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



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.


  1. 1

    Bendall, S. et al. A deep profiler's guide to cytometry. Trends Immunol. 33, 323–332 (2012).

  2. 2

    Chattopadhyay, P.K. et al. Cytometry: today's technology and tomorrow's horizons. Methods 57, 251–258 (2012).

  3. 3

    Chattopadhyay, P.K. et al. Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat. Med. 12, 972–977 (2006).

  4. 4

    Le Meur, N. et al. Data quality assessment of ungated flow cytometry data in high throughput experiments. Cytometry A 71, 393–403 (2007).

  5. 5

    Harvath, L. Quality control in clinical flow cytometry. Pathol. Immunopathol. Res. 7, 338–344 (1988).

  6. 6

    Horan, P.K., Muirhead, K.A. & Slezak, S.E. Standards and controls in flow cytometry. in Flow Cytometry and Sorting, 2nd edn. (eds. Melamed, M.R., Lindmo, T. & Mendelsohn M.L.) 397–414 (Wiley-Liss, 1990).

  7. 7

    Shapiro, H.M. Practical Flow Cytometry (Wiley-Liss, 2003).

  8. 8

    Perfetto, S.P., Ambrozak, D., Nguyen, R., Chattopadhyay, P. & Roederer, M. Quality assurance for polychromatic flow cytometry. Nat. Protoc. 1, 1522–1530 (2006).

  9. 9

    Schwartz, A., Marti, G.E., Poon, R., Gratama, J.W. & Fernandez-Repollet, E. Standardizing flow cytometry: a classification system of fluorescence standards used for flow cytometry. Cytometry 33, 106–114 (1998).

  10. 10

    Schwartz, A., Fernandez Repollet, E., Vogt, R. & Gratama, J.W. Standardizing flow cytometry: construction of a standardized fluorescence calibration plot using matching spectral calibrators. Cytometry 26, 22–31 (1996).

  11. 11

    Schwartz, A. et al. Formalization of the MESF unit of fluorescence intensity. Cytometry B Clin. Cytom. 57, 1–6 (2004).

  12. 12

    Zenger, V.E., Vogt, R., Mandy, F., Schwartz, A. & Marti, G.E. Quantitative flow cytometry: inter-laboratory variation. Cytometry 33, 138–145 (1998).

  13. 13

    Chase, E.S. & Hoffman, R.A. Resolution of dimly fluorescent particles: a practical measure of fluorescence sensitivity. Cytometry 33, 267–279 (1998).

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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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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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