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

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⁺/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.

SUBMITTER: Kim J

PROVIDER: S-EPMC11322860 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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

Kim James J Xu Shihan S Jung Seung-Ryoung SR Nguyen Alya A Cheng Yuanhua Y Zhao Mengxia M Fujimoto Bryant S BS Nelson Wyatt W Schiro Perry P Franklin Jeffrey L JL Higginbotham James N JN Coffey Robert J RJ Shi Min M Vojtech Lucia N LN Hladik Florian F Tewari Muneesh M Tigges John J Ghiran Ionita I Jovanovic-Talisman Tijana T Laurent Louise C LC Das Saumya S Gololobova Olesia O Witwer Kenneth W KW Xu Tuoye T Charest Al A Jensen Kendall Van Keuren KVK Raffai Robert L RL Jones Jennifer C JC Welsh Joshua A JA Nolan John P JP Chiu Daniel T DT

Journal of extracellular vesicles 20240801 8

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 d ...[more]

PMID: 39140467

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Project description:Extracellular vesicles (EVs) are commonly studied by flow cytometry. Due to their small size and low refractive index, the scatter intensity of most EVs is below the detection limit of common flow cytometers. Here, we aim to improve forward scatter (FSC) and side scatter (SSC) sensitivity of a common flow cytometer to detect single 100 nm EVs. The effects of the optical and fluidics configuration on scatter sensitivity of a FACSCanto (Becton Dickinson) were evaluated by the separation index (SI) and robust coefficient of variation (rCV) of polystyrene beads (BioCytex). Improvement is defined as increased SI and/or reduced rCV. Changing the obscuration bar improved the rCV 1.9-fold for FSC. A 10-fold increase in laser power improved the SI 19-fold for FSC and 4.4-fold for SSC, whereas the rCV worsened 0.8-fold and improved 1.5-fold, respectively. Confocalization worsened the SI 1.2-fold for FSC, and improved the SI 5.1-fold for SSC, while the rCV improved 1.1-fold and worsened 1.5-fold, respectively. Replacing the FSC photodiode with a photomultiplier tube improved the SI 66-fold and rCV 4.2-fold. A 2-fold reduction in sample stream width improved both SI and rCV for FSC by 1.8-fold, and for SSC by 1.3- and 2.2-fold, respectively. Decreasing the sample flow velocity worsened rCVs. Decreasing the flow channel dimensions and the pore size of the sheath filter did not substantially change the SI or rCV. Using the optimal optical configuration and fluidics settings, the SI improved 3.8∙104 -fold on FSC and 30-fold on SSC, resulting in estimated detection limits for EVs (assuming a refractive index of 1.40) of 246 and 91 nm on FSC and SSC, respectively. Although a 50-fold improvement on FSC is still necessary, these adaptions have produced an operator-friendly, high-throughput flow cytometer with a high sensitivity on both SSC and FSC. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Project description:The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages.

Dataset Information

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

Publications

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

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