Defining Biological Variability, Analytical Precision and Quantitative Biophysiochemical Characterization of Human Urinary Extracellular Vesicles

• Published in: Journal of extracellular vesicles Vol. 14; no. 5; pp. e70087 - n/a
• Main Authors: Aksamitiene, Edita, Park, Jaena, Marjanovic, Marina, Boppart, Stephen A.
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01.05.2025; John Wiley and Sons Inc; Wiley
• Subjects: Adult; analytical precision; biological variability; biomarker quantitation; Biomarkers; biophysiochemical properties; Cancer; Cellulose acetate; Centrifugation; coefficient of variation; Extracellular vesicles; Extracellular Vesicles - chemistry; Extracellular Vesicles - metabolism; Female; Humans; Immunoblotting; Light scattering; Male; Nanoparticles; Nanoparticles - chemistry; Polyethylene glycol; repeatability; Reproducibility; Reproducibility of Results; Silicon carbide; Technical Note; urinary extracellular vesicles; Urine - chemistry; repeatability; biological variability; biophysiochemical properties; urinary extracellular vesicles; analytical precision; biomarker quantitation; coefficient of variation
• ISSN: 2001-3078; 2001-3078
• DOI: 10.1002/jev2.70087

ABSTRACT The magnitude of combined analytical errors of urinary extracellular vesicle (uEV) preparation and measurement techniques (CVA) has not been thoroughly investigated to determine whether it exceeds biological variations. We utilized technical replicates of human urine to assess the repeatability of uEV concentration and size measurements by nanoparticle tracking analysis (NTA) following differential velocity centrifugation (DC), silicon carbide, or polyethylene glycol uEV isolation methods. The DC method attained the highest precision. Consequently, DC‐derived uEV size, most abundant protein levels, and optical redox ratio (ORR) were further assessed by dynamic light scattering (DLS), immunoblotting or multi‐photon (SLAM) microscopy. Procedural errors primarily affected uEV counting and uEV‐associated protein quantification, while instrumental errors contributed most to the total variability of NTA‐ and DLS‐mediated uEV sizing processes. The intra‐individual variability (CVI) of uEV counts assessed by NTA was smaller than inter‐individual variability (CVG), resulting in an estimated index of individuality IOI < 0.6, suggesting that personalized reference interval (RI) is more suitable for interpretation of changes in subject's test results. Population‐based RI was more appropriate for ORR (IOI > 1.4). The analytical performance of DC‐NTA and DC‐SLAM techniques met optimal CVA < 0.5 × CVI criteria, indicating their suitability for further testing in clinical laboratory settings. This study aimed to assess the short‐term measurement precision of the biophysiochemical properties of urinary extracellular vesicles (uEVs) isolated from fresh healthy human first‐morning urine using different methods, and to evaluate the analytical performance of preferred combined uEV isolation and downstream analysis technique by comparing the technical and biological variations. Differential centrifugation coupled to nanoparticle tracking analysis or simultaneous label‐free autofluorescence multi‐harmonic (SLAM) microscopy met optimal method performance criteria, indicating their suitability for further testing in clinical laboratory setting.