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J Extracell Vesicles. 2016 Sep 27;5:31242. doi: 10.3402/jev.v5.31242. eCollection 2016.

A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing.

Author information

1
School of Mathematics and Physics, The University of Queensland, St Lucia, QLD, Australia.
2
Izon Science Ltd., Burnside, Christchurch, New Zealand.
3
Laboratory of Experimental Clinical Chemistry, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
4
Department of Clinical Biochemistry and Clinical Medicine, Aalborg University Hospital, Aalborg, Denmark.
5
Department of Biochemistry, Duke University, Medical Centre, Durham, NC, USA.
6
Department of Neurosurgery and Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
7
Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary.
8
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
9
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia.
10
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, Australia.
11
Department of Clinical Biochemistry and Clinical Medicine, Aalborg University Hospital, Aalborg, Denmark; shp@rn.dk.

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.

KEYWORDS:

Coulter counter; EV; colloids; concentration; exosomes; extracellular vesicles; microparticles; micropores; nanoparticles; nanopores; resistive pulse sensing

Conflict of interest statement

and funding RV and MFB are contractors at Izon Science and their contributions to this paper were made as part of their contracts. AFH is funded by grants from the Australian National Health and Medical Research Council (grants 628946 and 400202; www.nhmrc.gov.au) and an Australian Research Council (www.arc.gov.au) Future Fellowship (grant FT100100560).

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