{"doi":"10.1002/cyto.a.24268","title":"MIFlowCyt‐EV: The Next Chapter in the Reporting and Reliability of Single Extracellular Vesicle Flow Cytometry Experiments","abstract":"Following in the footsteps of the minimum information to report about a flow cytometry experiment (MIFlowCyt), an extracellular vesicle (EV) flow cytometry working group composed of members from the international societies of extracellular vesicles (ISEV), advancement of cytometry (ISAC), and thrombosis and haemostasis (ISTH) developed the recent MIFlowCyt-EV framework position statement, with the aim to improve reporting and reliability of single EV flow cytometry experiments (1, 2). MIFlowCyt-EV was developed in response to the growing body of single EV flow cytometry literature that lacks rigor and standardization, thereby leading to irreproducible results. MIFlowCyt-EV builds upon pre-existing frameworks for reporting and best practices in both flow cytometry and EV research; MIFlowCyt and MISEV, respectively (2-4). A need for MIFlowCyt-EV exists because (1) single EV flow cytometry experiments face specific technical challenges, which may lead to artifacts that are not covered by MIFlowCyt, and (2) in contrast to Cytometry Part A, which requires manuscripts to be supplemented with a MIFlowCyt template, journals in the field of EVs lack a formal requirement tailored to EV flow cytometry. To emphasize the need of MIFlowCyt-EV, Table 1 shows differences between the technical challenges and opportunities involved in EV and cell detection. This is mainly because EVs have signal intensities just below and above the detection limit, signal intensities overlap with buffer contaminants and unstained reagents, swarm detection may occur, and sensitivity differences strongly affect measured EV concentrations. On the other hand, the morphological properties of EVs offer new flow cytometric opportunities, such as particle sizing and refractive index estimation (5-8). As the field has progressed, essential controls have been identified to ensure (1) the detection of single EVs, and (2) providing reassurance that the detected events are EVs and not artifacts, such as free fluorescent labels. In line with the controls, calibration methods have been identified that allow for data to be reported in comparable, standardized units with a quantifiable limit of detection, which allows validation and comparison of data on flow cytometers that differ in sensitivity. Despite the identification and acknowledged importance of essential controls and calibrations, these currently lack widespread adoption by the scientific community, as evidenced by the absence of utilization in the published literature. Hence, now it is time to disseminate the solutions to these EV specific technical challenges in the form of a field standard: MIFlowCyt-EV. The MIFlowCyt-EV reporting framework is composed of seven main components which include: (1) preanalytical variables and experimental design, (2) sample preparation, (3) assay controls, (4) instrument calibration and data acquisition, (5) EV characterization, (6) flow cytometry (FC) data reporting, and (7) FC data sharing. Together, these components ensure measurement of single EVs, data reproducibility, and the facilitation of data interpretation. Each component of the reporting framework aids in different areas of increasing reported data integrity. Components 1 and 2 provide the necessary details to repeat a reported assay. Component 3 provides evidence that single EVs of interest are being detected, and not labeling/purification artifacts or coincidence events. Components 4 and 5 allow for validation of assays irrespective of differences in instrument settings and/or limits of detection. Finally, Components 6 and 7 provide evidence of how the data was analyzed proof of raw data quality. Whereas the framework has a focus on EVs, it is also applicable to and useful for flow cytometry experiments on sub-micrometer particles other than EVs, such as viruses. First, the completion of the MIFlowCyt-EV components will aid the peer-review process by allowing reviewers and readers to have access to essential information ","journal":"Cytometry Part A","year":2020,"id":99560,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":30,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.7195,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":306821,"name":"Vera A. Tang","orcid":"0000-0003-3930-9139","position":1,"is_corresponding":false},{"id":226787,"name":"Edwin van der Pol","orcid":"0000-0002-9497-8426","position":2,"is_corresponding":false},{"id":226794,"name":"André Görgens","orcid":"0000-0001-9198-0857","position":3,"is_corresponding":false},{"id":215734,"name":"Joshua A Welsh","orcid":"0000-0002-1097-9756","position":0,"is_corresponding":true}],"reference_count":12,"raw_metadata":null,"created_at":"2026-07-18T22:38:09.754371Z","pmid":"33200505","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}