Intra-Individual Variability of Urinary EGF and Clusterin, and Effect of Frozen Storage on Stability: Results from UVALID.

Urinary epidermal growth factor (uEGF) and clusterin (uCLU) are emerging biomarkers in chronic kidney disease (CKD), but rigorous analytical validation is required before clinical implementation. We evaluated intra-individual variability and long-term storage stability of uEGF and uCLU in CKD. In the prospective, multicenter UVALID study, 60 adults with CKD stages 2-4 underwent urine sampling at three visits over 8 weeks. First-morning and 24-h urine samples were collected to assess intra-individual variability over 24 h, 3 days and 8 weeks. Biomarkers were measured in duplicate by ELISA and normalized to urinary creatinine (/Cr). Inter-laboratory performance was assessed using quality control samples. Stability after 12 and 15 months of storage at -20 °C and -80 °C and the influence of pH were evaluated. Over 24 h, 3 days, and 8 weeks, uEGF/Cr demonstrated low variability and remained stable after long-term storage at both temperatures. In contrast, uCLU/Cr showed greater variability and pronounced instability at -20 °C, whereas stability was preserved at -80 °C. Samples with pH > 6 partially preserved uCLU stability at -20 °C. Inter-laboratory reproducibility was acceptable for uEGF but suboptimal for uCLU at low concentrations. Thus, uEGF showed robust analytical performance, supporting its potential clinical applicability in CKD, whereas uCLU exhibited important analytical and pre-analytical limitations, warranting further assay optimization. These findings underscore the need for rigorous validation to facilitate biomarker implementation in clinical practice.