Analysis of Neat Biofluids Obtained During Cardiac Surgery Using Nanoparticle Tracking Analysis: Methodological Considerations.

Andrew I U Shearn,Pauline Carnell-Morris,Chantal Boulanger,Sezin Aday,Aled Clayton,Gianni D Angelini,Soumaya Ben-Aicha,Prakash Punjabi,Agnieszka Siupa,Giovanni Biglino,Costanza Emanueli

doi:10.3389/fcell.2020.00367

Abstract

Small extracellular vesicles (sEVs) are those nanovesicles 30–150 nm in size with a role in cell signalling and potential as biomarkers of disease. Nanoparticle tracking analysis (NTA) techniques are commonly used to measure sEV concentration in biofluids. However, this quantification technique can be susceptible to sample handing and machine settings. Moreover, some classes of lipoproteins are of similar sizes and could therefore confound sEV quantification, particularly in blood-derived preparations, such serum and plasma. Here we have provided methodological information on NTA measurements and systematically investigated potential factors that could interfere with the reliability and repeatability of results obtained when looking at neat biofluids (i.e., human serum and pericardial fluid) obtained from patients undergoing cardiac surgery and from healthy controls. Data suggest that variables that can affect vesicle quantification include the level of contamination from lipoproteins, number of sample freeze/thaw cycles, sample filtration, using saline-based diluents, video length and keeping the number of particles per frame within defined limits. Those parameters that are of less concern include focus, the “Maximum Jump” setting and the number of videos recorded. However, if these settings are clearly inappropriate the results obtained will be spurious. Similarly, good experimental practice suggests that multiple videos should be recorded. In conclusion, NTA is a perfectible, but still commonly used system for sEVs analyses. Provided users handle their samples with a highly robust and consistent protocol, and accurately report these aspects, they can obtain data that could potentially translate into new clinical biomarkers for diagnosis and monitoring of cardiovascular disease.

Highlights

Small extracellular vesicles are generally in the region of 30–150 nm in size and are released from all cells
For example, that dramatic changes in plasmatic sEV-size particle concentrations could be detected in patients undergoing coronary artery bypass graft surgery (CABG) under cardiopulmonary bypass (CPB)
Our study has investigated the way in which several different settings or preparation methods immediately prior to sample analysis have an effect on the particle concentration shown by Nanoparticle tracking analysis (NTA) using a NanoSight NS300 apparatus, seeking to compare the effect of the settings or preparations tested on the same sample

Summary

Introduction

Small extracellular vesicles (sEVs) are generally in the region of 30–150 nm in size and are released from all cells They are a heterogeneous group of nanoparticles of different cargo content and function, including exosomes (Giulietti et al, 2018). They have become of great interest due to their emerging role in cell-to-cell communication (Théry et al, 2002; Emanueli et al, 2015), through their ability to transfer their active molecular cargoes to recipient cells. Operator and software version were reported as other factors affecting the accuracy of NTA measurements (Vestad et al, 2017; Bachurski et al, 2019)

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Conclusion