Comparison of EV characterization by commercial high‐sensitivity flow cytometers and a custom single‐molecule flow cytometer

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<sup>+</sup>/PE<sup>+</sup> 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.

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Publication Details

Journal
Journal of Extracellular Vesicles
Published
2024-08-01
DOI
https://doi.org/10.1002/jev2.12498
Citations
37
Primary Topic
Extracellular vesicles in disease
Type
article
Field-Weighted Citation Impact
8.65
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article

Comparison of EV characterization by commercial high‐sensitivity flow cytometers and a custom single‐molecule flow cytometer

Олеся Гололобова, James N. Higginbotham, Ionita Ghiran, John P. Nolan et al.
37 citations
Journal of Extracellular Vesicles
Extracellular vesicles in disease
8.65
article

Comparison of EV characterization by commercial high‐sensitivity flow cytometers and a custom single‐molecule flow cytometer

Олеся Гололобова, James N. Higginbotham, Ionita Ghiran, John P. Nolan, Lucia Vojtech, James Kim, Min Shi, Saumya Das, Jeffrey L. Franklin, Yuanhua Cheng, John Tigges, Bryant S. Fujimoto, Daniel T. Chiu, Tuoye Xu, Joshua A Welsh, Robert J. Coffey, Tijana Jovanović‐Talisman, Seung‐Ryoung Jung, Louise C. Laurent, Perry G. Schiro, Florian Hladik, Kenneth Whitaker Witwer, Muneesh Tewari, Jennifer Jones, Robert L. Raffaı̈, Shihan Xu, Mengxia Zhao, Kendall Jensen, Al Charest, Wyatt C. Nelson, A. N. Nguyen
article en
37 citations

Abstract

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<sup>+</sup>/PE<sup>+</sup> 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.

Journal of Extracellular VesiclesVol. 13(8)
Translational Genomics Research Institute (US), National Institutes of Health (US), San Francisco VA Medical Center (US), Beth Israel Deaconess Medical Center (US), Harvard University (US), Johns Hopkins University (US), Cancer Research Institute (US), Vanderbilt University (US), University of Washington (US), University of Michigan (US), Johns Hopkins Medicine (US), University of California San Diego (US), Massachusetts General Hospital (US), Fred Hutch Cancer Center (US), Sanford Consortium for Regenerative Medicine (US), VA Ann Arbor Healthcare System (US), City of Hope (US), Michigan Center for Translational Pathology (US), National Cancer Institute (US), Center for Cancer Research (US), Scintillon Institute (US), Beckman Research Institute, Vanderbilt University Medical Center (US)
Openalex Percentile: Top 1%
Extracellular vesicles in disease
8.65
DOIOpen Access PDFOpenAlex
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