**4. Isolation of exosomes from plasma**

An existing barrier that has impeded the progress in EV research has been the lack of methods for their isolation from body fluids in a relatively "pure" form, i.e., without non-specifically attached plasma proteins, and in quantities sufficient for further studies. The current "gold standard" for the isolation of EVs has been the density gradient ultracentrifugation of pre-cleared plasma at 100,000x g for periods of time ranging from 12-24h [16]. Ultracentrifugation using iodixanol density gradients (24 spin time) is currently the preferred isolation method. However, for many reasons, including an inadequate recovery, vesicle aggregation and potential vesicle damage during prolonged ultracentrifugation as well as the isolation platform that does not lend itself to a high throughput required for clinical assessments, ultracentrifugation is being slowly replaced by other methods. The literature is replete in listing various technological advances for EV isolation from body fluids, including microfluidics and sophisticated ultrafiltration systems [17, 18]. Many factors need to be taken into account when selecting an EV isolation procedure, such as the volume of available fluid, desired recovery and purity of EVs and processing time. Among these various methods, size exclusion chromatography (SEC) emerges as the most efficient technique for the isolation of "purified" exosomes or sEVs from plasma [4, 19]. SEC is a readily applicable separation method based on differences in protein size, which removes unwanted soluble proteins from precleared plasma and allows for the recovery of partially "cleaned" exosomes in early fractions [4, 19]. Data from various studies indicate that upon sEV isolation by SEC, glycoprotein aggregates, albumin and other plasma proteins elute in the late fractions, while partly purified, tetraspaninpositive vesicles elute in the early fractions, allowing for a relatively simple, one-step separation of exosomes from most of "contaminating" plasma components. SEC outperforms various precipitation protocols which co-isolate contaminating plasma proteins [19]. It has been suggested that SEC has drawbacks, including relatively poor yield; however, as this results from removal of protein aggregates not a loss of vesicles, the lower yield is counterbalanced by increased sample purity. With relatively minor adjustments, SEC can be used for high throughput isolation of sEVs from serially collected body fluids, yielding partially purified sEVs in early fractions [4]. These exosomes retain their vesicular morphology and phenotypic as well as functional attributes, such as the ability to modify responses of recipient cells to exogenous signaling [4].

The use of SEC has facilitated the: (i) "cleaning" of exosomes from most, although not all, "contaminating' plasma proteins; (ii) separation of soluble Ags that might be weakly "associated" with exosomes from those embedded in or carried on the exosome membrane and (iii) recovery of morphologically intact, non-aggregated exosomes that retain their functional activity [4]. The isolation of non-aggregated vesicles is especially critical for the subsequent immunoaffinity capture of vesicles, because vesicle aggregation is likely to interfere with Ab-based capture. Equally important

is the fact that the recovered vesicles retain their functional activity, e.g., are able to induce apoptosis following a brief co-incubation with activated T cells, after removal of soluble plasma proteins. It is for these reasons, that immune capture of EVs from body fluids should be preceded by SEC and not be used for direct EV isolation from plasma.
