A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders
Abstract
Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil-derived extracellular vesicles (NDEVs) involved in immune and thrombo-inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID-19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.
Related
- The Role of Neutrophil Extracellular Traps (NETosis) in Systemic Lupus Erythematosus: Molecular Mechanisms, Pathogenic Roles, Diagnostic Biomarkers, and Emerging Therapeutic Strategies
- Helicobacter pylori Infection Remodels Neutrophil‐Derived Extracellular Vesicles, Promoting Stress and Apoptosis in Infected Epithelial Cells
- Multi-Omic Integration of the miRNome and Transcriptome in Dermal Fibroblasts from Hypermobile Ehlers–Danlos Syndrome and Hypermobility Spectrum Disorders Identifies Convergent Candidate miRNA-mRNA Regulatory Networks Linking Extracellular Matrix Remodeling and Inflammation
- Platelet-related extracellular vesicles: Therapeutic roles in musculoskeletal disorders.
- A Multidimensional Engineering Strategy Reprograms Microglia via Targeted and Sustained‐Release Extracellular Vesicles for Spinal Cord Injury Repair
BibTeX
@article{bonifay2026strategy,
title = {{A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders}},
author = {Amandine Bonifay and Stéphane Robert and Belinda Champagne and Paul‐rémi Petit and Aude Eugène and Corinne Chareyre and Anne‐claire Duchez and Mélanie Vélier and Shirley Fritz and Loris Vallier and Romaric Lacroix and Françoise Dignat‐george},
journal = {Journal of Extracellular Vesicles},
year = {2026},
month = sep,
doi = {10.1002/jev2.12204},
url = {https://hal.science/hal-03685896v1},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders
AU - Amandine Bonifay
AU - Stéphane Robert
AU - Belinda Champagne
AU - Paul‐rémi Petit
AU - Aude Eugène
AU - Corinne Chareyre
AU - Anne‐claire Duchez
AU - Mélanie Vélier
AU - Shirley Fritz
AU - Loris Vallier
AU - Romaric Lacroix
AU - Françoise Dignat‐george
PY - 2026
DA - 2026/09/17
JO - Journal of Extracellular Vesicles
DO - 10.1002/jev2.12204
UR - https://hal.science/hal-03685896v1
AB - Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil-derived extracellular vesicles (NDEVs) involved in immune and thrombo-inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID-19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.
SN - 2001-3078
ER -