Isolation of extracellular vesicles from human plasma samples: The importance of controls

Abstract

Background: Extracellular vesicles (EV) are enriched with proteins and RNA cargo, promoting cell-to-cell communication. Biofluid derived EV cargo is used for discovering disease specific markers for diagnosis and disease monitoring.Rational: Blood is a complex fluid with an abundance of protiens and thus isolation of EVs is challenging. Therefore, methods for EV isolation, including commercial kits use thromboplastin D (TP-D) for pretreatment of plasma to increase EV purity and yield. This pretreatment can introduce contaminants. Method and Results:We performed a comparative study to evaluate the effect of EV isolation methods focusing on (a) pretreatment of plasma with additives, which include: rabbit TP (rTP) versus human recombinant thromboplastin (huTP), to increase purity and yield (b) an additional centrifugation step prior to freezing plasma and (c) comparison of frozen versus fresh plasma EV isolations. Pretreatment with rTP generated a dynamic range of proteins, however, most of these proteins were contaminants, introduced from the rTP (99.1% purity). As an alternative, huTP was used, which did not introduce any significant contaminants, however, this did not increase yield or purity.Additionally, an extra 10,000 g centrifugation did not improve either EV yield or purity.Finally, comparison of fresh or frozen plasma showed no significant difference, an important factor when sourcing plasma from biobanks. Conclusion:Appropriate controlsare required when adding any additives during EV isolation as even a small percentage of contaminants can have a major effect on results. Furthermore, biobanked plasma can be used with no major changes to processing.

Standardized Method to Functionalize Plasma-Extracellular Vesicles via Copper-Free Click Chemistry for Targeted Drug Delivery Strategies

“…Therefore, developing a standardized methodology to functionalize plasma-derived EVs could have broader implications for targeted drug delivery and personalized medicine. Furthermore, it has to be taken into account that plasma is one of the most difficult body fluids to isolate EVs from due to the presence of a massive pattern of particles . Plasma proteins and protein aggregates, as well as the large set of lipidic nanoparticles, represent the most abundant contaminants .…”

“…Furthermore, it has to be taken into account that plasma is one of the most difficult body fluids to isolate EVs from due to the presence of a massive pattern of particles. 39 Plasma proteins and protein aggregates, as well as the large set of lipidic nanoparticles, represent the most abundant contaminants. 40 The commonly used methods to separate plasma-EVs are based on the size or density.…”

Comparing the Proteomic Profiles of Extracellular Vesicles Isolated using Different Methods from Long-term Stored Plasma Samples

“…Our results indicated that among the different isolation methods, the EVs-UC isolates were especially prone to protein aggregation and to show albumin expression. To improve the purity of isolated EVs, an albumin-depletion step is usually introduced [ 37 , 38 ]. However, this is not possible when handling limited plasma volumes.…”

Assessment of brain-derived extracellular vesicle enrichment for blood biomarker analysis in age-related neurodegenerative diseases: An international overview

“…Plasma is also considered the most physiological milieu to study blood EVs, taking into account that the number of EVs is higher in serum due to clot-induced platelet vesiculation [35]. Recently, comparisons of fresh versus frozen plasma found no significant difference in protein content of enriched EVs, which may further boost the use of biobanked plasma for EV research [37].…”

“…The protease thromboplastin-D converts prothrombin to thrombin during the clotting of blood, while the enzyme thrombin facilitates blood clotting by converting fibrinogen to fibrin. Relative to untreated plasma, pre-treatment with thromboplastin-D was reported to (i) remove clouding factors and prevent aggregation of ExoQuick enriched EVs [39], and (ii) not introduce contaminants, when using human recombinant thromboplastin and enriching for EVs using ultracentrifugation [37], though use of rabbit thromboplastin introduced exogenous tau contaminants [25]. Thrombin pre-treatment, unlike that observed with rabbit thromboplastin, lacked exogenous tau contaminants [25].…”