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Updated: May 25, 2026
Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry
Published on: March 17, 2015
High-sensitivity flow cytometry provides access to standardized measurement of small-size microparticles--brief
Stéphane Robert1, Romaric Lacroix, Philippe Poncelet
1UMR Institut National de la Santé et de la Recherche Médicale/Aix-Marseille Université, Faculté de Pharmacie, France.
Objective:
Cellular microparticles (MP) are promising biomarkers in many pathological situations. Although flow cytometry (FCM) is widely used for their measurement, it has raised controversies because the smallest MP size falls below the detection limit of standard FCM (sd-FCM). Following recent technological improvements leading to high sensitivity FCM (hs-FCM), our objectives were (1) to evaluate the potential of hs-FCM for extended MP detection, (2) to set up a standardized protocol for MP enumeration, and (3) to compare MP counts obtained with both sensitivity levels.
Methods And Results:
Compared with sd-FCM, hs-FCM displayed improved forward scatter resolution and lower background noise, allowing us to discriminate previously undetectable small MP in plasma samples. Using fluorescent beads with appropriate sizes (0.1/0.3/0.5/0.9 μm) and relative amounts, a new standardized hs-FCM MP protocol was set up and provided reproducible MP counts. Applied to coronary patient samples, it resulted into 8- to 20-fold increases in MP counts as compared with sd-FCM. Interestingly, the ratio between small and large MP varied according to clinical status but also depending on MP subset, suggesting access to new biological information.
Conclusions:
Recent improvements in FCM provide access to previously undetectable MP and represent a new opportunity to enhance their impact as biomarkers in clinical practice.
Insights
High-sensitivity flow cytometry (hs-FCM) significantly enhances the detection of microparticles (MPs), revealing previously undetectable populations. This advancement offers new opportunities for utilizing MPs as crucial biomarkers in clinical diagnostics.
Area of Science:
- Biomedical engineering
- Analytical chemistry
- Clinical diagnostics
Background:
- Cellular microparticles (MPs) are valuable biomarkers, but their detection is limited by standard flow cytometry (FCM) technology.
- The smallest MPs are often below the detection threshold of standard FCM (sd-FCM), leading to incomplete analysis.
- Recent advancements in FCM technology have led to high-sensitivity FCM (hs-FCM), improving detection capabilities.
Purpose of the Study:
- To assess the potential of hs-FCM for detecting a wider range of MPs.
- To establish a standardized protocol for enumerating MPs using hs-FCM.
- To compare MP counts obtained via hs-FCM with those from sd-FCM.
Main Methods:
- Utilized fluorescent beads (0.1/0.3/0.5/0.9 μm) to calibrate hs-FCM.
- Developed and validated a standardized hs-FCM protocol for MP enumeration.
- Applied hs-FCM to plasma samples from coronary patients.
Main Results:
- hs-FCM demonstrated superior forward scatter resolution and reduced background noise compared to sd-FCM.
- hs-FCM enabled the detection of small MPs previously undetectable by sd-FCM.
- MP counts in coronary patient samples were 8- to 20-fold higher with hs-FCM than with sd-FCM.
- The ratio of small to large MPs varied with clinical status and MP subset, indicating new biological insights.
Conclusions:
- Improvements in FCM technology, specifically hs-FCM, allow for the detection of previously inaccessible MPs.
- This enhanced detection capability presents a significant opportunity to improve the clinical utility of MPs as biomarkers.
- hs-FCM offers a more comprehensive approach to MP analysis, potentially leading to improved diagnostic and prognostic applications.
