Minimum information to report about a flow cytometry experiment on extracellular vesicles: communication from the ISTH SSC Subcommittee on Vascular Biology

Flow cytometry remains a commonly used methodology due to its ability to characterise multiple parameters on single particles in a high-throughput manner. In order to address limitations with lacking sensitivity of conventional flow cytometry to characterise extracellular vesicles (EVs), novel, highly sensitive platforms, such as high-resolution and imaging flow cytometers, have been developed. We provided comparative benchmarks of a conventional FACS Aria III, a high-resolution Apogee A60 Micro-PLUS and the ImageStream X Mk II imaging flow cytometry platform. Nanospheres were used to systematically characterise the abilities of each platform to detect and quantify populations with different sizes, refractive indices and fluorescence properties, and the repeatability in concentration determinations was reported for each population. We evaluated the ability of the three platforms to detect different EV phenotypes in blood plasma and the intra-day, inter-day and global variabilities in determining EV concentrations. By applying this or similar methodology to characterise methods, researchers would be able to make informed decisions on choice of platforms and thereby be able to match suitable flow cytometry platforms with projects based on the needs of each individual project. This would greatly contribute to improving the robustness and reproducibility of EV studies.

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Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results

Diagnostics ◽

10.3390/diagnostics11081320 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1320

Author(s):

Kristýna Pekárková ◽

Jakub Soukup ◽

Marie Kostelanská ◽

Jan Širc ◽

Zbyněk Straňák ◽

...

Keyword(s):

Flow Cytometry ◽

Cord Blood ◽

Extracellular Vesicles ◽

Correlation Coefficients ◽

Flow Cytometer ◽

Liquid Biopsies ◽

Physiological Conditions ◽

Flow Cytometric ◽

Term Births ◽

And Control

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods—one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

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Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry

PLoS ONE ◽

10.1371/journal.pone.0144678 ◽

2016 ◽

Vol 11 (1) ◽

pp. e0144678 ◽

Cited By ~ 33

Author(s):

Kirsty M. Danielson ◽

Jessica Estanislau ◽

John Tigges ◽

Vasilis Toxavidis ◽

Virginia Camacho ◽

...

Keyword(s):

Flow Cytometry ◽

Extracellular Vesicles ◽

Diurnal Variations

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Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

Scientific Reports ◽

10.1038/s41598-017-18227-8 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 14

Author(s):

Vera A. Tang ◽

Tyler M. Renner ◽

Anna K. Fritzsche ◽

Dylan Burger ◽

Marc-André Langlois

Keyword(s):

Flow Cytometry ◽

Extracellular Vesicles ◽

Single Particle ◽

Particle Discrimination

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Tetraspanin immunocapture phenotypes extracellular vesicles according to biofluid source but may limit identification of multiplexed cancer biomarkers

10.1101/2021.03.02.433595 ◽

2021 ◽

Author(s):

Rachel R. Mizenko ◽

Terza Brostoff ◽

Tatu Rojalin ◽

Hanna J. Koster ◽

Hila S. Swindell ◽

...

Keyword(s):

Flow Cytometry ◽

Extracellular Vesicles ◽

Fluorescence Detection ◽

Single Particle ◽

Cancer Biomarkers ◽

Surface Markers ◽

Isolation Method ◽

Standard Methods

AbstractTetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their general detection and classification from background contaminants. This common practice typically assumes a consistent expression of tetraspanins across EV sources, thus obscuring subpopulations of variable or limited tetraspanin expression. While some recent studies indicate differential expression of tetraspanins across bulk isolated EVs, here we present analysis of single EVs isolated using various field-standard methods from a variety of in vitro and in vivo sources to identify distinct patterns in colocalization of tetraspanin expression. We report an optimized method for the use of antibodycapture single particle interferometric reflectance imaging sensing (SP-IRIS) and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. Using SP-IRIS and immunofluorescence, we report that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles as measured by flow cytometry do not share similar trends, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers. Our findings may reveal key insights into the complexities of the EV biogenesis and signaling pathways and better inform EV capture and detection platforms for diagnostic or other downstream use.

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