ALL INFECTOLOGY COMMERCIALLY AVAILABLE DEVICES. and now BLOOD:

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1 ALL BLOOD: RBC, PLT, WBC ( /-/ ) (+ LDH, carbamide, ESR, periphery blasts, liver function deviation, renal function dev.) Classification: Morphology + genetics+ immunology! B-cell: T-cell: Dedifferentiated: rapid phenotyping with cytometry! INFECTOLOGY COMMERCIALLY AVAILABLE DEVICES White blood cells physiological bacterial infection: viral infection: flow cytometry Than flow sorting FACS: Fluorescence-Activated Cell Sorting and now neutrophils monocytes lymphocytes 1

2 Gucker, 1947 Wallace Coulter Coulter Orifice, 1956 Manuscript of the patent application Kamentsky, 1965 Photo by J.Paul Robinson Mack Fulwyler sorter Photo by J.Paul Robinson The first commercially availablecounter (Coulter Counter) Function of flow cytometry Principles of the measurements Technical conformation -Light source -Flow system -Optics -Detection system -Data collection - Data analysis -Sorting sample flow cell scatter sensor Setup Light source (laser) Fluidics system, hydrodynamic focusing Illumination of cells, generating scattering and fluorescent signal dicroic mirrors band filters laser Optics Photodiode detector for FSC (size) Electronics and computer system (data processing) PMT detectors for fluorsecence signals PMT detector for SSC (granularity) OPTICAL SETUP OF FLOW CYTOMETER, DETECTION OF OPTICAL SIGNALS Light source: exciting/illuminating system Lasers ( , 420, 457, 488, 514, 532, 600, 633 nm) Argon ion, Krypton ion, HeNe, HeCd, Yag, solid state Arc-lamps Mercury-vapour, Xenon THE SIGNIFICANCE AND PROPERTIES OF THE FLOW SPACE Flow cytometry cell Detecting system Photomultiplier tubes (PMTs) Formerly 1-2 tubes Currently tubes Photodiods Mainly for forward scatter (FSC) measurement Laser beam Analysis cell Fluorescent signals THE SIGNIFICANCE AND PROPERTIES OF THE FLOW SPACE Hydrodynamics system, hydrodynamical focusing CELL SEPARATION-SORTING 488 nm laser FSC sensor the last osculant drop charged plates fluorescent detector satellite droplet separated cells in test-tubes 2

3 CELL SEPARATION-SORTING FSC OPERATION PRINCIPLE OF FLOW CYTOMETER FSC Cells Laser FSC detector (photodiode) cells laser Cells Laser 90 scatter scattering SSC histogram 1D FSC histogram 1D SSC image 2D dot plot FSC image 3

4 U (voltage) FLUORESCENCE DETECTION Increasing brightness (time) DETECTION OF MORE THAN ONE FLUORESCENT SIGNAL 1. Analog signals DATA PROCESSING Numbers appear in the memory of a digital device Laser delay DATA PROCESSING DATA STORAGE Convert numbers to parameters FCS (flow cytometry standatrd) file contains all the data measured = list mode file Signal width: Peak height: Area: Number of the sample The biggest value Sum of the numbers No deadtime! area signal height width 4

5 1D 2D 3D Dot plot VISUALIZE DATA Density plot GATING Distribution of the whole FL4 measured Contour plot 3D (surface) Distribution of the whole FL4 measured through the red gate DIAGNOSTIC APPLICATIONS DIAGNOSTIC APPLICATIONS - AIDS DIAGNOSTIC APPLICATIONS - AIDS DIAGNOSTIC APPLICATIONS - ALL 5

6 DIAGNOSTIC APPLICATIONS IMMUNOLOLOGY Application field: Cancer cells -higher DNA content than normal cells -higher S and G2/M ratio activation test activation eg.diagnosis of chronic idiopathic urticaria Human diploid cell Human breast cancer cell line AVAILABLE ROUTINE LABORATORY TESTS POSSIBLE APPLICATION FIELDS Blood test ratio ratio ratio determination Determination of immunglobulin level Analysis of DNA content and cell proliferation Basophil activation test Leukaemia and lymphoma panels Medical diagnostics Infectology Immunology/allergology Hematology Oncology Drug discovery Immunogenicity assays Assessing drug effects Structural analysis DIAGNOSTIC APPLICATIONS INDUSTRIAL APPLICATIONS Clinical applications of flow cytometry 1. Quick detection of bacteria in drinking water and bathwater Calibrated automatic devices, fast and chip analysis Monitoring of large number of samples 2. Measure appropriate yeast concentration during winemaking 3. Survey of fertility in breeding animals Counting gametes Measure viability with DNA labeling 6

7 THE FUTURE IN VIVO FLOW CYTOMETRY Traceable tumour cells in lymp veins (in vivo) CLOSED SYSTEMS Allows morphological anamnesis: take images about flowing particles OPEN SYSTEMS Analysis of cells placed onto slides: morphological analysis and fluorescense registration cytometry Intravasal tracing of tumor cells Fluorescently labelled cells can be measured in living animal, same figures as flow cytometry TRANSITION BETWEEN MICROSCOPE AND CYTOMETER 7