Flow ~ cells in motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in a fluid stream. Ajdary S.

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1 What Is Flow Cytometry? Flow ~ cells in motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in a fluid stream

2 Flow cytometry provides rapid analysis of multiple characteristics of single cells. Characteristics that can be measured: Cell size Cytoplasmic complexity DNA or RNA content A wide range of membrane-bound and intracellular proteins variety of specimens: whole blood, bone marrow, serous cavity fluids, CSF, urine, and solid tissues.

3 much smaller, less expensive, more user-friendly, and well suited for high-volume operation BD Publication ns Medline Publications citing "Flow Cytometry" Year

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6 Advantages High speed analysis Measures single cells Measures large number of cells Simultaneous analysis of multiple parameters (up to 20) Identifies small subpopulations Quantification of fluorescence intensities

7 Disadvantages Very expensive and sophisticated instruments Need single particle Tissue architecture is lost Little information about intra-cellular distributions

8 Section I Background Information on Flow Cytometry

9 Flow Cytometry Instrumentation A typical flow cytometer consists of: Fluidics Light source: laser Optics and Detectors Computer system for analysis and storage of digitized dt data

10 The Fluidics System The Flow Cell The sample be ordered into a stream of single particles Fluidics system: Laminar flow Central sample fluid( hydrodynamic focusing) Outer sheath fluid

11 Light source Light source needs to be focused on the same point where cells are focused. On all flow lab instruments-lasers Light amplification i by stimulated emission i of radiation Lasers can provide: monochromatic light coherent light

12 Light Scatter excitation light is absorbed and then re-radiated by the particles

13 Forward Scatter Forward Scatter=FSC is scattered along the same axis the laser is traveling Laser Beam FSC Detector The intensity of this signal has been attributed to The intensity of this signal has been attributed to cell size

14 Side Scatter is scattered at the right angle to the laser beam Laser Beam FSC Detector The intensity of this signal is proportional p to the amount of cytosolic structure in the cell (eg. granules, cell inclusions, etc.) Collection Lens SSC Detector Detector

15 Why Look at FSC vs. SSC Since FSC ~ size and SSC ~ internal structure, allow for differentiation of cell types in a heterogenous cell population Lymphocytes Granulocytes SSC RBCs, Debris, Dead Cells Monocytes FSC

16 Fluorescence Fluorochromes are essentially dyes, which accept light energy (e.g. from a laser) at a given wavelength and reemit it at a longer wavelength

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18 Many fluorochromes possess overlapping emission wavelengths

19 Compensation This fluorescence interference can be corrected for by adjusting the measurement parameters of the flow cytometer. This correction is termed compensation

20 Fluorochromes in flow cytometry Fluorescent dyes may bind or intercalate with different cellular components such as DNA or RNA. Hoechst, Propidium iodide Antibodies conjugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells

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22 What can a Flow Cytometer tell us about a cell? Its relative size (Forward Scatter FSC) Its relative granularity or internal complexity (Side Scatter SSC) Its relative fluorescence intensity (FL1, FL2, FL3, FL4)

23 Fluidics Light source: laser Optics and Detectors Computer system

24 Filters and Detectors Laser Beam FSC Detector Collection Lens Fluorescence Detector A, B, C, etc

25 Optical Filters Optical filters are designed such that they absorb or reflect some wavelengths of light, while transmitting other.

26 Example Channel Layout Dt Detector#4 t Bandpass Filters Detector#2 Detector#3 Detector#1

27 Channel Layout Model SSC PE-Cy5 FITC <505nm >605nm 505nm-555nm 555nm-605nm PE

28 ELECTRONICS

29 Photo-detectors Detectors basically collect photons of light and convert them to electrical signal Photodiodes o Used for strong signals, Photomultiplier tubes (PMT) o More sensitive than a Photodiode,

30 Signal processing The electronics must process that light signal and convert the current to a digitized value that the computer can graph

31 Software data analysis software for instrument control, data acquisition, analysis and display Common programs include CellQuest Flowjo WinMDI FCS Express

32 software programs provide numerous options for displaying and analyzing flow cytometric data. Single Color Histogram Fluorescence intensity (FI) versus count Two Color Dot Plot FI of parameter 1 versus FI of Parameter 2 Two Color Contour Plot Concentric rings form around populations. The more dense the population, the closer the rings are to each other

33 Univariate histogram Data display Numbe er of eve ents Intensity of parameter

34 Bivariate display Contour Plot Dot Plot

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36 Gating Is used to isolate a subset of cells on a plot The goal is to mark the populations of interest so that they will be reported separately

37 Set positive/negative boundaries are cells positive or negative for a certain marker what proportion of cells is positive One parameter analysis

38 Two parameter analysis

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40 Immunofluorescence staining specific binding nonspecific binding

41 Isotype control

42 Isotype control

43 Quality assurance Pre-Analytical Analytical Post-Analytical Staff Sample processing Results record Sample Data Acquisition Results report Reagents Analysis Data back up Instrument maintenance Interpretation Quality improvement progrem

44 Staff The most important asset of laboratory Basic science graduate Should be trained appropriately (1 y) Exposure to work shops Competency assessment Direct observation, Proficiency test

45 Sample Correct collection Correct anticoagulant EDTA/ heparin Time between taking the sample and performing the test: Bone marrow/peripheral blood RT/O/N Fluids and FNA the same day/2-8 C

46 Sample processing Red cell lysis Ficoll-Hypaque density gradient Maximum recommended time before performance Cell viability Reagents The choice of fhigh haffinity, it specific antibodies Titration of Ab Appropriate flourochroms PE, APC, PE-cy7/ FITC, PerCP Cross Contamination with other Ab Contamination of sheath fluid and Ab

