Solga, M. | bioRxiv Science

bioRxiv2025

Temporal Dynamics of Urinary Extracellular Vesicle Excretion and Cargo in Healthy Subjects over 24 Hours

Urinary extracellular vesicles (uEVs) are promising non-invasive biomarkers of renal physiology and pathology, yet little is known about their temporal excretion patterns or optimal normalization strategies as urine concentrations vary significantly during the day. In this basic physiology study, we used a uniquely designed processing protocol of urine collection to characterize the variation of uEV excretion and cargo throughout a 24 hour period in healthy subjects. Thirteen healthy adults collected every individual void over 24 hours and these voids were independently analyzed along with a composite sample intended to recreate a 24 hour collection sample. uEVs were enriched by low-speed centrifugation with three low-ionic-strength washes to minimize uromodulin contamination. Size, concentration, morphology and cargo of uEVs were characterized with nanoparticle tracking analysis, cryo-electron microscopy, immunoblotting and single EV flow cytometry. We observed a stable urine creatinine excretion into the urine over the day. uEV concentrations varied up to 100-fold across the day between individuals with a decreasing trend over time with the result close to reaching statistical significance (p=0.06). uEV concentration was positively associated with urine creatinine, specific gravity, and TSG101, an EV marker. The association between urine flow and uEV excretion varied across the day. Most tested uEV cargo markers exhibited substantial within individual variation over the day without consistent trends in our healthy cohort. When grouped into predefined time periods to visualize temporal patterns, a transient dip at noon was observed, followed by a significant increase in the subsequent period, consistent with a pattern previously reported. These findings offer foundational insights into uEV excretion patterns over the course of a day in a healthy cohort and emphasize key methodological considerations essential for advancing uEV biomarker research.

physiology↗

bioRxiv2019

A Modified Injector and Sample Acquisition Protocol Can Improve Data Quality and Reduce Inter-Instrument Variability of the Helios Mass Cytometer

Mass cytometry is a powerful tool for high dimensional single cell characterization. Since the introduction of the first commercial CyTOF mass cytometer by DVS Sciences in 2009, mass cytometry technology has matured and become more widely utilized, with sequential platform upgrades designed to address specific limitations and to expand the capabilities of the platform. Fluidigms 3rd-generation Helios mass cytometer introduced a number of upgrades over the previous CyTOF2. One of these new features is a modified narrow bore sample injector that generates smaller ion clouds, which is expected to improve sensitivity and throughput. However, following rigorous testing we find that the narrow-bore sample injector may have unintended negative consequences on data quality and result in lower median signal intensities and higher coefficients of variation in antibody expression. We describe an alternative Helios acquisition protocol using a wider bore injector, which largely mitigates these data quality issues. We directly compare these two protocols in a multi-site study of 10 Helios instruments across 7 institutions and show that the modified protocol improves data quality and reduces inter-instrument variability. These findings highlight and address an important source of technical variability in mass cytometry experiments that is of particular relevance in the setting of multi-center studies.

immunology↗