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

ABSTRACT: 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. © 2018 International Society for Advancement of Cytometry.

SUBMITTER: Parks DR

PROVIDER: S-EPMC7901711 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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

Parks David R DR Moore Wayne A WA Brinkman Ryan R RR Chen Yong Y Condello Danilo D El Khettabi Faysal F Nolan John P JP Perfetto Stephen P SP Redelman Doug D Spidlen Josef J Van Dyke Jonathan J Wang Lili L Wood James C S JCS

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20180923 11

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 method ...[more]

PMID: 30244531

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Project description:High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.

Dataset Information

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

Publications

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

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