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Flow Cytometry

40. Flow Cytometry. Learning Objectives—Level I. At the end of this unit of study, the student should be able to: Describe the components of a flow cytometer and the principles of cell analysis. Illustrate by example the clinical applications of flow cytometry.

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Flow Cytometry

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  1. 40 Flow Cytometry

  2. Learning Objectives—Level I At the end of this unit of study, the student should be able to: • Describe the components of a flow cytometer and the principles of cell analysis. • Illustrate by example the clinical applications of flow cytometry. • Appraise the use of fluorochrome-labeled antibodies in immunophenotyping by flow cytometry. continued on next slide

  3. Learning Objectives—Level I At the end of this unit of study, the student should be able to: • Give examples of the clinical applications of immunophenotyping by flow cytometry, and interpret single dot plots. • Define clonality and identify methods for detecting a monoclonal population of cells by immunophenotyping. • List the specimens appropriate for immunophenotyping by flow cytometry. continued on next slide

  4. Learning Objectives—Level I At the end of this unit of study, the student should be able to: • Describe how flow cytometry can be used in cell quantitation. • Calculate and interpret the absolute CD4 count. • Explain how flow cytometry can be applied to DNA analysis. • List the cells positive for CD34, and explain the purpose of a CD34 count.

  5. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Compare and contrast the immunophenotyping results characteristic of chronic lymphocytic leukemia, hairy cell leukemia, and non-Hodgkin lymphoma. continued on next slide

  6. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Identify the pitfalls that can be encountered in immunophenotyping mature lymphoid malignancies by flow cytometry, and generate potential solutions. continued on next slide

  7. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Compare and contrast the immunophenotyping results characteristic of acute lymphoblastic leukemia and acute myelogenous leukemia. continued on next slide

  8. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Identify the pitfalls that can be encountered in immunophenotyping acute leukemia and lymphoma by flow cytometry, and generate potential solutions. continued on next slide

  9. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Compare and contrast the usefulness of the sucrose hemolysis test, Ham test, and flow cytometry immunophenotyping in diagnosing paroxysmal nocturnal hemoglobinuria. • Select and explain the quality measures that must be enforced in quantitative flow cytometry. continued on next slide

  10. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Compare and contrast the absolute CD4 count and HIV viral load in the surveillance of HIV infection. • Calculate and interpret the S phase fraction and the DNA index, and give the principle of DNA analysis by flow cytometry. continued on next slide

  11. Learning Objectives—Level II At the end of this unit of study, the student should be able to: • Assess the findings of flow cytometry cell analysis using two or more dot plots, and select the most likely cell represented. • Evaluate flow cytometry results to identify problems and generate solutions. • Summarize the uses of analysis for the CD34 antigen, and choose recommended procedures to analyze this antigen.

  12. Introduction • Flow cytometer • Instrument capable of detecting molecules on the surface of or inside individual particles such as cells • Particles/cells that possess the molecule of interest are labeled with a fluorescent marker that is specific for the molecule • Recognized by emission of fluorescent light following excitation

  13. Introduction • Current applications of flow cytometry in clinical laboratory • Immunophenotyping WBCs and RBCs • Counting CD4+ and CD34+ cells • Analyzing DNA

  14. Figure 40-1 Flow cytometer. The cells in the specimen are stained with fluorochrome-labeled antibodiesand separated into single cells that pass in front of a laser light source. Scattered and emitted light aredetected with photodectors and photomultiplier tubes (PMTs), undergo computer analysis, and are displayedas dot plots.APC = allophycocyanin; FITC = fluorescein isothiocyanate; PE = phycoerythrin; PerCP = peridinin chlorophyll

  15. Table 40-1 Applications of Flow Cytometry

  16. Principles of Flow Cytometry

  17. Isolation of Single Particles • Flow cytometry • Performed on particles in suspension • Leukocytes from peripheral blood and BM • Often analyzed after removal of RBCs by lysis

  18. Isolation of Single Particles • Cell suspension • Aspirated and injected into a flow chamber • Specimen handling area of a flow cytometer • Cells are forced into single file • Directed into path of a laser beam

  19. Isolation of Single Particles • Flow chamber • Contains two columns of fluid • Sample fluid and sheath fluid • Maintained at different pressures • Move through at different speeds • Gradient keeps fluid separate (laminar flow) • Controls diameter of the column of sample fluid continued on next slide

  20. Isolation of Single Particles • Flow chamber • Contains two columns of fluid • Central column of sample fluid is narrowed • Isolate single particles/cells that pass through the laser beam • Called hydrodynamic focusing continued on next slide

  21. Isolation of Single Particles • Flow chamber • Laser light focused on these single particles/cells • Scatters light • Measured by photodetectors (photodiodes) continued on next slide

  22. Isolation of Single Particles • Flow chamber • Fluorescent molecules attached to particles/cells • Laser excites the molecules • Emit light at a specific wavelength • Detected by photomultiplier tube (PMT)

  23. Light Scattering • Used to distinguish particles • Size, granularity, nuclear complexity • Side light scatter—90° angle • Related to internal complexity and granularity • ↑ in neutrophils due to cytoplasmic granules

