Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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).

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  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).

    Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  11. 11

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

    Article  Google Scholar 

  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).

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

Download references

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.

Corresponding author

Correspondence to Stephen P Perfetto.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing