Chapter 8 192 The development of reference materials such as HOBs reflects the ongoing efforts of the EV-field in cross-platform standardization. The benefit of using HOBs, rather than PS beads, is that HOBs with a given size will allow the gating of EVs of the same size without the need of Mie theory to correct light scattering signals into particle size 42. Therefore, HOBs are ideal reference beads with which to standardize optical measurements of EV concentrations within a predefined size range. Future improvements to HOBs could be fluorescent labelling (similar to PS beads with attributed MESF or ERF values), with the aim of creating a single reference reagent which allows simultaneous calibration of both light scattering and fluorescent signals. On a less technical note, the low flow rates and consequential long acquisition times, as well as the sizes of data files generated by IFCM may impair its clinical implication. Newer platforms, such as the recently developed CellStream, may solve these problems as this platform has an approximate 10-fold higher flow rate, and does not record images for each detected event – thus leading to smaller data files and shorter acquisition times. However, the downside of not having images associated with recorded events is that it would be impossible to 1) perform spot counts on individual events to determine potential coincidence detection (‘swarm’ effect) in individual samples, and 2) visually assess outlying events to determine whether these represent contaminating components such as cellular debris (which show brightfield images). Is the clinic ready for the technology? It is apparent that EV FC is state-of-the-art nanoparticle characterization and thereby more demanding that conventional (cellular) FC. At the moment of writing, this is largely due to the current technical limitations and rapid technological developments, which demand a high level of expertise – both on the level of sample acquisition as well as data analysis. Although the field is regularly updating its consensus on standardization efforts (through MISEV position papers), and more easy-to-use reference materials are being introduced, we have only just began to fully appreciate and recognize the biological significance of EVs in health and disease. The observation that ECD kidneys release EVs during NMP, and that subsets of EVs are correlated with well-established indicators of transplant outcome provides a stepping stone for future research into the potential of EVs as indicators of kidney quality prior to transplantation. For example, EV parameters such as concentration, size, and phenotype may be analyzed in perfusion fluids of both ECD and non-ECD
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