Abstract
The research field of extracellular vesicles (EVs) is increasing immensely and the potential uses of EVs seem endless. They are found in large numbers in various body fluids, and blood samples may well serve as liquid biopsies. However, these small membrane-derived entities of cellular origin are not straightforward to work with in regard to isolation and characterization.A broad range of relevant preanalytical issues was tested, with a focus on the phenotypic impact of smaller EVs. The influences of the i) blood collection tube used, ii) incubation time before the initial centrifugation, iii) transportation/physical stress, iv) storage temperature and time (short term and long term), v) choice of centrifugation protocol, vi) freeze-thaw cycles, and vii) exosome isolation procedure (ExoQuick™) were examined. To identify the impact of the preanalytical treatments, the relative amounts (detected signal intensities of CD9-, CD63- and/or CD81-positive) and phenotypes of small EVs were analyzed using the multiplexed antibody-based microarray technology, termed the EV Array. The analysis encompassed 15 surface- or surface-related markers, including CD9, CD63, CD81, CD142, and Annexin V.This study revealed that samples collected in different blood collection tubes suffered to varying degrees from the preanalytical treatments tested here. There is no unequivocal answer to the questions asked. However, in general, the period of time and prospective transportation before the initial centrifugation, choice of centrifugation protocol, and storage temperature were observed to have major impacts on the samples. On the contrary, long-term storage and freeze-thawing seemed to not have a critical influence. Hence, there are pros and cons of any choice regarding sample collection and preparation and may very well be analysis dependent. However, to compare samples and results, it is important to ensure that all samples are of the same type and have been handled similarly.
Highlights
In recent years, the interest in extracellular vesicles (EVs) has increased immensely (Lötvall et al, 2014) and several studies have shown the potential of utilizing them in a clinical setting, as diagnostic, prognostic and as therapeutic agents, as reviewed by Revenfeld et al (Revenfeld et al, 2014) and György et al (György et al, 2015)
Highly sensitive and high-throughput antibody-based platform of the EV Array, we have investigated a broad range of preanalytical factors that could be of importance when analyzing sEVs
We have only focused on sEVs positive for CD9, CD63, and/or CD81, as these molecules are used for detection with the EV Array in the current setup
Summary
The interest in extracellular vesicles (EVs) has increased immensely (Lötvall et al, 2014) and several studies have shown the potential of utilizing them in a clinical setting, as diagnostic, prognostic and as therapeutic agents, as reviewed by Revenfeld et al (Revenfeld et al, 2014) and György et al (György et al, 2015). EVs are small membrane-derived entities produced from a diverse range of cell types throughout the human body and, they are accessible in various body fluids (Caby et al, 2005; Admyre et al, 2007; Ogawa et al, 2008; Gonzales et al, 2009). These vesicles can be divided into several subgroups according to specific characteristics such as cellular origin,. When sorting using the latter characteristic, the major subgroups are exosomes (30– 100 nm in diameter), microvesicles (MVs, 100–1000 nm) and apoptotic bodies (500–4000 nm) (Pugholm et al, 2015), MVs are often denoted microparticles (MPs) (Colombo et al, 2014). Each of these subgroups possess numerous biological functions, and it is of interest to determine them thoroughly to fully understand and utilize the vesicular biology (Mulcahy et al, 2014)
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