Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

Abstract

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments.

Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

“…This demonstrates that CFF/PEG/Capto Core yields a more homogeneously sized population of EV than either PEG or UC. It is important to note that this method of EM dehydrates the vesicles, causing them to shrink in size [52]. However, given that all samples were prepared at the same time, we are confident in our statistical analyses with regards to EV size as measured by TEM.…”

Ultrafiltration and size exclusion chromatography combined with asymmetrical‐flow field‐flow fractionation for the isolation and characterisation of extracellular vesicles from urine

“…Despite the huge potential of EVs, their isolation is currently not optimal or standardised [10]. This research paper evaluates UF-SEC as a method for the enrichment of urinary EVs.…”

“…EVs from urine can reveal urological diseases or tumours and their progression [2,4,5]. Despite the potential applications of EVs, their detection, isolation and characterisation from different bodily fluids are currently not optimal or standardised due to their small size and low refractive index (RI) [6, 7, 8, 9, 10]. …”

Investigation of procoagulant activity in extracellular vesicles isolated by differential ultracentrifugation

“…In addition, we discern that washing of the pellets resulted in an increase in particles >100 nm and a reduction in the particles <100 nm in both 20K pellets and 100K pellets. Since the concentration of the smaller particles was significantly reduced, the increase in mean particle size could probably be partly attributed to the removal of lipoproteins, but possibly also aggregates of proteins or other plasma material (Figure 1 (c)), as it is well known that lipoproteins interfere with the particle quantitation by NTA [10,21]. This observation was in accordance with the reduced protein content shown by protein quantitation, and also the western blot findings, where we confirmed that the presence of apolipoprotein B, a marker for both lipoprotein fractions LDL, IDL, VLDL, and chylomicrons, disappeared upon washing.…”

“…Blood samples were collected from the antecubital vein with tourniquet and a 21-gauge needle in 6 ml 3.2% (0.105 M) citrated tubes (BD Vacutainer, BD Biosciences, San Jose, CA, USA). The blood samples were left 15 min at room temperature after collection, before platelet-free plasma was extracted by a double centrifugation at 2,500 ×  g for 15 min according to international recommendations [10,16], leaving 1 cm plasma both above the buffy coat and the pellet in the subsequent centrifugation step. Platelet-free-plasma was obtained within one hour from blood collection.…”