MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments

Abstract

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.

Keywords: Extracellular vesicles; flow cytometry; framework; reporting; standardization.

Figure 1.

Overview of the MIFlowCyt EV Reporting Framework. The left column shows each category of the reporting framework and the middle column shows the components within each category, the right-hand column shows the broad objective of each row. *Highlights the component that are broadly applicable to the majority of single-EV analysis experiments regardless of design or instrumentation. **Highlights the components that are only applicable in cases where certain reagents or protocols are used.

Figure 2.

Summary of a poll about what extracellular vesicles (EV) flow cytometry (FCM) working group (WG) members expect to be reported in scientific manuscripts on EV-FC. The top chart shows the number of working group members who have experience reviewing manuscripts from the ISEV, ISAC, ISTH journals (red) and those that have not (white). The bottom bar graph summarizes the personal expectations of all co-authors regarding components to be reported in EV-FC manuscript published in ISEV, ISAC and ISTH journals. The expectations fall into categories of all (blue), most (green) or select/specialized (yellow) manuscripts.

Figure 3.

(a) Example plot of reporting serial dilutions data, with event count per second on the left y-axis, median PE MESF intensity of the recorded data on the right y-axis and the dilution factor on the x-axis. (b) Example plot of reporting fluorescence calibration using regression. The fluorescent intensity in arbitrary units (channel number) is on the x-axis with the related fluorescence population reference units on the y-axis. (c) Example plot of light scatter calibration at 405 nm using FCM_PASS software default values. (d) Example plot of refractive index and diameter determination using Flow-SR. methodology.