Flow Cytometry in the Clinical Laboratory

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 move in a fluid past a stationary set of detectors.

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

4. Flow Cytometer Schematic PMT 4 PMT Dichroic 3 Filters Flow cell PMT 2 Bandpass Filters PMT 1 Laser Fluidics Purdue University Cytometry Laboratories

5. Fluidics • Cells in suspension in a sample fluidflow 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 (50-300 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 • defines the axis and dimensions of sheath and sample flow • defines the point of optimal hydrodynamic focusing

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

9. Laminar Flow: 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 Fluidics

10. Fluidics 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. V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3

11. Flow Cytometry Optics PMT 4 PMT Dichroic 3 Filters Flow cell PMT 2 Bandpass Filters PMT 1 Laser Optics Purdue University Cytometry Laboratories

12. Optics (Light source) • 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 Sheath fluid Fluorescence signals Interrogation point Purdue University Cytometry Laboratories

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

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

16. Optics - Light Source • Arc-lamps • provide mixtureof wavelengths that must be filteredto select desired wavelengths • provide incoherent light • provide milliwatts of light • inexpensive, air-cooled units

17. Flow Cytometer Schematic PMT 4 Optical Channels PMT Dichroic 3 Filters Flow cell PMT 2 Bandpass Filters PMT 1 Laser Purdue University Cytometry Laboratories

18. Optics - Optical Channels • 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

19. Properties Being Measured • Intrinsic Properties • Forward light scatter • Side light scatter • 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 intensity of forward scatter is proportional to the size and shapeof cells

21. Laser FALS Sensor Forward Angle Light Scatter 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 90o scatter channel • The intensity of side scatter is proportional to the complexity of cells

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

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

25. Extrinsic Properties Cell Wall Component (antigen site) Membrane Receptor Nuclear protein

26. Wavelength 400 nm 500 nm 600 nm 700 nm Excitation Emission Fluorescein (FITC) Fluorochromes Fluorescent dyes used to stain or label Purdue University Cytometry Laboratories

27. Fluorescent Dyes

28. 350 457 488 514 610 632 300 nm 400 nm 500 nm 600 nm 700 nm Common Laser Lines PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid 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 specificity of detection is controlled by the wavelength selectivity of optical filters and mirrors

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

33. Flow Cytometry Optics PMT 4 Filters PMT Dichroic 3 Filters Flow cell PMT 2 Bandpass Filters PMT 1 Laser Purdue University Cytometry Laboratories

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

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

36. Standard Band Pass Filters 630 nm Bandpass Filter White Light Source Transmitted Light 620 -640 nm Light Purdue University Cytometry Laboratories

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

38. Flow Cytometry- Detectors PMT 4 PMT Dichroic 3 Filters Flow cell PMT 2 Bandpass Filters PMT 1 Laser Photomultiplier Tubes (PMT’s) original from Purdue University Cytometry Laboratories; modified by R.F. Murphy

39. Detectors • Photomultiplier tube (PMT) • 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. Electronics • Processing of electrical signals from detectors • Preamplification • Strengthen signals so that they can travel from remote detectors to central electronics • Amplification • Adjust signal intensity • 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

43. Gating F A L SideScatter

44. Side Scatter Projection Neutrophils Scale 1000 200 100 50 40 Monocytes 30 20 15 Lymphocytes 8 200 400 600 800 1000 0 90 Degree Scatter 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 • Monoclonal antibodies: Developed against a specific component • 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. • Examples: • CD45 Common Leukocyte Antigen • CD2, CD3, CD5 T cells • CD19 and CD20 B cells • CD34 Stem cell marker

49. Indications for Flow Cytometry • Quantitation of lymphocyte subsets in immunodeficiencies, HIV monitoring • 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 CD34+ stem cells in peripheral blood and peripheral stem cell collection products

50. Flow is not particularly helpful in… • Chronic phase of CML • Hodgkin’s disease • 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