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Quality assurance for polychromatic flow cytometry using a suite of calibration beads

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Abstract

The quality assurance program presented here provides a means to maximize and maintain the performance of individual flow cytometers in a facility. To optimize performance, we recommend performing all three steps (optimization, calibration and standardization) in this program when a new flow cytometer is installed or whenever the flow cytometer's optical path is altered (e.g., lasers, filters or detectors are replaced). The complete process requires 3–4 h. On a more frequent basis, only a subset of these procedures need to be performed as a part of daily maintenance routines. The data generated can be tracked to monitor the instrument and determine whether service is needed. In addition, the data can provide a metric for whether repairs and upgrades have improved or harmed performance, and for future instrument-to-instrument comparisons. In sum, the procedures presented here represent an updated framework for optimizing, calibrating and standardizing a flow cytometer for daily use.

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Figure 1: Particles for a quality assurance program.
Figure 2: Determining a gain (voltage) range using the Gain Range Calibration Tool.
Figure 3: Determining the optimal PMT voltage using QCSBs.
Figure 4: Biological validation and COMP bead matrix.
Figure 5: Tolerance ranges for 1× Rainbow beads over time.
Figure 6: Monitoring of the R660 detector over time across five instruments.
Figure 7: Intra-instrument quality control data for the G560 detector.

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S.P.P. conceived and defined experiments, analyzed data and wrote the manuscript. D.A. and R.N. performed experiments and analyzed data. P.K.C. and M.R. designed experiments and wrote the manuscript.

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Correspondence to Stephen P Perfetto.

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The authors declare no competing financial interests.

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Perfetto, S., Ambrozak, D., Nguyen, R. et al. Quality assurance for polychromatic flow cytometry using a suite of calibration beads. Nat Protoc 7, 2067–2079 (2012). https://doi.org/10.1038/nprot.2012.126

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