FLOW CYTOMETRY

GUIDED BY DR. A. BASU PRESENTED BY : SHIVAM SAXENA & NAGENDRA SHARMA M.PHARM ( Ist SEM ) SOPS - RGPVFLOW CYTOMETRY

CONTENTINTRODUCTIONHISTORYPRINCIPLE & WORKINGDIAGRAMGRAPH OF FLOW CYTOMETRYINSTRUMENTATIONAPPLICATIONSFUTURE EXPECTATIONSSUMMARYREFERNCES

INTRODUCTIONFlow cytometryis alaser- orimpedance-based, biophysical technology employed incell counting,cell sorting,biomarkerdetection andprotein engineering, by suspendingcellsin a stream of fluid and passing them by an electronic detection apparatus.It allows simultaneousmultiparametric analysisof the physical andchemicalcharacteristics of up to thousands of particles per second.A common variation is to physically sort particles based on their properties, so as to purify populations of interest.

HISTORY [2]Wallace H. Coulter was discovered the first impedance-based flow cytometry device, using theCoulter principle in 1953.Mack Fulwyler was the inventor of the forerunner to today's flow cytometers - particularly the cell sorter in 1965.The first fluorescence-based flow cytometry device (ICP 11) was developed in 1968 by Wolfgang Gohde from theUniversity of Munster.

PRINCIPLE & WORKING[1,3]Flow cytometry measures optical and fluorescence characteristics of single cells (or any other particle , including nuclei, microorganisms, chromosome preparations, and latex beads). Physical properties, such as size (represented by forward angle light scatter) and internal complexity (represented by right-angle scatter) can resolve certain cell populations.Fluorescent dyes may bind or intercalate with different cellular components such as DNA or RNA.Antibodies conjugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells.

When labeled cells are passed by a light source, the fluorescent molecules are excited to a higher energy state.

Upon returning to their resting states, the fluorochromes emit light energy at higher wavelengths.

The use of multiple fluorochromes , each with similar excitation wavelengths and different emission wavelengths (or colors), allows several cell properties to be measured simultaneously.

Commonly used dyes include propidium iodide, phycoerythrin, and fluorescein, although many other dyes are available.

Tandem dyes with internal fluorescence resonance energy transfer can create even longer wavelengths and more colors.

Forward scatter Size of the cell Side scatter Shape & complexity of the cell

Direct beam stop

Laser

LightHigh angle scatter :(Side Scatter)Cell structure

Low angle scatter :(Forward Scatter)Cell size

Flow cellDirection of flow

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DIAGRAM [4]

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Fluorescence Assisted Cell Sorting (FACS)

GRAPH OF FLOW CYTOMETRY

ChargedPlatesCollection Tubes

INSTRUMENTATION A flow cytometer is made up of three main systems :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.

In the flow cytometer, particles are carried to the laser intercept in a fluid stream. Any suspended particle or cell from 0.2150 micrometers in size is suitable for analysis.Cells from solid tissue must be desegregated before 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.

APPLICATIONSDiagnosis of Hematological Malignancies [5]The identification and quantitation of cellular antigens with fluorochrome-labeled monoclonal antibodies (immunophenotyping) is one of the most important applications of the flow cytometer.Immunophenotypic analysis is critical to the initial diagnosis and classification of the acute leukemias , chronic lymphoproliferative diseases, and malignant lymphomas since treatment strategy often depends upon antigenic parameters.Immunophenotypic analysis provides prognostic information not available by other techniques, provides a sensitive means to monitor the progress of patients after chemotherapy or bone marrow transplantation.

Analysis of DNA Ploidy, the Cell Cycle, and Cell Death[6]The significant medical discovery of understanding the human cell cycle in combination with flow cytometric technology allowed for the development of DNA analysis of neoplasia by flow cytometry.A defined number of cells are stained with a known saturating amount of DNA-specific fluorescent dye under controlled conditions of temperature, pH, and ionic strength.The cells are then analyzed using a flow cytometer where, upon excitation by a light beam of the appropriate wavelength, the amount and intensity of fluorescent emission of the dye bound to DNA of each cell, is measured based on a statistically significant number of cells (i.e. 10,000) in a period of a few minutes.

The relative total DNA content in an unknown cell population is determined when compared to cells analyzed with known and constant DNA content.

In addition, small populations of cells can be detected in a heterogenous mixture, and cell populations with small variations in DNA content (~ 4% with a CV of 2%) can be detected.

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Analysis of a marine sample of photosynthetic picoplankton by flow cytometry showing three different populations (Prochlorococcus, Synechococcus, and picoeukaryotes)

Organ Transplantation and Hematopoietic Cell TherapyClinical applications of flow cytometry in solid organ transplantation include pre-transplant cross- matching, HLA antibody screening, and post-transplantation antibody monitoring.In bone marrow transplantation, the enumeration of CD34+ hematopoietic stem cells in the peripheral blood or bone marrow graft correlates with engraftment success and the length of hematopoietic recovery following stem cell transplantation.Other applications of flow cytometry in bone marrow transplantation include pre-transplantation determinations of the efficacy of ex-vivo T-cell graft depletion, and post-transplantation evaluation of immune recovery, graft rejection, graft-versus host disease, and the graft-versus-leukemia effect.

