3). Traditionally, a limited set of technologies has been used to characterize micro- and nano-vesicles with more techniques being available to the micro sized particles [32].

These include: flow cytometry, dynamic light scattering (DLS), electron microscopy [33] and [34] or enzyme linked immune-sorbent assays (ELISA) [35], [36] and [37]. Most widespread is flow cytometry; commercial flow cytometry typically has a lower practical size limit (for polystyrene beads) of around 300 nm at which point the signal is indistinguishable from the baseline noise level. Whilst this detection limit can be extended with the use of fluorescent labels, at lower sizes the ability to accurately size such particles is quite limited. Dynamic light scattering has also been

used in this application, but being an ensemble measurement, the results comprise either a simple z-average (intensity weighted) particle size and polydispersity, or a very limited-resolution particle selleck chemicals llc size distribution profile [38] and [39]. Electron microscopy is a useful research tool for studying micro- and nanovesicles but at the expense of capital running costs, extensive sample preparation [40]. Most frequently, EVS are counted in biological samples by flow cytometry [21], [41] and [42]. Several authors have pointed out that despite flow cytometry is the technique of choice for evaluation of EVS, it is limited by lack of adequate standardization [43]. Preanalytical as well as analytical issues have been evaluated in detail by Yuana et al. [44]. Preanalytical parameters GSK458 cell line were also studied in our laboratory, when measuring EVS in stored blood products: in addition to the flow cytometry parameters chosen for the numbering of EVS, we observed that many preanalytical variables have to be taken into account (diluents used,

temperature, vortexing duration, etc.) [45] and [46]. In addition, Mullier et al. recently evaluated many aspects of the prenalytical conditions as well as pending issue that has to be considered when measuring EVS in blood samples find more [47]. Because REVS express transmembrane proteins such as band 3, glycophorins, or blood group antigens, it is quite convenient to use specific antibodies raised against glycophorin A, CD47 or other blood group specific antigens for flow cytometric purposes. The expression of negatively charged phospholipids (surface phosphatidylserine) at the external part of the vesicles membrane can be explored by using annexin V as a ligand. However, standardization is mandatory in order to be able to compare data from different sets of experiments, to compare normal individuals with patients and to compare data from different laboratories. Recently, Xiong et al. presented a standardized approach based on quantitative flow cytometric technique [48]. The enumeration of REVS is made possible by using a fixed number of different-sized calibration beads spiked into each sample.