Freezing method affects the concentration and variability of urine proteins and the interpretation of data on microalbuminuria

Aims Microalbuminuria and, to a lesser extent, renal tubular proteins are w idely used in the early detection of incipient nephropathy in diabetes mell itus, Recent reports have indicated detrimental effects of storage at -20 d egrees C on urine proteins. This study investigated the effects of storage on the measurement of urine proteins and discusses implications interpretat ion of data. Methods Two-hundred and sixty-eight specimens, collected from children with Type 1 diabetes, split into duplicate aliquots and stored at -20 degrees C and -70 degrees C, respectively, for 6-8 months, were analysed for albumin , retinol binding protein, N-acetyl glucosaminidase and creatinine, in the same assays to eliminate inter-assay variability. Two independent non-diabe tic cohorts of children provided urine specimens, which were stored at -20 degrees C for one cohort and -70 degrees C for the other, to determine norm al ranges for urine proteins. Results Storage at -20 degrees C led to a variable underestimation of all t hree urine proteins in 20% of specimens. Creatinine was unaffected. This un derestimation was greater in more concentrated urine (r(2) = 0.38, P < 0.00 1, n = 262). Consequently storage at -20 degrees C increased the variance o f the albumin/creatinine ratio more than the variance of albumin concentrat ion. Temperature of storage affected the normal range, which was 0.1-2.1 mg /mmol at -20 degrees C compared to 0.3-3.1 mg/mmol at -70 degrees C. The pr evalence of microalbuminuria (> 2SD above the geometric mean in non-diabeti c specimens stored at -20 degrees C) was 27% after storage at -70 degrees C vs. 24% after -20 degrees C. The prevalence of microalbuminuria (> 2SD abo ve the geometric mean in nondiabetic specimens stored at -70 degrees C) was 21% after storage at -70 degrees C vs. 17% after -20 degrees C. Conclusions Urine proteins are significantly but variably underestimated af ter storage at -20 degrees C. These effects account for increased variance and differences in the normal range, but have less effect on the detection of microalbuminuria than might be predicted.