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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Author information

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.

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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