Flowcytometry.pdf

Flow Cytometery Flow Cytometers BD FACSCount™ Developed in the 1960s system FACS™: fluorescence- activated cell sorting BD FACSCalibur™ 2  Introduction The concept...

Flow Cytometery Flow Cytometers BD FACSCount™ Developed in the 1960s system FACS™: fluorescence- activated cell sorting BD FACSCalibur™ 2  Introduction The concept of flowcytometry has been in existence for more than five decades. Flowcytometric immunophenotyping (FCI) first appeared in clinical laboratories in the 1980s, in the wake of the AIDS epidemic. Initially utilized to assess CD4 T-cells, the technique was soon applied to lymphoid and eventually myeloid neoplasms. 3  Current flow cytometers have the capability of simultaneously measuring multiple parameters of individual cells in a cell suspension. – physical properties of cells(Intrinsic) the size, cytoplasmic granularity/internal complexity. – cell antigens/markers(Extrinsic)(surface, cytoplasmic, and nuclear) that can be recognized by specific antibodies. 4  Flow Cytometry Flow = stream(cells in motion) – Cyto = cells – Metry = measure Measuring cells in a flow system (suspension) instead of on a static microscope across a beam of light path. 5  Basic mechanism Flow Cytometry Biological sample Label it with a fluorescent marker Cells move in a linear stream through a focused light source (laser beam) Fluorescent molecule gets activated and emits light that is filtered and detected by sensitive light detectors (usually a photomultiplier tube) Conversion of analog fluorescent signals to digital signals 6  The basic components of a flow cytometer The Flow system (fluidics) Cells in suspension are brought in single file past The Optical system (light sensing) A focused laser which scatter light and emit fluorescence that is filtered and collected, direct the resulting light signals to the appropriate detectors. Includes lasers, optical filters and lenses The Electronic system (signal processing) converts the detected light signals into electronic signals that can be processed by the computer. Computer 7  The Flow System Transport particles in a fluid stream to the laser beam for interrogation When a sample is injected into a flow cytometer, it is ordered into a stream of single particles. The fluidic system consists of a FLOW CELL (Quartz Chamber): – Central channel/ core - through which the sample is injected, also called the sample core. – Outer sheath - contains faster flowing fluid , Sheath fluid (0.9% Saline / PBS) , enclosing the central core. 8  Hydrodynamic Focusing Once the sample is injected into a stream of sheath fluid within the flow chamber, they are forced into the center of the stream forming a single file by the PRINCIPLE OF HYDRODYNAMIC FOCUSING. 'Only one cell or particle can pass through the laser beam at a given moment.' 9  The sample pressure is always higher than the sheath fluid pressure, ensuring a high flow rate allowing more cells to enter the stream at a given moment. High Flow Rate - Immunophenotyping analysis of cells Low Flow Rate - DNA Analysis 10  OPTICS After the cell delivery system, the need is to excite the cells using a light source. The light source used in a flow cytometer: Laser (more commonly) – Why Lasers are more common?  They are highly coherent and uniform.  They can be easily focused on a very small area (like a sample stream).  They are monochromatic, emitting single wavelengths of light. Arc lamp 11  The site where a cell intersects a laser beam called 'interogation point' two events occur: a) light scattering b) emission of light (fluorescence ) The scattered and fluorescent light is collected by appropriately positioned lenses. filters steers /direct the resulting light signals to the appropriate detectors. The detectors produce electronic signals proportional to the optical signals striking them. 12  FORWARD SCATTER (FSC) Light that is scattered in the forward direction (along the same axis the laser is traveling , typically up to 20° offset from the laser beam’s axis) is detected in the Forward Scatter Channel. The intensity of this signal(FSC) roughly equates to the particle’s size and can also be used to distinguish between cellular debris and living cells. 13  SIDE SCATTER (SSC) Laser light that is scattered at 90 degrees to the axis of the laser path is detected in the Side Scatter Channel. The intensity of this signal is proportional to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, etc.) 14  Both FSC and SSC are unique for every particle and a combination of the two may be used to differentiate different cell types in a heterogeneous sample. 15 Study of FSC and SSC allows us to know the differentiation of certain types of cells. Granulocytes Lymphocytes SSC Monocytes RBCs, Debris, Dead Cells FSC 16  IMMUNOPHENOTYPING ANALYSIS Requires – Antibodies. – Fluorochromes. 17  ANTIBODY Highly specific monoclonal antibodies are used for identification and distinction of cell surface antigens (cluster of differentiation (CD)). Using CD system we can identify cells by the presence or absence of particular surface markers for e.g. CD3- or CD4+ etc. 18  FLUOROCHROMES Fluorochromes are substances that can be excited by certain light source (such as laser) and emit a fluorescent signal at a single wavelength. Fluorescent dyes can directly bind to certain cellular content, such as DNA and RNA, and allow us to perform quantitative analysis on individual cells. However, in most cases fluorochromes are conjugated with monoclonal antibodies, which specifically target cellular antigens/markers. 19  IMMUNOPHENOTYPING ANALYSIS 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. 20  Simultaneous detection of multiple cell antigens/markers. Multiple cell antigens ( Ag ) are recognized by fluorochrome conjugated specific antibodies ( Ab ). Because different fluorochromes have different emission wavelengths/colors, they can be simultaneously detected by a flow cytometer. FITC fluorescein isothiocyanate; PE phycoerythrin; PerCP peridinin chlorophyll protein; PE-T Red PE-Texas Red.21 22  ELECTRONICS The electronic subsystem converts photons to photoelectrons (voltage pulse). Measures amplitude, area and width of photoelectron pulse. It amplifies pulse and then digitalizing the amplified pulse. 23 Electronics- Creation of a Voltage Pulse 24  Gating Electronically isolating a population for further analysis or Selection of only a certain population of cells for analysis on a plot. Allows the ability to look at parameters specific to only that subset. – The Th lymphocyte gating strategy uses the CD3+ T cell gating.(CD3+,CD4+). 25  CD45/Side scatter gating Granulocytes (N, E) Monocytes Lymphocytes Lymphocytes are defined as CD45 bright, low side scatter 26  Application detection of antigens/markers on cell surface Provide multiple intrinsic parametres of cells (neoplastic tissue, benign,reactive and normal) Detect cell lineage (B lymph vs. T lymph) Degree of maturation (Pre- T cell/ mature T cell 27  Reference 1. Naville J. Bryant Laboratory Immunology and Serology 3rd edition. Serological services Ltd.Toronto,Ontario,Canada,1992 2. Tizard. Immunology an introduction,4th edition ,Saunders publishing,1994 3. Mary Louise.Immunology and Serology in Laboratory medicine 3rd edition 28