Because procedures of handling and storage of body fluids affect numbers and composition of extracellular vesicles (EVs), standardization is important to ensure reliable and comparable measurements of EVs in a clinical environment. We aimed to develop standard protocols for handling and storage of human body fluids for EV analysis. Conditions such as centrifugation, single freeze–thaw cycle, effect of time delay between blood collection and plasma preparation and storage were investigated. Plasma is the most commonly studied body fluid in EV research. We mainly focused on EVs originating from platelets and erythrocytes and investigated the behaviour of these 2 types of EVs independently as well as in plasma samples of healthy subjects. EVs in urine and saliva were also studied for comparison. All samples were analysed simultaneously before and after freeze–thawing by resistive pulse sensing, nanoparticle tracking analysis, conventional flow cytometry (FCM) and transmission (scanning) electron microscopy. Our main finding is that the effect of centrifugation markedly depends on the cellular origin of EVs. Whereas erythrocyte EVs remain present as single EVs after centrifugation, platelet EVs form aggregates, which affect their measured concentration in plasma. Single erythrocyte and platelet EVs are present mainly in the range of 100–200 nm, far below the lower limit of what can be measured by conventional FCM. Furthermore, the effects of single freeze–thaw cycle, time delay between blood collection and plasma preparation up to 1 hour and storage up to 1 year are insignificant (p>0.05) on the measured concentration and diameter of EVs from erythrocyte and platelet concentrates and EVs in plasma, urine and saliva. In conclusion, in standard protocols for EV studies, centrifugation to isolate EVs from collected body fluids should be avoided. Freezing and storage of collected body fluids, albeit their insignificant effects, should be performed identically for comparative EV studies and to create reliable biorepositories.

Transmission electron microscopy (TEM) and transmission scanning electron Microscopy (TSEM), which denotes application of a scanning electron microscope (SEM) in the transmission mode, have been used to detect and characterize particles down to an imaging resolution of ~1 nm. In the field of EVs, TEM also has been valued for its capability to detect and characterize single EV. Furthermore, employing immunogold labeling in TEM could give information regarding biochemical properties of EV surface proteins. Significant shortcomings in TEM such as dehydration, chemical fixation, and/or staining of the biological specimens are eluded by the use of cryo-TEM. In cryo-TEM imaging, samples are directly applied onto an EM grid, vitrified and visualized, thus allowing for characterization of EVs near its native state. In this chapter, we describe a step-by-step guide for preparing EVs on the grid before TEM and cryo-TEM imaging. Finally, we provide a guide to an automated image-processing analysis to provide the size distribution of EVs.

Resolution is a key performance metric, which often defines the quality of a scanning electron microscope (SEM). Traditionally, there is the subjective measurement of the distance between two points on special "resolution" samples and there are several computer-based resolution-calculation methods. These computer-based resolution-calculation methods are much more precise than direct measurement, but none of them can currently be considered an objective way of measuring the resolution. The methods are still under development; therefore, objective testing is necessary. One approach to algorithm testing is to use simulated images. Simulated images are very useful for this purpose because they can be well-defined in all parameters unlike the real SEM images. Simulated images can be generated that closely mimic the gold-on-carbon SEM test sample images that usually consist of bright grains on a dark background. Simulation can account for edge effect, roughness of the substrate, different focusing, drift and vibration, and noise. Shapes, positions, and sizes of the grain structures are random. The simulated images can be then used for testing the resolution-calculation methods, especially for finding how the particular properties of SEM images affect the resultant instrument performance and image resolution. To support this testing, NIST has developed and made available a reference set of simulated SEM images generated using the methods described in this article.

In future, measurements of extracellular vesicles in body fluids could become a standard diagnostic tool in medicine. For this purpose, reliable and traceable methods, which can be easily applied in hospitals, have to be established. Within the European Metrological Research Project (EMRP) 'Metrological characterization of micro-vesicles from body fluids as noninvasive diagnostic biomarkers' (www.metves.eu), various nanoparticle reference materials were developed and characterized. We present results of an international comparison among four national metrology institutes and a university hospital. The size distributions of five monodisperse and two bimodal spherical particle samples with diameters ranging from 50 nm to 315 nm made out of silica and polystyrene were compared. Furthermore, the stability of the samples was verified over a period of 18 months. While monodisperse reference particle samples above a certain size level lead to good agreements of the size measurements among the different methods, small and bimodal samples show the limitations of current 'clinical' methods. All samples proved to be stable within the uncertainty of the applied methods.

In this article, a new scanning electron microscopy (SEM) image composition technique is described, which can significantly reduce drift related image corruptions. Drift distortion commonly causes blur and distortions in the SEM images. Such corruption ordinarily appears when conventional image-acquisition methods, i.e., "slow scan" and "fast scan," are applied. The damage is often very significant; it may render images unusable for metrology applications, especially where subnanometer accuracy is required. The described correction technique works with a large number of quickly taken frames, which are properly aligned and then composed into a single image. Such image contains much less noise than the individual frames, while the blur and deformation is minimized. This technique also provides useful information about changes of the sample position in time, which may be applied to investigate the drift properties of the instrument without a need of additional equipment.