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Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry
Published on: March 17, 2015
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Preventing swarm detection in extracellular vesicle flow cytometry: a clinically applicable procedure
Naomi C Buntsma1,2,3,4,5, Mona Shahsavari1,2,4,6, Aleksandra Gąsecka1,2,4,7
1Amsterdam Vesicle Center and Laboratory of Experimental Clinical Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands.
Research and Practice in Thrombosis and Haemostasis
|June 7, 2023
Summary
To avoid swarm detection in extracellular vesicle flow cytometry, dilute plasma samples at least 110-fold and maintain a count rate below 11,000 events/s. This ensures accurate particle concentration measurements in clinical research.
Area of Science:
- Biomedical Engineering
- Analytical Chemistry
- Cell Biology
Background:
- Flow cytometry is crucial for detecting cell-derived extracellular vesicles (EVs) in biofluids like plasma.
- Swarm detection, an artifact of simultaneous particle illumination, leads to inaccurate EV concentration measurements.
- Current methods for preventing swarm detection, such as sample dilution, are impractical for routine clinical analysis due to varying sample concentrations.
Purpose of the Study:
- To develop a practical method for determining optimal plasma sample dilution for EV flow cytometry.
- To ensure accurate EV quantification in clinical research by mitigating swarm detection.
Main Methods:
- Measured dilution series of five plasma samples using flow cytometry (Apogee A60-Micro), with particle concentrations ranging from 2.5 × 10^9 to 2.1 × 10^11 mL^-1.
- Analyzed data to identify conditions preventing swarm detection while maintaining statistically significant particle counts.
Main Results:
- Swarm detection was eliminated at dilutions ≥1.1 × 10^3-fold or count rates <3.0 × 10^3 events·s^-1.
- However, these individual criteria often resulted in insufficient particle counts.
- Combining minimal dilution with a maximum count rate proved most effective for preventing swarm detection and maintaining significant particle counts.
Conclusions:
- A practical procedure using the measurement count rate of a single diluted plasma sample can determine the optimal dilution factor.
- For the studied conditions, an optimal dilution factor of ≥1.1 × 10^2-fold combined with a count rate <1.1 × 10^4 events·s^-1 prevents swarm detection.
