Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry

Area of Science:

• Biochemistry
• Cell Biology
• Biotechnology

Background:

• Extracellular vesicles (EVs) in plasma are crucial in physiological and pathological processes, but their study is hindered by isolation and characterization challenges.
• Conventional flow cytometry (FCM) using light scattering is limited in detecting sub-micrometer EVs.

Purpose of the Study:

• To apply a fluorescence triggering FCM approach for enumerating and phenotyping EVs in platelet-free plasma (PFP).
• To investigate the origins (platelet, erythrocyte) and phosphatidylserine (PS) exposure of EVs.
• To assess the impact of freeze-thawing on EV detection.

Main Methods:

• Utilized a fluorescence triggering FCM method to detect Annexin-A5 binding EVs (Anx5+ EVs), CD41+ EVs, and CD235a+ EVs in PFP.
• Compared fluorescence triggering with conventional light scattering triggering.
• Analyzed fresh and freeze-thawed PFP samples.

Main Results:

• Fluorescence triggering detected substantially higher concentrations of Anx5+ EVs (40×), CD41+ EVs (75×), and CD235a+ EVs (15×) compared to light scattering.
• Approximately 30% of Anx5+ EVs were platelet-derived, and 3% were erythrocyte-derived.
• A majority of platelet and erythrocyte EVs did not expose PS.
• Freeze-thawing increased Anx5+ EVs by ~35% without affecting other EV phenotypes.

Conclusions:

• The fluorescence triggering FCM method is a simple, sensitive, and reliable approach for EV detection and phenotyping.
• This method offers improved insights into EV formation mechanisms and functions.
• It holds potential for the development of novel EV-based biomarkers for disease diagnosis and monitoring.