1 Flow Cytometry in the Clinical Laboratory Patricia Aoun, M.D., M. P. H. Jean Bailey, MT-ASCP Kellie Neth, MT-ASCP The Nebraska Medical Center

2 Definition Flow Cytometry is the measurement of the cellular properties of cells/particles as they Flow Cytometry is the measurement of the cellular properties of cells/particles as they move in a fluid past a stationary set of detectors.

3 Basics of Flow Cytometry Fluidics Optics Electronics Cells in suspension flow in a single -file through an illuminated volume... where they scatter light and emit fluorescence that is collected and filtered... then converted to digital values that are stored on a computer.

4 PMT Dichroic Filters Bandpass Filters Flow Cytometer Schematic Laser Flow cell Purdue University Cytometry Laboratories Fluidics

5 FluidicsFluidics Cells in suspension in a sample fluid flow Cells in suspension in a sample fluid flow single file through an illuminated flow cell single file through an illuminated flow cell In most instruments, accomplished by injecting the sample fluid into a sheath fluid as it passes through a small ( µm) orifice In most instruments, accomplished by injecting the sample fluid into a sheath fluid as it passes through a small ( µm) orifice

6 Fluidics Sample Fluid: An isotonic fluid in which the cells are suspended and in which they are injected into the sheath fluid stream Sheath Fluid: A different isotonic fluid that is forced under pressure through a conical nozzle assembly designed to produce laminar flow

7 Fluidics - Flow Chamber The flow cell The flow cell defines the axis and dimensions of sheath and sample flow defines the axis and dimensions of sheath and sample flow defines the point of optimal hydrodynamic focusing defines the point of optimal hydrodynamic focusing

8 Flow Cell Injector Tip Fluorescence signals Focused laser beam Sheath fluid Purdue University Cytometry Laboratories

9 Fluidics Laminar Flow: If the flow is set up correctly, the sheath fluid and the sample fluid will not mix, and laminar flow will result. If the flow is set up correctly, the sheath fluid and the sample fluid will not mix, and laminar flow will result. Hydrodynamic Focusing: Focusing effect of laminar flow which places the sample stream in the center of the sheath stream

10 FluidicsFluidics V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3 Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions. Notice also how the position of the inner ink stream is influenced by the position of the ink source.

11 PMT Dichroic Filters Bandpass Filters Flow Cytometry Optics Laser Flow cell Purdue University Cytometry Laboratories Optics

12 Optics (Light source) The light source used must be The light source used must be focused on the same point where the cells have been focused (The Interrogation Point)

13 Flow Cell Injector Tip Fluorescence signals Interrogation point Sheath fluid Purdue University Cytometry Laboratories

14 Optics (Light Source) Two types of light sources are used Two types of light sources are used Lasers Lasers Arc-lamps Arc-lamps

15 Optics - Light Source Lasers Lasers can provide a single wavelength of coherent light (a laser line) can provide a single wavelength of coherent light (a laser line) can provide from milliwatts to watts of light can provide from milliwatts to watts of light can be inexpensive, air-cooled units or can be inexpensive, air-cooled units or expensive, water-cooled units

16 Optics - Light Source Arc-lamps Arc-lamps provide mixture of wavelengths that must be filtered to select desired wavelengths provide mixture of wavelengths that must be filtered to select desired wavelengths provide incoherent light provide incoherent light provide milliwatts of light provide milliwatts of light inexpensive, air-cooled units inexpensive, air-cooled units

17 PMT Dichroic Filters Bandpass Filters Flow Cytometer Schematic Laser Flow cell Purdue University Cytometry Laboratories Optical Channels

18 Optics - Optical Channels An optical channel is a path that light can follow from the illuminated volume to a detector An optical channel is a path that light can follow from the illuminated volume to a detector Certain types of optical elements (filters, mirrors) provide separation of channels and wavelength selection Certain types of optical elements (filters, mirrors) provide separation of channels and wavelength selection

19 Properties Being Measured Intrinsic Properties Intrinsic Properties Forward light scatter Forward light scatter Side light scatter Side light scatter Extrinsic Properties Extrinsic Properties

20 Optics - Forward Scatter Channel The amount of light scattered in the forward direction (along the same axis that the laser light is traveling) is detected in the forward scatter channel The amount of light scattered in the forward direction (along the same axis that the laser light is traveling) is detected in the forward scatter channel The intensity of forward scatter is proportional to the size and shape of cells The intensity of forward scatter is proportional to the size and shape of cells

21 Forward Angle Light Scatter FALS Sensor Laser Purdue University Cytometry Laboratories

22 Optics - Side Scatter Channel The amount of light scattered to the side (perpendicular to the axis that the laser light is traveling) is detected in the side scatter or 90 o scatter channel The amount of light scattered to the side (perpendicular to the axis that the laser light is traveling) is detected in the side scatter or 90 o scatter channel The intensity of side scatter is proportional to the complexity of cells The intensity of side scatter is proportional to the complexity of cells

23 90 Degree Light Scatter FALS Sensor 90LS Sensor Laser Purdue University Cytometry Laboratories

24 Properties Being Measured Properties Being Measured Intrinsic properties Intrinsic properties Extrinsic properties Extrinsic properties Cellular components detected by fluorescently-labeled antibodies specific to the component Cellular components detected by fluorescently-labeled antibodies specific to the component (ex. antigen site) (ex. antigen site)

25 Extrinsic Properties Cell Wall Component (antigen site) (antigen site) MembraneReceptor Nuclear protein

26 Fluorochromes Fluorescent dyes used to stain or label Fluorescein (FITC) 400 nm600 nm700 nm Wavelength 500 nm Excitation Emission Purdue University Cytometry Laboratories

