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 White blood cells physiological flow cytometry bacterial infection: flow sorting FACS: Fluorescence-Activated Cell Sorting viral infection: Than Gucker, 1947 Wallace Coulter Coulter Orifice, 1956 and now neutrophils Manuscript of the patent application Photo by J.Paul Robinson Photo by J.Paul Robinson The first commercially available counter (Coulter Counter) lymphocytes monocytes Kamentsky, 1965 Mack Fulwyler sorter - 1965 1
Function of flow cytometry Principles of the measurements Technical conformation -Light source -Flow system -Optics -Detection system -Data collection - Data analysis -Sorting sample Setup Light source (laser) Fluidics system, hydrodynamic focusing Illumination of cells, generating scattering and fluorescent signal dicroic mirrors 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 (350-363, 420, 457, 488, 514, 532, 600, 633 nm) Argon ion, Krypton ion, HeNe, HeCd, Yag, solid state Arc-lamps Mercury-vapour, Xenon Detecting system Photomultiplier tubes (PMTs) Formerly 1-2 tubes Currently 12-15 tubes flow cell scatter sensor band filters laser Photodiods Mainly for forward scatter (FSC) measurement THE SIGNIFICANCE AND PROPERTIES OF THE FLOW SPACE Flow cytometry cell THE SIGNIFICANCE AND PROPERTIES OF THE FLOW SPACE Hydrodynamics system, hydrodynamical focusing Fluorescent signals Laser beam Analysis cell CELL SEPARATION-SORTING CELL SEPARATION-SORTING 488 nm laser charged plates FSC sensor fluorescent detector the last osculant drop satellite droplet separated cells in test-tubes 2
OPERATION PRINCIPLE OF FLOW CYTOMETER FSC FSC Laser Cells FSC detector (photodiode) Cells Laser scattering SSC histogram 1D FSC histogram 1D cells laser 90 scatter SSC image 2D dot plot FSC image 3
U (voltage) 2016.05.06. FLUORESCENCE DETECTION DETECTION OF MORE THAN ONE FLUORESCENT SIGNAL 1. 2. 1. Increasing brightness 2. 3. 4. 3. (time) DATA PROCESSING DATA PROCESSING Analog signals Convert numbers to parameters Signal width: Peak height: Area: Number of the sample The biggest value Sum of the numbers Numbers appear in the memory of a digital device No deadtime! area signal height Laser delay width DATA STORAGE FCS (flow cytometry standatrd) file contains all the data measured = list mode file 1D 2D 3D VISUALIZE DATA Dot plot Density plot Contour plot 3D (surface) 4
GATING DIAGNOSTIC APPLICATIONS Distribution of the whole FL4 measured Distribution of the whole FL4 measured through the red gate DIAGNOSTIC APPLICATIONS - AIDS DIAGNOSTIC APPLICATIONS - ALL 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 5
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 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 6
TRANSITION BETWEEN MICROSCOPE AND CYTOMETER 7