47 Quality control in Flow cytometry External quality control Internal quality control instrument reagents staff

48 Quality control of the instrument Qualified service personnel (1-2 /year) Operator (each start-up of the instrument) Setting PMT voltages using an unstained sample Compensation Set positive/negative boundaries Calibration of the fluorescence channels commercially prepared samples Beads

49 Calibration each microbead having a predefined level of fluorescence intensity

50 Check the stability of the cytometer over time Number of events 00 Number R1 Number Once a protocol is defined for a particular standard 1.37 NR1bead 1.93 solution Number FS Lin 1.99 R3 N Number R FL1 Lin R4 Beads must always be plotted in the same regions FL2 Lin Parameter intensity (au) FL3 Lin

51 APPLICATION

52 APPLICATION CELLULAR ANTIGENS cytokines structure t enzymes Adhesion Metabolic Receptors

53 CD Markers used for Primary Diagnosis T-Cell Markers B-Cell Markers Myeloid Markers CD 2 CD10 sigg CD11b CD3 CD19 sigm CD11c CD4 CD20 Kappa CD13 CD5 CD22 Lambda CD14 CD6 CD23 CD33 CD7 CD25

54 APPLICATION Research Laboratories Immune function studies Proliferation assay by CFSE analysis of T-cell subsets Counting TCR specific T cells by MHC tetramer Analysis of DNA Ploidy, the Cell Cycle, and Cell Death Hematopoietic stem cells Multi-drug resistance studies (cancer) Kinetics studies (cell function) Applications in Microbiology

55 Applications in Clinical Laboratories Immunophenotyping Applications in Hematology

56 leukocyte analysis Leukemia and lymphoma phenotyping Accurate, rapid diagnoses Classical l morphology Immunophenotyping Cytogenetic Molecular genetic

57 Myelomonocytic Antigen Distribution CFU-GM PROGRANULOCYTE CDw13 MY8 CD11b CD16 MYELOBLAST MYELOCYTE META- MYELOCYTE BAND PMN HLA-Dr CD34 CD33 CD38 CD71

58 Hematopoietic stem cells: CD34, HLA-DR pan-b-cell panel: CD19, CD20, CD22 pan-t-cell: CD2, CD3, CD4 and/or CD7 AML:CD13, CD33, CD117, Monocytic leukemia: CD4, CD11b, CD11c, CD14, CD36, CD64, or CD68. Megakaryocytic lineage: CD41 and CD61

59 Leukocyte analysis very effective in distinguishing myeloid and lymphoid lineages in acute leukemias and minimally differentiated leukemias

60 AML

61 Hematolymphoid neoplasms B and T lineages B-cell, pre-b cell, early B-precursor treatment strategy often depends upon antigenic i parameters: Various CD20+ B cell malignancies are now commonly treated t with anti-cd20 Abs A subset of CD52-expressing T cell and B cell malignancies may be treated with anti-cd52 (alemtuzumab)

62 leukocyte analysis Detection of minimal residual disease after therapy Staging and management of HIV-l infected patients Determination of the numbers of CD4 + T-cells Measurement of CD38 expression on CD8+ T-cells Autoimmune neutropenias Severe combined immune deficiency

63 Functional deficiencies of leukocytes Diagnosis of congenital immune deficiency syndromes (LAD) T and B cell mitogen responses Oxidative Burst (CGD) Phagocytic function

64 Neutrophil Oxidative Burst Hydroethidine HE O 2 - EB NADPH Oxidase Phagocytic Vacuole NADPH NADP O 2 O 2 - EB O 2 - H 2 O 2 HE SOD H2 O 2 OH - HE Example: Neutrophil Oxidative Burst

65 Neutrophil Oxidative Burst Unstimulated Neutrophils PMA-Stimulated Neutrophils 80 Control PMA-stimulated PMN 60 cou unts log FITC Fluorescence

66 Phagocytosis FITC-Labeled Bacteria

67 Erythrocyte analysis Foeto-maternal haemorrhage Fluorescently labeled antibodies to The rhesus (D) antigen Hemoglobin F (antibody to the g chain)

68 Reticulocyte enumeration To monitor bone marrow function important indicator of effective erythropoiesis much more accurate, precise, and cost-effective reticulocyte maturity index (RMI) parameter is the earliest indicator of bone marrow early indicator of the effect of erythropoietin in hemodialysis patients,

69 PNH greater precision, sensitivity, and reproducibility than the traditional i method (Ham s acid hemolysis test)

70 Organ Transplantation and Hematopoietic Cell Therapy bone marrow transplantation solid organ transplantation pre-transplant cross- matching, HLA antibody screening, post-transplantation antibody monitoring

71 Platelet analysis Congenital platelet function disorders: Glanzmann s Thrombasthenia (GPIIb/IIIa). Bernard Soulier Syndrome (GPIb). Auto/alloimmune thrombocytopenic purpura Determintion of the rate of thrombopoiesis : stress / reticulated p. platelet activation stored dblood components cardiopulmonary bypass, renal dialysis, the treatment of patients with myocardial infarction

72 Questions? Thank you Soheila Ajdary, Ph.D. Immunology Dept., Pasteur Institute of Iran

73 APPLICATION Defining the cell of origin of specific neoplasms Cell cycle and ploidy analysis of tumors Quantification of hematopoietic stem cells Flow cross-matching (organ transplantation) Postoperative monitoring Detection of autoantibodies

74 TYPES OF MEASUREMENTS DNA content & cell cycle analysis Aneuploidy and/or elevated S-phase fraction have been shown to be ominous prognostic indicators in breast, colon, rectal, prostate, and bladder tumours.

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