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Quality assurance for polychromatic flow cytometry.

Stephen P Perfetto1, David Ambrozak, Richard Nguyen

  • 1Flow Cytometry Core Facility, Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, 40 Convent Drive, Bethesda, Maryland 20892, USA. sperfetto@mail.nih.gov

Nature Protocols
|April 5, 2007
PubMed
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This protocol details a quality assurance program for polychromatic flow cytometry. It ensures accurate cell measurement by optimizing, calibrating, and monitoring instruments with multiple fluorochromes.

Area of Science:

  • Biotechnology
  • Analytical Chemistry
  • Cell Biology

Background:

  • Polychromatic flow cytometry enables multi-parameter analysis of cells using five or more fluorochromes.
  • Ensuring instrument performance is critical for reliable and reproducible results in complex cell analysis.

Purpose of the Study:

  • To establish a comprehensive three-part quality assurance program for polychromatic flow cytometers.
  • To optimize, calibrate, and monitor instrument performance for accurate fluorescent signal detection.

Main Methods:

  • System optimization: Configuring lasers, mirrors, and filters for optimal fluorescent signal generation and transmission.
  • Calibration: Measuring fluorescence across a range of photomultiplier tube (PMT) voltages to determine sensitivity, dynamic range, and linearity.

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  • Monitoring: Implementing procedures to track precision, accuracy, and sensitivity over time.

    • Main Results:

    • The protocol provides a structured approach to instrument setup and validation.
    • It defines specific steps for optimizing optical components and calibrating detectors.
    • Procedures are outlined for ongoing performance monitoring to ensure data integrity.

    Conclusions:

    • This quality assurance program is essential for maintaining the reliability of polychromatic flow cytometry.
    • Implementation upon installation or after optical path changes is recommended.
    • Routine monitoring ensures consistent and accurate cell measurement over time.