Intermethod Characterization of Commercially Available Extracellular Vesicles as Reference Materials

Area of Science:

• Biotechnology
• Nanotechnology
• Analytical Chemistry

Background:

• Extracellular vesicles (EVs) are critical biomarkers, but their characterization lacks standardization.
• The National Institute of Standards and Technology (NIST) is developing reference materials (RMs) for EVs.
• Orthogonal analytical methods are needed to accurately measure EV properties like size and concentration.

Purpose of the Study:

• To evaluate and compare particle size distributions (PSDs) and particle number concentrations (PNCs) of EVs from different cell sources using orthogonal techniques.
• To assess the proteomic and microRNA (miRNA) content of EVs.
• To identify variability across measurement platforms and support the development of standardized EV RMs.

Main Methods:

• Orthogonal techniques including cryogenic electron microscopy (Cryo-EM), particle tracking analysis (PTA), asymmetrical flow field-flow fractionation (AF⁴), and microfluidic resistive pulse sensing (MRPS) were employed.
• Proteomic profiling was performed using mass spectrometry (MS).
• MicroRNA (miRNA) content was analyzed using small RNA sequencing (RNA-seq).

Main Results:

• All tested methods showed discrepancies in PSDs and PNCs compared to Cryo-EM, with MRPS being closest and AF⁴ most divergent.
• Quantitative differences in PNCs reached up to two orders of magnitude across methods and cell sources.
• Mass spectrometry identified key EV proteins, while RNA-seq demonstrated significant inter-laboratory variation.

Conclusions:

• Significant variability exists across different analytical platforms for EV characterization.
• The findings underscore the critical need for reproducible methods and standardized reference materials for EVs.
• NIST's ongoing efforts are vital for advancing reliable EV measurements in research and clinical applications.