Fluorescent Activated Cell Sorting: Diagnosis of HIV Infection
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This presentation is prepared for Gene and Tissue Culture Technology. The tittle is discuss one application of FACS.
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Fluorescent Activated Cell Sorting: Diagnosis of HIV Infection
- 1. FLUORESCENT ACTIVATED CELL SORTING Present by, Muhammad Ashraf Bin Mazlan Calvin Lim Lai Hock Soon Tsuey Ning Chia Kay Veng Liew Lee Bing
- 2. 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
- 3. Principles of Fluorescent Activated Cell Sorting
- 4. 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
- 5. Applications of FACS in Diagnosis of HIV infection
- 6. 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
- 7. Monoclonal antibody cloning using FACS Immunotherapy for HIV infection ● Broad neutralizing antibodies. ● Protect against SIVs that express HIV-1 envelope glycoproteins
- 9. Similarities FLOW CYTOMETRY FACS - Fluorescent labels, cellular properties Differences 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
- 10. Current Development Derived of smaller and lower cost equipments PanLeucogating technology Anchor marker Validation of small instruments Cell count error Outline of the procedures Challenges
- 11. 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.
- 12. References Abcam, 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]
Hinweis der Redaktion
- FACS is an adaptation of flow cytometer which allows sorting or separating cell populations according to the fluorescence intensity and cell size.
- Flow Cytometry : http://flowbook.denovosoftware.com/chapter-1-introduction-0 - As the name implies, flow cytometry is the measurement of cells in a flow system, which delivers the cells singly past a point of measurement. - Light is focused at the point of measurement. The scattered light and fluorescence of different wavelengths are then recorded. Typically, light scatter at two different angles and from one to six or more fluorescences will be measured. - Fluorescent chemicals may used to label cell components, such as DNA, directly; others are attached to antibodies against a wide variety of cellular proteins. FACS : http://www.abcam.com/protocols/fluorescence-activated-cell-sorting-of-live-cells Effective - sort as many as 50 000 cells per second - not limited to one single sorting criterion Multiple lasers and detectors can be used to simultaneously sort by more than one factor.
- http://www.bio.davidson.edu/courses/genomics/method/facs.html The process begins by placing the cells into a flask and forcing the cells to enter a small nozzle one at a time (figure 1). The cells travel down the nozzle which is vibrated at an optimal frequency to produce drops at fixed distance from the nozzle. As the cells flow down the stream of liquid, they are scanned by a laser (blue light in figure 1). Some of the laser light is scattered (red cone emanating from the red cell) by the cells and this is used to count the cells. This scattered light can also be used to measure the size of the cells. If you wanted to separate a subpopulation of cells, you could do so by tagging those of interest with an antibody linked to a fluorescent dye. The antibody is bound to a protein that is uniquely expressed in the cells you want to separate. The laser light excites the dye which emits a color of light that is detected by the photomultiplier tube, or light detector. By collecting the information from the light (scatter and fluorescence) a computer can determine which cells are to be separated and collected. The final step is sorting the cells which is accomplished by electrical charge. The computer determines how the cells will be sorted before the drop forms at the end of the stream. As the drop forms, an electrical charge is applied to the stream and the newly formed drop will form with a charge. This charged drop is then deflected left or right by charged electrodes and into waiting sample tubes. Drops that contain Figure 1. Diagram of FACS machine. Cells have been fluorescently tagged with either red or green antibodies, though not every cell expresses the epitope and therefore some are not tagged either color.
- HIV which causes AIDS killed CD4 T cells Membrane proteins expressed by two subsets of these T cells AIDS began to spread through the populations of the world, and with it, the first major clinical application of immunophenotyping by flow cytometry While immunophenotyping was initially directed to the measurement of membrane markers on cells, it became apparent that intracellular markers could also be measured. The ability to identify a cell population by membrane markers and simultaneously determine the cell’s function had now become a powerful application of flow cytometry.
- This application involved determination of the ratio of CD4+(helper) and CD8+ T cells (cytotoxic) may be a direct indicator of HIV progression in HIV positive patients.
- - It can be used for separation of subsets of T-lymphocytes, which is important for the diagnosis of immunodeficiency disease such as AIDS. - When a fluorescent marker is conjugated to a MAb, it can be used to identify a particular cell type based on the individual antigenic markers of the cell. This can be done by adding the conjugated Ab’s to a mixture of cells/particles. - Since it allows the sorting of cells that express specific antigen, monoclonal antibodies are used to determine CD4+ T cell counts after HIV infection. patients with HIV disease has a lower CD4+ (helper) T cells to CD8+ (cytotoxic) T cells ratio. This is because HIV specifically targets and binds to the CD4 antigen on CD4+ T cells to replicate, ultimately causing the destruction and deterioration of the immune system. In normal humans, CD4+ T cells are greater in number which is approx. 1000cells/μL. The monitoring of CD4+ T cells during the course of HIV infection is therefore a crucial indicator of when to initiate ARV therapy. Frequency of CD4+ T-cell monitoring can delay or even prevent ARV initiation.
- So they came up with immunotherapy for HIV infection by cloning monoclonal antibody using FACS. Few years ago, they proved that transfer of first generation broad neutralization antibodies (bNAbs) including b12, 2G12, 2F5 or 4E10 is able to protect against simian immunodeficiency viruses (SIVs) that express the HIV-1 envelope glycoproteins infection.
- single memory B cells were isolated using FACS based on surface expressed markers such as CD3, CD27 and CD19. Immunoglobulin (Ig) genes of isolated single B cells were amplified using single cell reverse transcription and polymerase chain reaction (RT-PCR). Amplified heavy- and light-chain antibody regions were cloned into eukaryotic expression vectors and transfected into human embryonic kidney (HEK) 293 or 293 T cells to produce monoclonal human antibodies of the same specificity in vitro. Recombinant antibodies were purified from supernatants and tested for antigen specificity at the end using ELISA. Recent clinical trial demonstrated that a single infusion of an experimental antibody 3BNC17 significantly reduced HIV levels in infected people for as long as 28 days
- Advantage : Cells are identified using fluorescent labels or cellular properties like relative cell size and complexity. Current-generation fluorescence-activated cell sorters are now smaller and have many automated features that make them more accessible to typical research labs. Like modern flow cytometers, they are easier to use and have intuitive software interfaces requiring less expertise to run. Differences : Flow cytometers are found commonly in research labs, user-friendly. Cell sorters more personalized and designed for the researcher Cell sorters can separate cells from unwanted cells based on properties, into heterogeneous populations for downstream studies.
- Derived of smaller and lower cost equipments for resource-constrained countries To overcome the issue of sampling aging Anchor marker : obtain an accurate and consistent gate check, enable analysis blood samples for longer periods post-phlebotomy, enabling samples to be transported over longer distance before analysis Challenges Increase number in non-leukocyte contamination, sample more than 36 hours old are hard to analyse Validation of small instruments is still await Outline of the procedure must be standardized https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728050/ http://onlinelibrary.wiley.com/doi/10.1002/cyto.10073/pdf