Principles of Flowcytometry

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1 Objectives Introduction to Cell Markers: Principles of Flowcytometry Michelle Petrasich NZIMLS Scientific Meeting August 24, 2010, Paihia What are cell markers How do we detect them Production of Monoclonal anti bodies Their role in diagnosing Haematological disorders Principle of a simple flow cytometer Theo ry of fluorescence Fluo rochrom es an d compensation Cell Markers Antigens Protein found on or in the cell: - Cell m embrane = su rface markers - cytoplasm - nucleus or nuclear membrane Cell Markers Profile of detected antigens for a cell is called the Immunophenotype Proteins are classified according to CD nomenclature system May be general ( pan ) or very specific eg. CD45 all white cells CD3 - all T cells CD4 - T helper subset CD45RA+ -T Naive CD45RO+ -T Memory Cell Markers Some markers can be expressed at different stages of development CD Nomenclature System CD = Cluster of Differentiation Classification for the pro teins and the monoclonal antibodies that are used to detect them 1982 : International Workshop on Human Leucocyte Differentiation Antigens (HLDA) CD : characterisation of the protein + 2 specific monocloncal ab. bind to mo lecule WHO,

2 CD Nomenclature System HCDM Work shop (Human Cell differentiation Molecules) 2010 March Work shop 363 CD antigens Estimated 2,500 leucocyte su rface molecules CD Nomenclature System CD4 MW : 55 kda protein Expression: T helper cells, monocytes, granulocytes Function: Co-receptor for MHC Class II,signal transduction; also a receptor used by HIV to enter CD4+ cells Clones: OKT4, Sk3, MT310, RPA-T4 CD20 MW: kda protein Expression: B cells, minor subset of T cells Function: Type III trans-membrane protein that forms a calcium channel in the cell membrane allowing for the influx of calcium required for cell activation Clones: B1; 2H7; LEU-16; L26, L27 Monoclonal Antibodies (Mab) Use highly specific antibodies that bind to specific antigens on the cell Specific site on the p rotein antigen is called the epitope (6 or mo re amino acids) Monoclonal Antibodies Mab are product of mouse hybridoma technology (single clone of B cells) Kohler and Milstein Antibodies are bought conjugated with fluorescent dyes - fluo rochromes eg. FITC, PE, PerCP, APC, APC-H7 % positivity: propo rtion of cells that are positive for that marker eg. 52% CD19: 52% of the cells ar e positive for CD19 Monoclonal Antibody Production Monoclonal Antibodies Predominantly IgG, a few are IgM molecules Fluorochrome is conjugated to Fc portion Monoclonal antibody to B cell marker CD19 Mab is conjugated with FITC fluorochrome (glow green) Fluor ochrome Source: National Health Museum Resource Centre,

3 Cells + Monoclonal Antibodies Why Labelled cells Flowcytometry allows simultaneous addition of a 8-10 antibodies to 1 tube -all with different fluorochromes! Why Why Role of Cell markers Flowcytometry Haemato logy: Diagnosis an d Monitoring Identify the lineage eg. blasts Identify a malignant population/clone eg. lymphocytes Identify the absence or presence of specific protein eg. PNH (rbc, wc, plat.) Quantitation of a specific cell subtype eg. CD34 stem cell harvest collects CD4 monitoring in HIV patients CD20 after Immunotherapy with Rituximab HbF feotal RBC 3

4 Flowcytometry Multi-parameter assessment of single cells using a laser beam Lasers simultaneously assesses each cell: -Size (Forward Scatter) - Internal complexity/granularity (Side Scatter) - Up to 10 antigens using fluorescently labelled Mab Flowcytometer Flow cell Components Components Fluidics: H ydrodynamic focusing- single cells through the flow cell Lasers (2-3): each pro duces a single wavelength of light : scatter characteristics of cell (FS, SS) an d fluorescence emission Dichroic and bandpass filters : split light (spatial separation), an d block or pass on light of selec ted waveleng ths Light Detectors: PMT tubes and silicon photodiodes: detec t light, pro duce elec trical pulses Electronics :filtering and amplification of signals Software: data presentation Features: Assay large numbers of cell >10 6 cells/tube Fast: assay up to 10,000 cells/second Subpopulations can be analy sed sep arately Light Theory Electromagnetic energy that travels in waves Length of the waves determines colou r of light (visible range = 380nm -700nm) Detected by P MTs and p hotodiodes Identify rare populations eg. stem cells, dendritic cells Data storage Electromagnetic Spectrum 4

5 Theory of Fluorescence Mab is labelled with a fluorochrome Fluo rescen t molecule: Fluorochromes 3 laser Flowcytom eter: 488nm,633nm,405nm Absorbs light of one wavelength (excitation) Atom of a fluorescent molecule Emits light of a longer wavelength (emission): Stokes shift Eg. FITC : Excitation 488nm laser FITC : Emits: 525nm Emission Spectra Fluorescence Compensation Emission spectra of FITC (Fluoroscein Isothiocyanate) Spectral Overlap A B Flurochrome A flows into filter for B : FL2 - % FL1 eg. 17% Flurochrome B flows into filter for A : FL1 - % FL2 eg. 1.5% Nuclear Fluorescence - TDT Surface Fluorescence CD19 5

6 Nuclear Protein : APML Plots.... Fluorescence Plots.... Conclusion Protein markers detected using Mab Flo wcytometry: Multi-parameter approach Use the web excellent resource!! 6

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