title: Freezing method affects the concentration and variability of urine proteins and the interpretation of data on microalbuminuria. The Oxford Regional Prospective Study Group.
abstract: OBJECTIVE
  Microalbuminuria and, to a lesser extent, renal tubular proteins are widely used in the early detection of incipient nephropathy in diabetes mellitus. Recent reports have indicated detrimental effects of storage at -20 degrees C on urine proteins. This study investigated the effects of storage on the measurement of urine proteins and discusses implications for the interpretation 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-diabetic 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 normal ranges for urine proteins.
  
  
  RESULTS
  Storage at -20 degrees C led to a variable underestimation of all three urine proteins in 20% of specimens. Creatinine was unaffected. This underestimation was greater in more concentrated urine (r2 = 0.38, P < 0.001, n = 262). Consequently storage at -20 degrees C increased the variance of the albumin/ creatinine ratio more than the variance of albumin concentration. 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 prevalence of microalbuminuria (> 2SD above the geometric mean in non-diabetic 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 above 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 after 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.
authors.affiliation: Division of Public Health and Primary Health Care, Institute of Health Sciences, University of Oxford, UK.
authors.forename: C J
authors.initials: CJ
authors.lastname: Schultz
authors.forename: R N
authors.initials: RN
authors.lastname: Dalton
authors.forename: C
authors.initials: C
authors.lastname: Turner
authors.forename: H A
authors.initials: HA
authors.lastname: Neil
authors.forename: D B
authors.initials: DB
authors.lastname: Dunger
mesh_terms: D000118:Acetylglucosaminidase / Q000652:urine; D000293:Adolescent; D000419:Albuminuria; D002648:Child; D003404:Creatinine / Q000097:blood; D003922:Diabetes Mellitus, Type 1 / Q000652:urine*; D003928:Diabetic Nephropathies / Q000175:diagnosis* / Q000652:urine; D005260:Female; D005615:Freezing; D006801:Humans; D008297:Male; D011507:Proteinuria; D012016:Reference Values; D015203:Reproducibility of Results; D012177:Retinol-Binding Proteins / Q000652:urine; D013048:Specimen Handling / Q000379:methods*; D016482:Urinalysis / Q000379:methods*
chemical_list: D012177:Retinol-Binding Proteins; D003404:Creatinine; D000118:Acetylglucosaminidase