Impact of preanalytical conditions on plasma concentration and size distribution of extracellular vesicles using Nanoparticle Tracking Analysis

Simin Jamaly,Nadezhda Latysheva,Sigrid K Brækkan,Cathrine Ramberg,Randi Olsen,Paul Webster,Timofey Sovershaev,John-Bjarne Hansen

doi:10.1038/s41598-018-35401-8

Abstract

Optimal pre-analytical handling is essential for valid measurements of plasma concentration and size distribution of extracellular vesicles (EVs). We investigated the impact of plasma preparation, various anticoagulants (Citrate, EDTA, CTAD, Heparin), and fasting status on concentration and size distribution of EVs measured by Nanoparticle Tracking Analysis (NTA). Blood was drawn from 10 healthy volunteers to investigate the impact of plasma preparation and anticoagulants, and from 40 individuals from a population-based study to investigate the impact of postprandial lipidemia. Plasma concentration of EVs was measured by NTA after isolation by high-speed centrifugation, and size distribution of EVs was determined using NTA and scanning electron microscopy (SEM). Plasma concentrations and size distributions of EVs were essentially similar for the various anticoagulants. Transmission electron microscopy (TEM) confirmed the presence of EVs. TEM and SEM-analyses showed that the EVs retained spherical morphology after high-speed centrifugation. Plasma EVs were not changed in postprandial lipidemia, but the mean sizes of VLDL particles were increased and interfered with EV measurements (explained 66% of the variation in EVs-concentration in the postprandial phase). Optimization of procedures for separating VLDL particles and EVs is therefore needed before NTA-assessment of EVs can be used as biomarkers of disease.

Highlights

Extracellular vesicles (EVs), including exosomes (30–100 nm in diameter) and microvesicles (100–1000 nm in diameter), are bilayer membrane vesicles released from various cells into their surroundings[1]
The impact of freezing platelet poor plasma (PPP) compared to platelet free plasma (PFP) on concentration and size distribution of plasma-derived extracellular vesicles (EVs) is shown in Fig. 1, panel A–D
There were no statistical differences in total concentration and size distribution of EVs between plasmas prepared as PPP and PFP before freezing in any of the anticoagulants used (Fig. 1)

Summary

Introduction

Extracellular vesicles (EVs), including exosomes (30–100 nm in diameter) and microvesicles (100–1000 nm in diameter), are bilayer membrane vesicles released from various cells into their surroundings[1]. High-speed centrifugation to achieve platelet free plasma (PFP) from PPP prior to freezing has been shown to lower the concentration of platelet-derived EVs compared to plasma subjected only to lower speed centrifugation (PPP) before freezing[23] It is not known whether high-speed centrifugation after thawing of PPP would affect concentration and size distribution of plasma derived EVs compared to PFP prepared by double centrifugation (including one low and one high-speed centrifugation step) before freezing. Pre-analytical conditions such as centrifugation steps, choice of anticoagulant, and fasting status may impact the plasma concentration and size distribution of EVs determined by NTA. We aimed to investigate the impact of plasma preparation, assessed by freezing plasma before (PPP) or after (PFP) a second high-speed centrifugation, various anticoagulants in commercial blood collection tubes (Citrate, EDTA, CTAD, and Heparin), and fasting status on plasma concentration and size distribution of EVs using NTA and SEM

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