**2.3 Methods for EVs isolation**

Biological fluids containing EVs, which serve as potential minimally invasive liquid biopsies, have shifted its proteomic and genomic profiling research towards identification of biomarkers for disease diagnosis, prognosis and longitudinal monitoring. Studying EVs and their cargo typically requires separation from a biological matrix (such as a complex fluid or tissue) to analyze the unique EV components. However, isolating EVs from different sources presents certain challenges. For example, in serum and plasma the main challenge is to separate EVs from highly abundant non-EV proteins, such as albumin and globulins and non-EV lipid particles, such as lipoproteins and chylomicrons [21]. These co-purified contaminants pose a challenge for the isolation, analysis, and application of EVs. Correct interpretation and detailed reporting of the nature of EV samples and sample handling including storage, isolation, and analytical procedures for the analysis of EVs is required [18]. Many approaches have been used, including differential ultracentrifugation, density gradient ultracentrifugation, size exclusion chromatography, and affinity/immunoaffinity capture methods. All these approaches have their limitations and advantages, which are challenged by both the source and quantity of starting material and the downstream application [21]. Serial centrifugation enables the separation of EVs from cells, cell debris and larger vesicles such as apoptotic bodies. Ultracentrifugation (UC) exploits high centrifugal speed (100.000 x g) for a sufficient time to allow EVs to pellet. It separates particles based on their size, shape, and flotation density and is less efficient for smaller and less dense particles. Repeated centrifugation can reduce the amount of non-EVs particles, but also reduces the yield and may damage the EVs [21]. Density centrifugation or density ultracentrifugation uses a density gradient medium or cushion of denser solution (e.g. sucrose cushion; sUC) [22] to separate particles of a similar density. This technique takes advantage of the fact that particles denser than the solvent sediment in the suspension, while particles less dense float up. This increases the purity of samples and reduces the potential of mechanical damage to the vesicles [23]. Density gradient ultracentrifugation is successful in separating chylomicrons, very low-density, low-density and intermediate density lipoproteins present in plasma. However, particles of similar density, such as high-density lipoproteins, are co-isolated with the EVs [21]. Size exclusion chromatography (SEC) is a chromatographic method that allows vesicles of a particular size to be separated where EVs retain their structure and physiological function [24]. When performing SEC protein contaminants and aggregates of similar size, are often still present. In addition, the sample has to be further concentrated because of the different pooled fractions, decreasing the yield of isolation. Holcar et al. have investigated the purity of the samples by comparing sUC and SEC; the two most commonly used methods for the isolation of EVs. Transmission electron microscopy (TEM) of EVs isolated with SEC showed increased levels of lipoproteins. This was further confirmed by determining a significant increase of ApoA1 (found in high-density lipoproteins) and ApoB100

*Extracellular Vesicles: Intercellular Communication Mediators in Antiphospholipid Syndrome DOI: http://dx.doi.org/10.5772/intechopen.97412*

(found in very low-density, low-density and intermediate-density lipoproteins) [22]. Based on their results, the presence of lipoproteins in SEC isolates could have a significant impact on downstream analysis. Polymer-based precipitation uses volume-excluding polymers to lower the solubility of EVs and similarly sized non-EV particles which are isolated via low speed centrifugation. The main problem using this method is that protein removal kits must be used [21]. The highest purity of isolated EVs is achieved by using different immunopurification methods, such as immunomagnetic isolation. This method separates EVs on the basis of an antigen– antibody interactions where the antibodies linked to the matrix (e.g. magnets) are directed against a specific antigen of interest on EVs [25]. Using this methodology, a specific EV subpopulation is investigated, however, the information about the general vesicle population is lost. In addition, when using the immunopurification method, EVs stay bound to the matrix, which makes them incompatible with certain downstream analyses (**Table 1**).
