulses that are recorded by the computer.  However, each labeled cell will also emit light of specific wavelength as a result of the fluorescent label. For instance, CD4 cells will emit green fluorescent light of wavelength 525-530 nm while CD8 cells emit orange light of wavelength 560 nm. These fluorescent signals pass through the Photomultiplier tubes and generate voltage pulses.  The software integrates all the information for a particular cell allowing characterization of individual cells. References: Kuby Immunology, 7th Edition
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  Clinical Applications: DNA ContentAnalysis  Investigators are currently using techniques of DNA flow cytometry to measure ploidy status (DNA content) and proliferative potential (S phase fraction) in a wide variety of solid tumors. These measurements have shown relevance for diagnosis, prognosis, and treatment for patients with cancer.  The measurement of cellular DNA content by flow cytometry uses fluorescent dyes, such as propidium iodide, that intercalate into the DNA helical structure. The fluorescent signal is directly proportional to the amount of DNA in the nucleus and can identify gross gains or losses in DNA.  Abnormal DNA content, also known as “DNA content aneuploidy”, can be determined in a tumor cell population. DNA aneuploidy generally is associated with malignancy; however, certain benign conditions may appear aneuploid.  Cell Cycle Analysis: This technique is based on the premise that cells in G0 or G1 phases of the cell cycle possess a normal diploid chromosomal, and hence DNA content (2n) whereas cells in G2 and just prior to mitosis (M) contain exactly twice this amount (4n). As DNA is synthesized during S-phase, cells are found with a DNA content ranging between 2n and 4n. A histogram plot of DNA content against cell numbers gives the classical DNA profile for a proliferating cell culture. References: http://europepmc.org/abstract/med/2645625 http://www.clinchem.org/content/46/8/1221.full http://www.icms.qmul.ac.uk/flowcytometry/uses/cellcycleanalysis/cellcycle/index.html
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  Flow Cytometry andEcology  Assessments of diversity, abundance, and activity of water column microorganisms are fundamental to studies in aquatic microbiology.  Currently, most applications of flow cytometry to environmental samples make use of various morphological and physiological characteristics of the cells (e.g., size and pigment content of photosynthetic organisms).  These criteria generally are not sufficient for identification at the genus or species level. Staining with DNA-specific fluorochromes offers information about numbers of bacterial cells but not about their identity.  The combined use of dyes that bind preferentially to G- C or A. T base pairs has been used to distinguish organisms of different G+C content References: Appl.%20Environ.%20Microbiol.-1990-Amann-1919-25.pdf
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  Flow Cytometry andCancer Research  The prognosis of patients with cancer is largely determined by the specific histological diagnosis, tumor mass stage, and host performance status.  Quantitative cytology in the form of flow cytometry has greatly advanced the objective elucidation of tumor cell heterogeneity by using probes that discriminate tumor and normal cells and assess differentiate as well as proliferative tumor cell properties.  Both DNA content analysis and FACS can be utilised in cancer research.  Abnormal nuclear DMA content is a conclusive marker of malignancy and is found with increasing frequency in leukemia (23% among 793 patients), in lymphoma (53% among 360 patients), and in myeloma (76% among 177 patients), as well as in solid tumors (75% among 3611 patients), for an overall incidence of 67% in 4941 patients.  Flow cytometric immunophenotyping (FCI) aids in the differentiation of chronic lymphocytic leukemia (CLL) from mantle cell lymphoma (MCL); however, overlapping phenotypes may occur. CD11c expression has been reported in up to 90% of CLL cases but has rarely been reported in MCL. Whether CD11c can be used to exclude MCL has not been directly addressed. FCI reports were reviewed for 90 MCL cases (44 patients) and 355 CLL/small lymphocytic lymphoma (SLL) cases (158 patients). References: http://ajcp.ascpjournals.org/content/134/2/271.full.pdf+html http://cancerres.aacrjournals.org/content/43/9/3982.full.pdf+html
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  References  http://flowcytometry.berkeley.edu/pdfs/Basic%20Flow%20Cytometry.pdf  http://www.azom.com/article.aspx?ArticleID=6020 https://www.beckmancoulter.com/wsrportal/wsr/industrial/particle-technologies/coulter-principle/index.htm  http://www.cyto.purdue.edu/cdroms/cyto2/6/coulter/ss000103.htm  http://ajcp.ascpjournals.org/content/134/2/271.full.pdf+html  http://cancerres.aacrjournals.org/content/43/9/3982.full.pdf+html  Appl.%20Environ.%20Microbiol.-1990-Amann-1919-25.pdf  http://europepmc.org/abstract/med/2645625  http://www.clinchem.org/content/46/8/1221.full  http://www.icms.qmul.ac.uk/flowcytometry/uses/cellcycleanalysis/cellcycle/index.html  Kuby Immunology, 7th Edition  http://www.clinchem.org/content/46/8/1221.full  http://www.seattlechildrens.org/research/cores/flow-cytometry/applications-of-flow-cytometry  http://www.d.umn.edu/~biomed/flowcytometry/introflowcytometry.pdf
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  Thank You.