fluorescent activated cell sorting: diagnosis of hiv infection
TRANSCRIPT
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FLUORESCENT ACTIVATED CELL
SORTING Present by,
Muhammad Ashraf Bin MazlanCalvin Lim Lai Hock
Soon Tsuey NingChia Kay Veng
Liew Lee Bing
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BASIC CONCEPTS OF FACS
Measurement of cells in a flow system - delivers the cell singly past a point of measurement- light is focused
FACS enables sorting of a population of cells into subpopulations.- based on fluorescent labeling- sorting involves mechanisms in flow cytometer
Antibodies bond with specific protein are coated with a fluorescent dye.
Very effective
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Principles of Fluorescent Activated Cell Sorting
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History
- In 1980s, immunophenotyping rise as CD4 T cells was killed.
- Antibodies for CD3, CD4 and CD8 are among the first characterised monoclonal antibodies
- AIDS First major clinical application Flow cytometry
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Applications of FACS in Diagnosis of HIV
infection
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Diagnosis of Immunodeficiency disease
● Separation of T-lymphocytes subsets
● Fluorochrome + monoclonal antibodies + antigen → fluorescence
● Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cells ratio.
● AIDS development CD4+ T cells (~200 cells/μL)
● Normal CD4+ T cells level = 1000 cells/μL
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Monoclonal antibody cloning using FACS
Immunotherapy for HIV infection● Broad neutralizing antibodies.
● Protect against SIVs that express HIV-1 envelope glycoproteins
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SimilaritiesFLOW CYTOMETRY FACS
- Fluorescent labels, cellular propertiesDifferences
Commonly found in research labs, user-friendly
More personalized, designed for researcher
Cannot sort cells Sort heterogeneous mixture of cells into different populations
Intuitive software interfaces
Automated features
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Current Development
Derived of smaller and lower cost equipments
PanLeucogating technologyAnchor marker
Validation of small instrumentsCell count errorOutline of the procedures
Challenges
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Conclusion➔ Fluorescent activated cell sorting
(FACS) is a specialized type of flow cytometry, It allows multiparametric analysis of heterogeneous cells populations based on their cellular characteristics
➔ Diagnosis of HIV Infection.
◆ separation of subsets of T-lymphocytes
◆ Fluorescent marker ---> identify cell type ---> individual antigenic marker
◆ Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cells ratio.
➔ Monoclonal antibody cloning.
◆ immunotherapy for HIV infection.
➔ Current development
◆ Producing compact and cheap equipments.
◆ PanLeucogating technology
◆ Anchor marker
➔ Challenges
◆ cell count error
◆ outline of the procedures
◆ validation of small instrumets.
➔ FACS can be utilize in analysis and treating HIV infection.
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ReferencesAbcam, 2011, Fluorescence activated cell sorting of live cells. Available from:
http://www.abcam.com/protocols/fluorescence-activated-cell-sorting-of-live-cells [23 September 2016]Flowbook.denovosoftware.com, 2016, Chapter 1: Introduction | Flow Cytometry - A Basic Introduction.
Available at: http://flowbook.denovosoftware.com/chapter-1-introduction-0 [26 September 2016]Mukherjee A, 2011, Fluorescent-Activated Cell Sorting. Available from:
http://www.biotecharticles.com/Applications-Article/Fluorescence-Activated-Cell-Sorting-570.html [29 September 2016]
Parks D & Perez O, 2002, The History and Future of the Fluorescence Activated Cell Sorter and Flow Cytometry: A View from Stanford. Available from: http://clinchem.aaccjnls.org/content/48/10/1819 [29 September 2016]
Robinson R, Pellenz S, 2013, What is flow cytometry (FACS analysis)?, Available from: http://www.antibodies-online.com/resources/17/1247/what-is-flow-cytometry-facs-analysis. [24 September 2016]
VenturiOne, 2012, Flow Cytometry. Available from: http://www.appliedcytometry.com/flow_cytometry.php [29 September 2016]