Urinary extracellular vesicles: Assessment of pre-analytical variables and development of a quality control with focus on transcriptomic biomarker research.

Urinary extracellular vesicles (uEV) are a topical source of non-invasive biomarkers for health and diseases of the urogenital system. However, several challenges have become evident in the standardization of uEV pipelines from collection of urine to biomarker analysis. Here, we studied the effect of pre-analytical variables and developed means of quality control for uEV isolates to be used in transcriptomic biomarker research. We included urine samples from healthy controls and individuals with type 1 or type 2 diabetes and normo-, micro- or macroalbuminuria and isolated uEV by ultracentrifugation. We studied the effect of storage temperature (-20°C vs. -80°C), time (up to 4 years) and storage format (urine or isolated uEV) on quality of uEV by nanoparticle tracking analysis, electron microscopy, Western blotting and qPCR. Urinary EV RNA was compared in terms of quantity, quality, and by mRNA or miRNA sequencing. To study the stability of miRNA levels in samples isolated by different methods, we created and tested a list of miRNAs commonly enriched in uEV isolates. uEV and their transcriptome were preserved in urine or as isolated uEV even after long-term storage at -80°C. However, storage at -20°C degraded particularly the GC-rich part of the transcriptome and EV protein markers. Transcriptome was preserved in RNA samples extracted with and without DNAse, but read distributions still showed some differences in e.g. intergenic and intronic reads. MiRNAs commonly enriched in uEV isolates were stable and concordant between different EV isolation methods. Analysis of never frozen uEV helped to identify surface characteristics of particles by EM. In addition to uEV, qPCR assays demonstrated that uEV isolates commonly contained polyoma viruses. Based on our results, we present recommendations how to store and handle uEV isolates for transcriptomics studies that may help to expedite standardization of the EV biomarker field.

Assessment of Time-Dependent Platelet Activation Using Extracellular Vesicles, CD62P Exposure, and Soluble Glycoprotein V Content of Platelet Concentrates with Two Different Platelet Additive Solutions.

Novel analytical measures are needed to accurately monitor the properties of platelet concentrates (PCs). Since activated platelets produce platelet-derived extracellular vesicles (EVs), analyzing EVs of PCs may provide additional information about the condition of platelets. The prospect of using EVs as an auxiliary measure of platelet activation state was investigated by examining the effect of platelet additive solutions (PASs) on EV formation and platelet activation during PC storage. The time-dependent activation of platelets in PCs with PAS-B or with the further developed PAS-E was compared by measuring the exposure of CD62P by flow cytometry and the content of soluble glycoprotein V (sGPV) of PCs by an immunoassay. Changes in the concentration and size distribution of EVs were determined using nanoparticle tracking analysis. A time-dependent increase in platelet activation in PCs was demonstrated by increased CD62P ex-posure, sGPV content, and EV concentration. Using these strongly correlating parameters, PAS-B platelets were shown to be more activated compared to PAS-E platelets. Since the EV concentration correlated well with the established platelet activation markers CD62P and sGPV, it could potentially be used as a complementary parameter for platelet activation for PCs. More detailed characterization of the resulting EVs could help to understand how the PC components contribute the functional effects of transfused PCs.