• Document: Cellular phenotyping and application of cytometry for diagnostics purposes Part I. dr n. med. Karolina Bukowska-Straková
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“Cellular phenotyping and application of cytometry for diagnostics purposes” Part I dr n. med. Karolina Bukowska-Straková Flow cytometry - wikipedia definition ;-) Flow cytometry (abbreviated: FCM) is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other applications in both research and clinical practice. A common variation is to physically sort particles based on their properties, so as to purify populations of interest. Forward scatter - FSC Forward scatter - FSC Forward scatter - FSC Forward scatter - FSC Side scatter - SSC Side scatter - SSC FSC versus SSC Monoclonal antibodies – conjugated with fluorochrome Monoclonal antibodies (mAbs or moAbs) Monoclonal antibodies (mAbs or moAbs): - made by identical immune cells that are all clones of a unique parent cell - monospecific → monovalent affinity bind to the same epitope. José M. Casasnovas, Mykol Larvie and Thilo StehleThe EMBO Journal (1999) 18, 2911 - 2922 Monoclonal antibodies (mAbs or moAbs): The same: isotype, allotype, idiotype Idiotype - differences in Ag recognition Allotype- Individual differences in Isotype - amino acid different heavy sequence chains: (polymorphisms) µ, δ, γ, ε, α or light chains: κ, λ Fluorochrome excitation Flow cytometry Lasers / fluorochroms Fluorochrome choose Mean fluorescence intensity Data presentation Our instrument – LSR II 10 colors Immunophenotyping of leukocytes • Flow cytometric immunophenotyping with (monoclonal) antibodies allows the recognition of leukocyte subsets. • The CD nomenclature has created clarity in the field of membrane bound leukocyte antigens (but not for intracellular markers) • These CD antibodies recognise many different types of markers (lineage specific, differentiation stage specific, etc) and thereby allow the recognition of many different (immature and mature) leukocyte subsets. • Usage of multiparameter analyses allow the dissection of differentiation and maturation pathways as well as the detecion of specific proteins (see: Van Lochem et al. Cytometry 2004;60B:1-13). Classical leukocyte markers Immature markers T-cell markers CD34: precursor marker CD1: common thymocyte marker TdT: terminal deoxnucleotidyl transferase CD2: pan-T-cell marker CD117: myeloid precursor marker CD4: helper T-cell marker HLA-DR: precursor cells (and APC cell) CD8: cytotoxic T-cell marker CD3: mature T-cell marker B-cell markers TCR: T-cell receptor CD10: immature B-cell marker CD19: pan-B-cell marker Myeloid markers CD20: mature B-cell marker CD13 and CD33: pan-myeloid SmIg: membrane bound Ig CD14: monocytic marker CyIg: cytoplasmic Ig CD15: granulocytic marker Hematopoiesis Physiological balance of hematopoiesis Its regulation depends on: cytokines, celluler interactions, transcription and metabolic factors Hematopoietic cells may be missing or do not function properly Immuno- deficiencies Flow cytometry in primary immunodeficiency Flow cytometric immunphenotyping and functional studies of blood / bone marrow / other material Targeted analysis of immune cells: 1) are all cells present in normal frequencies; 2) do they have all relevant proteins expressed; 3) is their function normal? Primary immunodeficiencies (PID) DNA repair defects Defects in innate immunity Congenital defects in fagocyte number or/and function Complement deficiencies Other well defined immunodeficiency Alternative complement syndromes pathway defects Diseases of immune Regulatoty proteins of dysregulaton comp

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