The Dilution Paradox in Extracellular Vesicle Flow Cytometry

Area of Science:

• Biomedical engineering
• Analytical chemistry
• Cell biology

Background:

• Extracellular vesicles (EVs) are crucial biomarkers.
• Flow cytometry is a common method for EV detection.
• Accurate EV quantification is essential for clinical applications.

Purpose of the Study:

• To investigate the impact of sample dilution on EV detection by flow cytometry.
• To identify challenges in reliable EV measurement using flow cytometry.
• To evaluate the reliability of previous clinical EV studies.

Main Methods:

• Measurement of EVs in human plasma with varying lipoprotein concentrations.
• Analysis of different sample dilutions using scatter-triggering and fluorescence-triggering.
• Evaluation of two published clinical EV studies using an analytical model.

Main Results:

• A 'dilution paradox' was identified where optimal dilution for light scattering conflicts with fluorescence detection.
• This paradox can lead to unreliable detection of both EV signals.
• 30% and 65% of measurements in two clinical studies were found to be unreliable.

Conclusions:

• The 'dilution paradox' challenges the reliability of current flow cytometry-based EV detection methods.
• Previous clinical study results may require re-evaluation.
• Strategies like fixed dilution with fluorescence-triggering and sample/reagent purification can potentially overcome this paradox.