FLOW CYTOMETRY: PRINCIPLES AND APPLICATIONS. By: Douaa Moh. Sayed

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2 FLOW CYTOMETRY: PRINCIPLES AND APPLICATIONS By: Douaa Moh. Sayed

3 Definition Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus

4 BASIC PRINCIPLES Flow cytometry involves the analysis of the fluorescence and light scatter properties of single particles (e.g. cells, nuclei, chromosomes) during their passage within a narrow, precisely defined liquid stream.

5 Flow Cytometry - Principles Target: Evaluation of single cell Solution: hydrodynamic Focusing

7 When conditions are right, sample fluid flows in a central core that does not mix with sheath fluid. This termed Laminar flow.

8 Lasers Produce a single wavelength of high intensity Nearly all instruments fitted with an argon laser Gives blue light at 488 nm. Second or third laser may be fitted: - He-Ne- red light at 633 nm - He-Cd- UV at 325 nm

9 Light Scatter Forward angle light scatter: in a narrow angle from the direction of the laser beam (FALS or FS). Forward Scatter (FSC) < 10 detection Laser ~ Cell size

10 Light Scatter Side Scatter (SSC) 90 deflection ~ Cell structures Laser Right angle light scatter at right angles to the laser beam (RALS or SS).

11 Light Scatter FS tends to be more sensitive to the size and surface properties can be used to distinguish live from dead cells. SS tends to be more sensitive to inclusions within cells can be used to distinguish granulated cells from non-granulated

12 Scatter Diagram: Morphology

13 Instrument Parameters Side Scatter (SSC) 90 deflection ~ Cell structures Forward Scatter (FSC) < 10 detection Laser Y ~ Cell size Fluorescence Intensity Antigen Density

14 Fluorochromes Dye Excitation Emission Molecular Weight FITC 488 nm 520 nm 389 Da PE 488 nm 578 nm Da ECD 488 nm 613 nm Da PC5 488 nm 668 nm da PerCP 488 nm 688 nm Da APC 613 nm 665 nm Da

15 Common Fluorochromes for 488nm Excitation LASER 488 FITC 520 PE 575 ECD PI PC5 665 <390 λ λ λ λ λ λ λ >750 λ ultra- violet blue green yellow orange red infraviolet red FITC PE ECD PI PC5 = Fluorescein Isothiocyanate = Phycoerythrin (RD1) = Energy Coupled Dye = Propidium Iodide = Phycoerythrin Cyanin 5 (PC5)

17 Laminar Fluidic Sheath Core Sheath FITC FL PE FL Outer Sheath 488nm Sct

18 Each cell generates a quanta of fluorescence Photomultiplier Tubes (PMT s) PE FL FITC FL 488nm Sct Discriminating Filters Dichroic Lenses Confocal Lens Forward Light Scattering Detector

19 Multiparametric Analysis Simultaneous Detection of up to 20 Parameters Y Cell size Cell structure Dyes Time (follow enzyme reactions)

20 Flow Cytometry - Principles A typical flow cytometer consists of: Light source: argon laser 488 Fluidics Detectors/Computer system for analysis and storage of digitized data.

21 Amplification The electrical pulses originating from light detected by the PMTs are then processed by a series of linear and log amplifiers. Logarithmic amplification is most often used to measure fluorescence in cells. This type of amplification expands the scale for weak signals and compresses the scale for strong or specific fluorescence signals.

22 Data display Univariate histogram Dot plot (Bivariate display)

23 Data display Density plot Contour plot

24 Surface Plot The surface Plotmay be custom oriented smoothed rescaled Zoomed in and out by using the scroll button

25 Tomogram The Tomogram may be: rotated viewed in color precedence or density mode

26 Light Scattering, 2 Parameter Histogram Bigger 90 degree Light Scatter Y Axis Apoptotic Cells Dead Cells Bigger Cells More Granular X Axis Forward Light Scatter (FLS) Live Cells

27 1 Parameter Histogram Negative Positive Count Dimmer Brighter Channel Number Fluorescence picked up from the FITC PMT

28 Statistics Signal Analysis % Negative & % Positive Cells Antigen density ~ Mean Fluorescence Intensity ~ Channel number % negative cells mean fluorescence intensity % positive cells 0 Channel Number 1024

29 Single Positive PI Population 2 Parameter Histogram Double Positive Population PE FL Negative Population FITC FL Single Positive FITC Population

30 Compensation Spectral overlap of emission spectra s of different dyes

31 Compensation Simple rule for Adjustment of Color Compensation B1 B2 Compensation: FL1 versus FL2 Fluorescence 2 B3 B4 Mean-Channel Region B3 = B4 Compensation: FL2 versus FL1 Fluorescence 1 Mean-Channel Region B1 = B3

