The EV-TRACK platform

Why?

Extracellular vesicles (EVs) have emerged as having important (patho)physiological roles, and as such they have been the focus of intense study over the past decade. Despite substantial progress, the complexity and challenges associated with EV research remain considerable. EVs released from different cell types (and even from a single cell type) are heterogeneous in size and in protein, nucleic acid and lipid content. The separation of EV populations is frequently complicated by the presence of other extracellular macromolecular structures with comparable features. Different separation methods enrich for single or multiple EV subtypes with diverse composition and variable purity, thus identifying method-dependent EV content and function. Each detection and characterization method has its own accuracy and precision in measuring EVs.

In EV research, as in any field, the implementation of different methods requires validated controls and adequate reporting of experimental parameters. Failure to follow these principles can result in data that are difficult to interpret and reproduce. Similar to minimal information checklists in other fields, the International Society for Extracellular Vesicles (ISEV) introduced minimal informations for studies on EVs (MISEV) guidelines. Nevertheless, EV research is unfolding at a rate that impedes widespread adoption of these guidelines.

What?

We developed the EV-TRACK knowledgebase to record experimental parameters of EV-related studies with the aim to:

donecreate an informed dialog among researchers about relevant experimental parameters

doneimprove the reproducibility and interpretation of experiments studying EVs

donerecord the evolution of EV research

doneenable queries of published EV experiments

The final goal of the EV-TRACK consortium is to facilitate standardization of EV research through increased systematic reporting on EV biology and methodology.

How?

The EV-TRACK online toolset consists of seven enabling features to coach researchers through the use of the EV-METRIC, to centralize data on EV characteristics and methods, to query research articles and to involve researchers in decision-making on future improvements to EV-TRACK and its EV-METRIC.

The EV-METRIC is a key feature, designed to reflect the level of transparency in reporting of experimental parameters. It is presented as a percentage of fulfilled components from a list of nine, which were argued by the EV-TRACK consortium to be indispensable for unambiguous interpretation and independent reproduction of EV experiments. Researchers are encouraged to obtain this EV-METRIC prior to submitting their manuscript for peer review. After uploading requested experimental parameters, an EV-TRACK ID will be assigned and a preliminary EV-METRIC is calculated. The authors can implement this metric to improve the transparency of their experiments. When including the EV-TRACK ID in the material and methods section of a manuscript, journal editors and reviewers will also be able to access the corresponding EV-TRACK data entry, which provides them with a comprehensive overview of the presented data. Upon publication, the data submitted in EV-TRACK are curated by the EV-TRACK administrators, the final EV-METRIC is calculated, and the experiment(s) is(are) included and searchable in the public knowledgebase.

EV-TRACK also allows uploading of experimental parameters of already published experiments. A unique feature of EV-TRACK is the possibility to add unpublished experimental parameters of an already published EV experiment. For example, since the publication of EV-TRACK, researchers might realize that they forgot to include important experimental information. Since this information is generally available in the lab, it can be added post-publication to increase the reporting transparency.

Publications loaded in the EV-TRACK knowledgebase can be queried for specific experimental parameters that are not easily searchable in any current biomedical literature database. By centralizing this information, the EV-TRACK knowledgebase contributes to creating a better understanding of EV biology and methodology, which is needed to develop the next generation of experimental guidelines, if and when they are required.

The EV-METRIC
The EV-METRIC is currently composed of these nine experimental parameters.
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Experimental parameters related to EV characterization include:
Protein analysis
EV-enriched proteins: analysis of three or more EV-enriched proteins
non EV-enriched protein: assessment of at least one non-EV-enriched protein
antibody specifics: antibody clone/reference number and dilution. Antibody specifics can be considered ‘not applicable’ depending on the implemented characterization methods in a particular study
lysate preparation: lysis buffer composition provided, or reference number for commercial lysis buffers. Lysate preparation can be considered ‘not applicable’ depending on the implemented characterization methods in a particular study
Particle analysis
qualitative and quantitative analysis: implementation of both qualitative (example: EM, AFM) and quantitative methods (example: NTA, DLS, TRPS, high resolution flow cytometry). For the quantitative method, reporting of particle concentration is expected.
electron Microscopy images: inclusion of a wide-field and a close-up electron microscopy image
Experimental parameters related to EV separation include:
density gradient: density gradient performed, at least as validation if described results are attributed to EVs
EV density: reporting of obtained EV density
ultracentrifugation specifics: reporting of g-forces, duration and rotor type of ultracentrifugation steps. Ultracentrifugation specifics can be considered ‘not applicable’ depending on separation method implemented in a particular study

The EV-METRIC for each study in the EV-TRACK knowledgebase is calculated by expressing the proportion of reported parameters as a (rounded-up) percentage. It is introduced to raise awareness about common issues with reporting of EV separation and characterization. While a high EV-METRIC implies well-annotated published data, it should not be seen as a stand-alone ‘score’ that directly relates to the quality of any particular study.

Conclusion

EV-TRACK and the EV-METRIC are tools to enhance transparency and interpretation of EV experiments. An increase in reporting transparency will benefit overall reproducibility in EV studies. Most importantly, EV-TRACK is driven by both the input and consensus of researchers that perform the experiments.

Reference

If you make use of the EV-TRACK platform, please cite it as:
Van Deun J, et al. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nature methods. 2017;14(3):228-32.

SharedIt link to paper.

Version

Current EV-TRACK version: 19.48

EV-TRACK version history