27 Fluorescent Dyes

28 Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm300 nm500 nm700 nm400 nm Common Laser Lines Purdue University Cytometry Laboratories

29 Spectral Overlap Like the colors in a rainbow, the fluorescent antibodies we use overlap/blend together. Need to correct for this spectral overlap in order to detect distinct cell populations Can be done by several methods

30 ADC (Advanced Digital Compensation) Color Compensation Grid Coulter FC500

31 Optics - Fluorescence Channels The fluorescence emitted by each fluorochrome is usually detected in a unique fluorescence channel The fluorescence emitted by each fluorochrome is usually detected in a unique fluorescence channel The specificity of detection is controlled by the wavelength selectivity of optical filters and mirrors The specificity of detection is controlled by the wavelength selectivity of optical filters and mirrors

32 Laser Fluorescence Detectors Fluorescence FALS Sensor Fluorescence detector (PMT3, PMT4 etc.) Purdue University Cytometry Laboratories

33 PMT Dichroic Filters Bandpass Filters Flow Cytometry Optics Laser Flow cell Purdue University Cytometry Laboratories Filters

34 Optics - Filter Properties Long pass filters transmit wavelengths above a cut-on wavelength Long pass filters transmit wavelengths above a cut-on wavelength Short pass filters transmit wavelengths below a cut-off wavelength Short pass filters transmit wavelengths below a cut-off wavelength Band pass filters transmit wavelengths in a narrow range around a specified wavelength Band pass filters transmit wavelengths in a narrow range around a specified wavelength Band width can be specified Band width can be specified

35 Standard Long Pass Filters Transmitted Light Light Source 520 nm Long Pass Filter >520 nm Light Transmitted Light Light Source 575 nm Short Pass Filter <575 nm Light Standard Short Pass Filters Purdue University Cytometry Laboratories

36 Standard Band Pass Filters Transmitted Light White Light Source 630 nm Bandpass Filter nm Light Purdue University Cytometry Laboratories

37 Dichroic Filter/Mirror Filter placed at 45 o Reflected light Transmitted LightLight Source original from Purdue University Cytometry Laboratories; modified by R.F. Murphy

38 PMT Dichroic Filters Bandpass Filters Flow Cytometry- Detectors Laser Flow cell original from Purdue University Cytometry Laboratories; modified by R.F. Murphy Photomultiplier Tubes (PMT’s)

39 DetectorsDetectors Photomultiplier tube (PMT) Photomultiplier tube (PMT) The PMT converts the light to a voltage pulse which rises and falls with the amount of light entering. The PMT converts the light to a voltage pulse which rises and falls with the amount of light entering.

40 PMTs with Filters on the Coulter FC500 series Notice the close proximity of the detectors, signal stays stronger

41 ElectronicsElectronics Processing of electrical signals from detectors Processing of electrical signals from detectors Preamplification Preamplification Strengthen signals so that they can travel from remote detectors to central electronics Strengthen signals so that they can travel from remote detectors to central electronics Amplification Amplification Adjust signal intensity Adjust signal intensity Conversion of analog electrical signals to digital signals Conversion of analog electrical signals to digital signals

42 Data Acquisition Digital Signals are collected and stored by a computer, and can be displayed in a variety of formats Digital Signals are collected and stored by a computer, and can be displayed in a variety of formats

43 Side Scatter FALFAL Gating

44 90 Degree Scatter Lymphocytes Monocytes Neutrophils Side Scatter Projection Scale Purdue University Cytometry Laboratories

45 A Picture is Worth a Thousand Words

46 Clinical Applications of Flow Cytometry

47 General Principles Cellular antigens: Membrane, cytoplasmic or nuclear components Cellular antigens: Membrane, cytoplasmic or nuclear components Monoclonal antibodies: Developed against a specific component Monoclonal antibodies: Developed against a specific component CD ( cluster designation ) numbers: Universal system of numbers for grouping different monoclonal antibodies against the same antigen CD ( cluster designation ) numbers: Universal system of numbers for grouping different monoclonal antibodies against the same antigen

48 CD Nomenclature The system used for leukocyte surface molecules, as identified by monoclonal antibodies. The system used for leukocyte surface molecules, as identified by monoclonal antibodies. Examples: Examples: CD45Common Leukocyte Antigen CD45Common Leukocyte Antigen CD2, CD3, CD5T cells CD2, CD3, CD5T cells CD19and CD20 B cells CD19and CD20 B cells CD34 Stem cell marker CD34 Stem cell marker

49 Indications for Flow Cytometry Quantitation of lymphocyte subsets in immunodeficiencies, HIV monitoring Quantitation of lymphocyte subsets in immunodeficiencies, HIV monitoring Determination of cell type and stage of differentiation in acute leukemias, including blast crisis of CML Determination of cell type and stage of differentiation in acute leukemias, including blast crisis of CML Detection of lymphoma cells in blood, bone marrow, fluids and tissues Detection of lymphoma cells in blood, bone marrow, fluids and tissues Detection of CD34+ stem cells in peripheral blood and peripheral stem cell collection products Detection of CD34+ stem cells in peripheral blood and peripheral stem cell collection products

50 Flow is not particularly helpful in… Chronic phase of CML Chronic phase of CML Hodgkin’s disease Hodgkin’s disease Low grade myelodysplastic syndromes Low grade myelodysplastic syndromes (better when able to use antigen mapping technique) Screening of peripheral blood in patients who do not have a sustained lymphocytosis or abnormal findings on the smear Screening of peripheral blood in patients who do not have a sustained lymphocytosis or abnormal findings on the smear