Future Expectations1. Multispectral imaging of hematopoietic cells; where flow meets morphology- normal and abnormal cells are typically analyzed by either histologic or flow cytometric approaches. Histology allows morphological examination of complex visual traits but with relatively limited number of cells. Flow cytometry can quantify multiple flourocent parameters on millions of cells , but lacks morphological or sub cellular spatial detail . In this we can present a how a new flow technology ,the imagestream ,blends morphology and cytometry can be used to analyse cell populations -six stages of erythroid maturation-levels of fluorescent intensity can be find out.

The Patent Description & Claims data below is fromUSPTO Patent Application , Blood velocity measurement using correlative spectrally encoded flow cytometry.-In the article entitled Flow cytometry usingspectrally encoded confocal microscopy by L.Golan and D. Yelin, there is described a techniquetermed spectrally encoded flow cytometry (SEFC),which has been shown effective for noninvasive,high-resolution imaging of blood flowing in themicrocirculation. There therefore exists a need for a method ofmeasuring blood flow velocity in an SEFC system,which overcomes at least some of thedisadvantages of prior art systems and methods.

Spectrally encoded flow cytometry

Blood flow velocities in small mesenteryvessels are estimated by spatially spectrallyencoded an imaging beam, such as by dispersionthrough a diffraction grating, and splitting thedispersed beam into separate paths, which arethen focused in the form of spectrally encodedlines onto two closely positioned locations acrossthe flow path of the blood stream in themeasurement region. By measuring temporalcorrelations of the flow patterns obtained from thelight patterns reflected from the two line locationsalong the vessel, accurate velocity measurement ofthe imaged cells can be made. An advantage ofthis correlative SEFC method for measuring bloodvelocity is that it relies on high-resolution confocalimages that allow effective extraction ofmicroscopic flow.

Separation of type of cells-The classification and separation of one celltype or particle from others is a fundamentaltask in many areas of science. Numeroustechniques are available to perform this task;however, electrostatic cell sorting has gainedeminence over others because, when combinedwith the analysis capabilities of flow cytometryit provides flexible separations based onmultiple parameters. Unlike competingtechnologies, such as gradient or magneticseparations that offer much larger totalthroughput, flow cytometric cell sorting permitsselections based on various levels offluorescent reporters, rather the completepresence or absence of the reporter.

Microfluidic impedance flow cytometry enablinghigh-throughput single-cell electrical property characterization. Recent developments in micro fluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included under it:- (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) micro fluid impedance flow cytometry withenhanced sensitivity; (3) microfluidic impedance and optical flow cytometry forsingle-cell analysis (4) integrated point of care system based on micro fluidic impedance flow cytometry.

Bead based immunoassay Cytometric Bead Array (CBA) solutions measure a variety of soluble and intracellular proteins, including cytokines, chemokines, growth factors, and phosphorylated cell signaling proteins using flow cytometry. BD CBA solutions enable analysis of up to 30 proteins using just 25 to 50 L of sample in comparison to other methods such as ELISA and Western blot, which enable only one protein to be analyzed per sample.

CBA Flex Sets provide an open and configurable method of detection, so that researchers can build their own multiplexes for protein quantization as low as 10 pg/mL. CBA Enhanced Sensitivity Flex Sets are capable of detecting cytokine concentrations as low as 0.274 pg/mL.CBA Kits are preconfigured for achieving consistent results for routine panels.

SUMMARYFlow cytometry is cellular technique in which cell-counting and cell separation perform.The main principle is based on forward scatted and side scatted of laser beam.The flow cytometer consists of fluidics system, optics system & electronic system. It have applications in diagnosis of hematological malignancies, analysis of cellular events & organ transplantation.Flow cytometry can be helpful in immunological interactions among body fluids.In marine biology, the autofluorescent properties of photosynthetic plankton can be exploited by flow cytometry in order to characterise abundance and community structure.

REFERNCEShttps://youtu.be/ZBqg0ShradQ.Fulwyler MJ (1965). "Electronic separation of biological cells by volume".Science.150(3698): 910911.Chapman GV. Instrumentation for flow cytometry. J Immunol Methods. 2000;243:3-12.Alamo AL, Melnick SJ. Clinical application of four and five-color flow cytometry lymphocyte subset immunophenotyping. Cytometry. 2000;42:363-70.Riley RS. Cellular proliferation markers in the evaluation of human cancer. Clin Lab Med. 1992;12:163-99.Kathleen e. mcgrath ,timothy p. bushnell james palis, journal of immunological methods 31 July 2008.

7.In the article entitled Flow cytometry usingspectrally encoded confocal microscopy by L.Golan and D. Yelin, one of the present inventors,and published in Optics letters, 2010. Vol. 35(13),pp. 2218-2220

8. http://www.haematologica.org/content/97/11/1648