The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray

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.

Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives

“…Anticoagulants seem to be an important pre‐analytical step since they limit platelet activation during blood collection and therefore the release of EVs. In the literature, several anticoagulants have been compared for EVs analysis (e.g., EDTA and heparin) . However, sodium citrate is considered the most commonly used for EVs analysis and has been recommended by ISTH …”

“…Samples must be aliquoted appropriately since freeze–thaw cycles significantly alter the number of EVs . Indeed, a single freeze–thaw cycle and storage up to 1 year (−80°) is the recommended guideline since this procedure has a low impact on the concentration and size of particles …”

Mesenchymal stromal cell‐derived extracellular vesicles as cell‐free biologics for the ex vivo expansion of hematopoietic stem cells

“…(2) Their bilayered lipid vesicular structure and nanosize are more beneficial for a long‐term circulatory and therapeutic action (Lösche et al, 2004; Biancone et al, 2012; Karp and Leng Teo, 2009). (3) In addition, EVs show prolonged stability at low temperatures such as −20, −80, and −196°C (Jeyaram and Jay, 2018) and are more resistant to freezing‐thawing cycles (up to four cycles) (Bæk et al, 2016) making them more suitable for biomanufacturing and eventual clinical administration (Lamparski et al, 2002; Yu et al, 2014; Akers et al, 2016; Bæk et al, 2016).…”

Extracellular Vesicles in Oncology: Progress and Pitfalls in the Methods of Isolation and Analysis

“…Moreover, hemolyzed samples should not be used and if included in the analysis the obtained results should be used with care . For the plasma collection, the choice of anticoagulants (e.g., EDTA, citrate, and heparin) remains an open question . It is recommended to choose the anticoagulant on the basis of specific research questions.…”

“…A study of Bæk and coworkers showed that the main factors impacting on EVs isolation are the storage temperature, the period of time and prospective transportation before the initial centrifugation, and the centrifugation protocol. On the contrary, long‐term storage and freeze‐thawing did not appear to have a critical influence.…”