32 Compensation

33 Compensation

34 Compensation

35 Compensation

36 Quality Control - CDC-Panel Internal Checks Internal Consistency X % CD3 + cells if all tubes +/- 3 % Lymphocytes sums (Purity?) % CD3 + cells + % CD19 + cells + % CD16 + /CD56 + cells n lymphocyte gate = 100 % (+/- 5%) % CD3 + /CD4 + cells + % CD3 + /CD8 + cells = % CD3 + cells

38 Gating Set a region on a histogram or cytogram If cell in region then show another property cell selection

39 Gating-Option ungated Dual Parameter Correlation: FSC = Cell size SSC = Cell structure gated Analysis of Cell Sub-populations i.e. Lymphocytes

40 Flow Cytometry Data Smaller Region, Live cells mostly Larger Region includes all cells

41 Cell sorting Flow cytometry can be used to select and purify a specific subset of cells within a population cell sorting based on physical, biochemical and antigenic traits.

42 Flow Cytometry and sorting

43 Measurable parameters volume and morphological complexity of cells cell pigments such as chlorophyll or phycoerythrin DNA (cell cycle analysis, cell kinetics, proliferation etc.) RNA chromosome analysis and sorting (library construction, chromosome paint) protein expression and localization transgenic products in vivo, particularly the Green fluorescent protein or related fluorescent proteins cell surface antigens (Cluster of differentiation (CD) markers) intracellular antigens (various cytokines, secondary mediators etc.) nuclear antigens

44 Measurable parameters enzymatic activity ph, intracellular ionized calcium, magnesium, membrane potential membrane fluidity apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity) cell viability monitoring electropermeabilization of cells oxidative burst characterising multidrug resistance (MDR) in cancer cells glutathione various combinations (DNA/surface antigens etc.)

45 Flow Cytometry Applications molecular biology, The specific antibodies bind to antigens on the target cells and help to give information on specific characteristics of the cells pathology, immunology, plant biology and marine biology: the auto-fluorescent properties of photosynthetic plankton can be exploited by flow cytometry in order to characterise abundance and community structure. protein engineering: flow cytometry is used in conjunction with yeast display and bacterial display to identify cell surface-displayed protein variants with desired properties.

46 Clinical applications in flow cytometry Immunophenotyping of leukemia and lymphoma DNA & cell cycle analysis Minimal residual disease PNH-diagnostics RNA content (reticulocytes) Immune-deficiencies

47 Clinical applications in flow cytometry Platelet Function Analysis: Platelet associated immunoglobulins platelet-associated IgG quantitation for the diagnosis of immune thrombocytopenias platelet cross-matching in transfusion reticulated platelet assay to detect stress platelets fibrinogen receptor occupancy studies for monitoring the clinical efficacy of plateletdirected anticoagulation in thrombosis detection of activated platelet surface markers, cytoplasmic calcium ion measurements, and platelet microparticles for the assessment of hypercoagulable states.

48 Clinical applications in flow cytometry Measurement of the Efficacy of Cancer Chemotherapy Multi-Drug-Resistance assays of proliferative survival using bromodeoxyuridine (BrdU) incorporation Ligand, antigen, or molecule-targeted biological therapy utilizing monoclonal antibodies

49 Clinical applications in flow cytometry Cell Function Analysis every event that occurs during the process of lymphocyte activation can be measured by flow cytometry [tyrosine phosphorylation (multiplex bead technology), calcium flux, oxidative metabolism, neoantigen expression (CD11b/CD18 and CD154), and cellular proliferation) Applications in Transfusion Medicine Fetal red cells in maternal blood Quality control of blood products

50 Clinical applications in flow cytometry Organ Transplantation and Hematopoietic Cell Therapy HLA-typing and cross- matching, enumeration of CD34+ hematopoietic stem cells pre-transplantation determinations of the efficacy of ex vivo T-cell graft depletion, post-transplantation evaluation of immune recovery, graft rejection, graft-versus host disease, and the graft-versus-leukemia effect.

51 Clinical applications in flow cytometry Applications in Microbiology detection of bacteria, fungi, parasites and viruses quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity sperm sorting in IVF Apoptosis PI incorporation TdT Assay Annexin V APO2.7

53 Advantages high speed analysis (> s -1 ) Measures single cells Measures large number of cells simultaneous analysis of multiple parameters (up to 20) Identifies small subpopulations quantification of fluorescence intensities sorting of predefined cell populations (up to s -1

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

55 THANKS

56 THANKS

57 THANK YOU FOR YOUR ATTENTION

Introduction to flow cytometry

Introduction to flow cytometry Flow cytometry is a popular laser-based technology. Discover more with our introduction to flow cytometry. Flow cytometry is now a widely used method for analyzing the expression