  24. Light Scattering • Forward light scatter • Related to particle size • Larger cells produce more than small cells

  25. Figure 40-2 Flow cytometry histograms. Data acquired using flow cytometry can be displayed in a variety of plots. The plot of forward light scatter (FSC-A) versus side light scatter (SSC-A) (top left) distinguishes granulocytes (black) with high side scatter from other cells but cannot distinguish blasts (blue) from lymphocytes (red). The plot of CD45 versus SSC-A (top right) distinguishes blasts with weak intensity CD45 expression (blue) from lymphocytes with bright intensity CD45 expression (red). Data can also be displayed as single-color histograms (lower plot) but are less useful for distinguishing cells types.Histograms generated using FACS DIVA software BD Biosciences, San Jose, CA.

  26. Detection of Fluorochromes • Flow cytometer detects fluorochromes • Bound fluorescent markers • Molecules excited by one wavelength • Emit light of a different wavelength • Fluorochromes are used to • Label detection antibodies • Bind stoichiometrically to DNA or RNA

  27. Detection of Fluorochromes • Flow cytometer • Use light of a single wavelength • Generated by an argon laser • Excite fluorochromes bound to particle of interest • Light emitted from fluorochromes • Separated from incident laser light • Detected and quantified by photomultiplier tube

  28. Detection of Fluorochromes • Clinical flow cytometers • Use two or three laser light sources • ID four to eight antigens on each cell

  29. Detection of Fluorochromes • Compensation • Overlap between the light emitted from different fluorochromes • Overlap compensated by: • Adjusting settings of flow cytometer • Performing a math correction before or after data collected

  30. Detection of Fluorochromes • Some FDA-approved kits available (e.g., CD4 or CD8)

  31. Detection of Fluorochromes • Clinical laboratories • Responsible for development of labeled antibodies to meet specific needs which include • Identifying properties of interest • Fluorochrome reagent selection and titering • Testing for precision, sensitivity, and specificity

  32. Table 40-2 Example Fluorochromes Used in Flow Cytometry

  33. Immunophenotyping by Flow Cytometry

  34. Immunophenotyping • ID of antigens using detection antibodies • Antibodies • Bind specifically to antigens • Can be labeled with fluorochromes • Provide a sensitive and specific detection method continued on next slide

  35. Immunophenotyping • ID of antigens using detection antibodies • Detection antibodies • Polyclonal • Monoclonal

  36. Immunophenotyping • Polyclonal antibodies • Antibodies made by injecting antigen into animals • Rabbit antihuman antibodies • Animal produces many antibodies directed against different portions of the antigen continued on next slide

  37. Immunophenotyping • Polyclonal antibodies • Allows antigen to be recognized even if some parts are abnormal • Difficult to standardize and prone to specific nonbinding

  38. Immunophenotyping • Monoclonal antibodies • Directed against a single portion of the antigen • Produced in myeloma/tumor cell lines • High purity and reproducibility

  39. Immunophenotyping • CD designation • Antibodies are grouped together when recognizing the same antigen • All antibodies that recognize the same antigen are given the same cluster of differentiation (CD) number

  40. Table 40-3 Antibodies Used for Immunophenotyping by Flow Cytometry continued on next slide

  41. Table 40-3 (continued) Antibodies Used for Immunophenotyping by Flow Cytometry continued on next slide

  42. Table 40-3 (continued) Antibodies Used for Immunophenotyping by Flow Cytometry

  43. Specimen Requirements and Preparation for Immunophenotyping • Requires a suspension of individual cells • Samples already contain cells in suspension • Anticoagulated blood or bone marrow aspirate • Body fluid specimens • Fine needle aspiration continued on next slide

  44. Specimen Requirements and Preparation for Immunophenotyping • Requires a suspension of individual cells • Leukocytes isolated either by • RBC lysis • Density gradient centrifugation • Cells can also be isolated from fresh tissue biopsy

  45. Specimen Requirements and Preparation for Immunophenotyping • Suspension of leukocytes with lysed RBCs • Stained using fluorochrome-labeled antibodies • Aspirated into the flow cytometer continued on next slide

  46. Specimen Requirements and Preparation for Immunophenotyping • Suspension of leukocytes with lysed RBCs • Record for every cell identified includes: • Amount of scattered light • Intensity of each florescent signal • Graphic display on histograms or dot plots

  47. Specimen Requirements and Preparation for Immunophenotyping • Dot plots • Identify cells with similar staining properties • Highlighted with different colors • Plots are divided into quadrants • Cells labeled with • One fluorochrome • Other fluorochrome • Both fluorochromes

  48. Specimen Requirements and Preparation for Immunophenotyping • Cells are positive for antigen if: • Fluorescence intensity > than either • Negative control or negative population of cells in same analysis tube • Intensity of light emitted is compared to known range of intensities • Dim or weak, intermediate, bright or strong • Related to density of antigens

  49. Table 40-4 Specimen Requirements for Immunophenotyping by Flow Cytometry

  50. Gating • Isolate cells of interest • Separated during • Specimen processing • Data analysis

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