Introduction to Flow Cytometry

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A basic understanding of flow cytometry and its use in cancer diagnostics.

A basic understanding of flow cytometry and its use in cancer diagnostics.

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  • 1. CSI Labs Training Seminar 2007 Flow Cytometry: Principles and Application Eric Diersen CSI Labs Atlanta, Georgia
  • 2. Agenda
    • Principles of flow cytometry
      • Definition
      • Basics: Fluidics, Optics, Electronics
    • Application of flow cytometry
      • Leukemia and lymphoma diagnostics
      • Zap-70
      • Case Studies
  • 3. Definition
    • Flow cytometry is the technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid.
    • It allows simultaneous multi-parametric analysis of the physical and or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus.
  • 4. Flow Cytometry Diagram
  • 5. What does flow tell us?
    • Relative cell (particle) size
    • Cellular complexity
    • Cellular characteristics
  • 6. Common uses of flow cytometry
    • Quantitative:
      • CD4+ T-cell (HIV infection)
      • Stem cell harvest
  • 7. Common uses of flow cytometry
    • Qualitative:
      • Immunophenotyping
        • Leukemia
        • Lymphoma
        • PNH
      • Subclassifying hematopoietic malignancies beyond the capabilities of traditional morphologic and cytochemical techniques
  • 8. Principles of Flow: Fluidics
    • Cells must be in suspension to flow in single file through the flow cell.
    • The sample is injected into sheath fluid as it passes through a small (50-300µm) orifice.
    • When done correctly, the sample fluid flows in central core that does not mix with the sheath fluid (Laminar flow).
  • 9. Principles of Flow: Fluidics
    • Hydrodynamic Focusing:
      • The introduction of a large volume into a small volume in such a way that becomes “focused” along an axis.
    *Notice how the ink is focused into a tight stream as it is drawn into the tube under laminar flow conditions.
  • 10. Principles of Flow: Optics
    • Light source: Laser
      • Provides single wavelengh of light.
      • Usually achieved with argon filter at a wavelength of 488nm.
  • 11. Principles of Flow: Optics
    • Forward Scatter (FSC)
      • With a laser source, the amount of light scattered in a forward direction (along the same axis that the laser travels) detected in forward scatter channel.
      • Forward scatter (FSC) is more sensitive to size.
      • Forward scatter can distinguish living from dead cells.
  • 12. Principles of Flow: Optics
    • Forward Scatter (FSC)
  • 13. Principles of Flow: Optics
    • Side Scatter (SSC)
      • With a laser source, the amount of light scattered to the side (perpendicular to the axis that the laser travels) is detected as the side or 90º scatter.
      • Side scatter (SSC) is more sensitive to inclusions and cell complexity.
      • Side scatter can distinguish granulated cells from non-granulated cells.
  • 14. Principles of Flow: Optics
    • Side Scatter (SSC)
      • Major morphologic discrimination by light scatter is lymphocytes, monocytes, and granulocytes.
      • Lymphocytes have low granularity; therefore, low side scatter.
      • Granulocytes are multi-nucleated with many granules; therefore, high side scatter.
  • 15. Principles of Flow: Optics
    • Immunofluorescence:
      • Fluorescent dye bound to an antibody.
      • When excited by laser, the dye emitts at a specific wavelengh that is captured by detectors.
      • Different cellular characteristics may be probed for by simply changing the antibodies.
  • 16. Principles of Flow: Optics
    • Fluorescence Channels
  • 17. Principles of Flow: Optics
    • Filters
      • Simultaneous measurement requires multiple channels (multiple detectors)
      • Considerations
        • Spectral properties of fluorochromes
        • Proper order of filters and mirrors
  • 18. Principles of Flow: Electronics
      • The electonics process the signals coming from the photodetectors.
      • This process includes signal shaping, amplification, and conversion from analog to digital format.
      • Two types of photodetectors are used:
        • Photodiodes
        • Photomultiplier tubes (PMT)
  • 19. Principles of Flow: Electronics Photodetectors Display
  • 20. Summary
    • Flow cytometry simultaneously measures multiple characteristics of particles or cells.
    • These characteristics (parameters) are quantified using an optical-to-electronic coupling system.
    • The electronic system measures how cells (particles) interact with a fixed beam of laser light.
    • Data from each event (cell or particle) is collected and stored for analysis.
  • 21. Leukemia and Lymphoma Diagnostics
    • ALL – Acute Lymphoblastic Leukemia
    • CLL – Chronic Lymphoblastic Leukemia
    • AML – Acute Myelogenous Leukemia
    • CML – Chronic Myelogenous Leukemia
    • MDS – Myelodysplasia
    • HCL – Hairy Cell Leukemia
  • 22. Zap-70 CLL Prognostics
    • Overview
      • Zap-70 is a pivotal prognostic marker for patients with CLL
      • Zap-70 is a labile, intracellur antigen presented by certain malignant B-cell lymphocytes.
      • Due to the aggressive nature of these malignant populations, identification is critical for treatment.
  • 23. Case Study #1:
    • A 61-year-old female with a past medical history of chronic lymphocytic leukemia (CLL) for 10 years.
    • Flow cytometric immunophenotypic studies show a population of large cells (see arrow).
    • This population coexpresses CD5 and CD19, is dim CD20 positive, CD22 positive, CD10 negative, FMC7 negative, ZAP-70 positive, and CD38 positive.
    • Recavarren et al, 2005.
  • 24. Case Study #2
    • 57 year old female who noticed weakness and easy bruising over a one month period.
    • Flow cytometry results of bone marrow.
    • Cell surface markers CD45, CD15, CD7, and CD14 are bound with corresponding antibodies.
    • Expression of these markers are indicative of hematopoietic cell lineages.
    • Borowitz, et al, 1998.
    Diagnosis: M2-AML *Antigen Profile: Positive for CD71, CD33, HLADR, CD7, CD38, CD13; partly positive for CD11b, CD15 .
  • 25.  
  • 26. References
    • Bray, R. & Gebel, H. (n.d.). “Basic Principles of Flow Cytometry”. Department of Pathology, Emory University Hospital, Atlanta, GA.
    • Owens, M. & Loken, M.(1995). Flow Cytometry Principles for Clinical Laboratory Practice: Quality Assurance for Quantitative Immunophenotyping . New York, NY: Wiley-Liss.
    • Recavarren, R., Contis, L., & Hu, J. (2005). Case 446 -- A 61-year-old woman with chronic lymphocytic leukemia, pancytopenia and abdominal pain . Retrieved September 13, 2007 from path.upmc.edu/cases/case446.html.
    • Borowitz, M. & Silberman, M. (1998). Flow Cytometry of Leukemia. Department of Pathology, Johns Hopkins Medical Institutes. Retrieved September 13, 2007 from 162.129.103.34/cgi-win/front.exe/Instr?3