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Cytometry A. 2018 Nov;93(11):1087-1091. doi: 10.1002/cyto.a.23605. Epub 2018 Sep 23.

Methodology for evaluating and comparing flow cytometers: A multisite study of 23 instruments.

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Shared FACS Facility and Department of Genetics, Stanford University, Stanford, California.
Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia.
Beckman Coulter Life Sciences, Miami, Florida.
Scintillon Institute, San Diego, California.
Consultant, Vancouver, British Columbia.
Vaccine Research Center, NIH, Bethesda, Maryland.
University of Nevada, Reno, Nevada.
FlowJo, LLC, Ashland, Oregon.
University of California, Davis, California.
National Institute of Standards and Technology, Gaithersburg, Maryland.
Wake Forest University Baptist Medical Center, Comprehensive Cancer Center and Department of Cancer Biology, Winston-Salem, North Carolina.


We demonstrate improved methods for making valid and accurate comparisons of fluorescence measurement capabilities among instruments tested at different sites and times. We designed a suite of measurements and automated data processing methods to obtain consistent objective results and applied them to a selection of 23 instruments at nine sites to provide a range of instruments as well as multiple instances of similar instruments. As far as we know, this study represents the most accurate methods and results so far demonstrated for this purpose. The first component of the study reporting improved methods for photoelectron scale (Spe) evaluations, which was published previously (Parks, El Khettabi, Chase, Hoffman, Perfetto, Spidlen, Wood, Moore, and Brinkman: Cytometry A 91 (2017) 232-249). Those results which were within themselves are not sufficient for instrument comparisons, so here, we use the Spe scale results for the 23 cytometers and combine them with additional information from the analysis suite to obtain the metrics actually needed for instrument evaluations and comparisons. We adopted what we call the 2+2SD limit of resolution as a maximally informative metric, for evaluating and comparing dye measurement sensitivity among different instruments and measurement channels. Our results demonstrate substantial differences among different classes of instruments in both dye response and detection sensitivity and some surprisingly large differences among similar instruments, even among instruments with nominally identical configurations. On some instruments, we detected defective measurement channels needing service. The system can be applied in shared resource laboratories and other facilities as an aspect of quality assurance, and accurate instrument comparisons can be valuable for selecting instruments for particular purposes and for making informed instrument acquisition decisions. An institutionally supported program could serve the cytometry community by facilitating access to materials, and analysis and maintaining an archive of results.


LED; automated data analysis; instrumentation; limit of detection; microspheres; photoelectron scale; resolution limit; sensitivity; standardization; flow cytometry

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