Review of techniques in imaging and cytometry. Peter Meeus Onze Lieve Vrouw Ziekenhuis, Aalst 7 may 2009, SCK/CEN Mol

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1 Review of techniques in imaging and cytometry Peter Meeus Onze Lieve Vrouw Ziekenhuis, Aalst 7 may 2009, SCK/CEN Mol

2 Cytometry: The counting and measuring of cells,... Mosby's Medical Dictionary, 8th edition. 2009, Elsevier. The measurement of physical characteristics of cells. McGraw-Hill Concise Dictionary of Modern Medicine by The McGraw-Hill Companies, Inc.

3 Workshops BVAC/ABCA: Microscopy Microscope settings and capture of digital images Leucocyte differentiation with images (DM96) Dysplasia in MDS

4 Automatic count & screening Manual confirmation

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6 Computer assisted confirmation

7 Cytophotometric factors causing apparent differences between Feulgen DNA contents of different leukocyte types Bedi, K. S.; Goldstein, D. J. Nature, Volume 251, Issue 5474, pp (1974). Nuclear cytomorphometry in feline mammary gland epithelial tumours R. Simeonov and G. Simeonova Accepted 6 September Available online 25 October 2007.

8 Computer asssisted imaging

9 Laser Scanning Microscope

10 Computer assisted screening

11 Workshops BVAC/ABCA: Flow Cytometry Basic settings, calibration and compensation Multicolor protocols and validation Gating algorithms, rare event analysis and MRD Cell sorting

12 # CD34 counting using the ISHAGE protocol Intracytoplasmic and cytokine multicolour protocols Workshops BVAC/ABCA: Flow Cytometry Microparticles and platelets Apoptosis and DNA repair Multiplex Array Assay Plant analysis by flow cytometry Microbiology and flow cytometry

13 BD FACSCanto II

14 Fluidics Cells in suspension flow in single-file Optics through an illuminated volume where they scatter light and emit fluorescence Electronics that is collected, filtered and converted to digital values that are stored on a computer

15 Injector Tip Sheath fluid Fluorescence signals Focused laser beam

16 Flow Cytometry Optics PMT 4 Flow cell Dichroic Filters PD 1 Bandpass Filters PD 2 Laser PMT 3

17 Detectors FSC Detector Photodiode with488/10bp SSC Detector PMTwith 488/10BP FluorescenceDetectors: 6 PMTs 488nm Laser FITC, PE, PerCP or PerCP-Cy5.5, PE-Cy7 633nm Laser APC, APC-Cy7 Detector Bands 488nm Laser: 530/30;585/42; >670;780/60 nm 633nm Laser: 660/20;780/60 nm

18 8-Color Option with 3 Lasers Lasers Air-cooled: 405-nm solid state diode, 30-mW fiber power output 488-nm solid state, 20-mW laser output 633-nm HeNe, 17-mW laser output Fluorescence Detectors 8 PMTs in configuration Laser Dyes Violet: Pacific Blue, AmCyan (455, 488 nm) Blue: FITC, PE, PerCP or PerCP-Cy5.5, PE-Cy7 (525, 575, 678, or 695, 785 nm) Red: APC, APC-Cy7 (660, 785 nm) Detector Bands Violet: 450/50; 502 to 525 nm Blue: 530/30; 585/42; >670; 780/60 nm Red: 660/20; 780/60 nm 8-Color Option with 2 Lasers Lasers Air-cooled: 488-nm solid state, 20-mW output 633-nm HeNe, 17-mW output Fluorescence Detectors 8 PMTs in 5-3 configuration Laser Dyes Blue: FITC, PE, PE-Texas Red, PerCP or PerCP-Cy5.5, PE-Cy7 (525, 575, 615, 678 or 695, 785 nm) Red: APC, Alexa Fluor 700, APC-Cy7 (660, 720, 785 nm) Detector Bands Blue: 530/30; 585/42; 616/23; >670; 780/60 nm Red: 660/20; 712/21; 780/60 nm

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22 Cytometric Bead Array Rotor 1-Tube_040 APC-Cy7-A PE-A

23 Detection of fusion proteins

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25 Combine flow- and imagecytometry

26 Cell sorters

27 Magnetic sorting

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29 Indications: role of MB? Immunology: B,T,4/8, Th1/Th2, Memory B HIV/AIDS: CD4 Interstitial lung diseases (B.A.L.-fluid) (Hematologic) Malignancy: diagnosis, classification, staging and follow-up (MRD) (Stemcell) Transplant (CD34, ) HLA typing Microparticles PLT-Leukocyt adhesion, activation Interleukines etc. (CBA)

30 Indications: role of MB? Trombocytes (Glanzman, Bernard-Soulier) Reticulocytes Apoptosis DNA-content, S-phase, FISH (Flow-)Karyotyping Cell-tracking Rare event analysis (e.g. Circulating tumor cells) Cell signaling Fusion proteïnes (e.g. bcr-abl) Microbiology