Quality assurance for polychromatic flow cytometry


This protocol outlines a three-part quality assurance program to optimize, calibrate and monitor flow cytometers used to measure cells labeled with five or more fluorochromes (a practice known as polychromatic flow cytometry). The initial steps of this program (system optimization) ensure that the instrument's lasers, mirrors and filters are optimally configured for the generation and transmission of multiple fluorescent signals. To determine the sensitivity and dynamic range of each fluorescence detector, the system is then calibrated by measuring fluorescence over a range of photomultiplier tube (PMT) voltages by determining the PMT voltage range and linearity (Steps 2–10) and validating the PMT voltage (Steps 11–17). Finally, to ensure consistent performance, we provide procedures to monitor the precision, accuracy and sensitivity of fluorescence measurements over time. All three aspects of this program should be performed upon installation, or whenever changes occur along the flow cytometer's optical path. However, only a few of these procedures need to be carried out on a routine basis.

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Figure 1: Quality assurance particles.
Figure 2: Voltage series applied to eight-peak Rainbow beads and unlabeled CompBeads.
Figure 3: Determining the linear PMT voltage range using a plot of CV, PMT linearity and ratio.
Figure 4: Sample graphic illustrating CV, PMT linearity and signal-to-background ratio.
Figure 5: Compensation profiles of a selected group of detectors.
Figure 6: Time plots of accuracy, precision and sensitivity.


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We would like to thank D. Parks (Stanford University School of Medicine) for his expert advice and knowledge and J. Trotter (BD Biosciences) for his conceptual work in quality controls and technical support.

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Correspondence to Stephen P Perfetto.

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Perfetto, S., Ambrozak, D., Nguyen, R. et al. Quality assurance for polychromatic flow cytometry. Nat Protoc 1, 1522–1530 (2006). https://doi.org/10.1038/nprot.2006.250

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