A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing.
Abstract
BACKGROUND: Understanding the pathogenic role of extracellular vesicles (EVs) in disease
and their potential diagnostic and therapeutic utility is extremely reliant on in-depth
quantification, measurement and identification of EV sub-populations. Quantification
of EVs has presented several challenges, predominantly due to the small size of vesicles
such as exosomes and the availability of various technologies to measure nanosized
particles, each technology having its own limitations. MATERIALS AND METHODS: A standardized
methodology to measure the concentration of extracellular vesicles (EVs) has been
developed and tested. The method is based on measuring the EV concentration as a function
of a defined size range. Blood plasma EVs are isolated and purified using size exclusion
columns (qEV) and consecutively measured with tunable resistive pulse sensing (TRPS).
Six independent research groups measured liposome and EV samples with the aim to evaluate
the developed methodology. Each group measured identical samples using up to 5 nanopores
with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate
data analysis with principal component analysis (PCA) were used to evaluate reproducibility
across the groups and to explore and visualise possible patterns and outliers in EV
and liposome data sets. RESULTS: PCA revealed good reproducibility within and between
laboratories, with few minor outlying samples. Measured mean liposome (not filtered
with qEV) and EV (filtered with qEV) concentrations had coefficients of variance of
23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements
could be attributed to the use of qEVs and the polydisperse nature of EVs. CONCLUSION:
The results of this study demonstrate the feasibility of this standardized methodology
to facilitate comparable and reproducible EV concentration measurements.
Type
Journal articleSubject
Coulter counterEV
colloids
concentration
exosomes
extracellular vesicles
microparticles
micropores
nanoparticles
nanopores
resistive pulse sensing
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Show full item recordScholars@Duke
Margarethe Joanna Kuehn
Associate Professor of Biochemistry
Enterotoxigenic E. coli (ETEC) causes traveler's diarrhea and infant mortality in
underdeveloped countries, and Pseudomonas aeruginosa is an opportunistic pathogen
for immunocompromised patients. Like all gram negative bacteria studied to date, ETEC
and P. aeruginosa produce small outer membrane vesicles that can serve as delivery
"bombs" to host tissues. Vesicles contain a subset of outer membrane and
soluble periplasmic proteins and lipids. In tissues and sera of infected hosts,

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