Decreases in Albumin/Creatinine and N-Acetylglucosaminidase/Creatinine Ratios in Urine Samples Stored At -20 Degrees C

Abstract

The effects of storage for 6 months or 2 years at -20 degrees C were studied in urine samples from Type II diabetic patients by assaying albumin by immunoturbidity, N-acetylglucosaminidase (EC 3.2.1.30) by methoxynitrovinylphenol release, and creatinine by the Jaffé method. There were significant decreases (P < 0.001) in albumin/creatinine ratios from 1.14 (0.63-2.98) to 0.83 (0.32-2.12) g/mol (median + interquartile ranges) after 6 months (n = 97), and from 1.64 (0.74-5.72) to 1.00 (0.37-4.54) g/mol after 2 years (n = 89). The percentage of samples with results below the detection limit of the albumin assay (2 mg/L) increased from 5% to 21% after 6 months and from 0% to 34% after 2 years. N-Acetylglucosaminidase/creatinine ratios decreased (P < 0.001) from 520 (358-832) to 380 (263-695) U/mol after 6 months and from 520 (330-865) to 258 (82-462) U/mol after 2 years. The effect of storage was greater in samples with concentrations in the normal range (< 2.5 g/mol for albumin/creatinine, < 500 U/mol for N-acetylglucosaminidase/creatinine). Samples with albumin concentrations more than twice the normal range were still detected as abnormal after storage at -20 degrees C; e.g., 18% were > 5 g/mol (albumin/creatinine) initially, with 17% > 5 g/mol after 6 months of storage. We therefore recommend storage of urine samples at 4 degrees C for no longer than 7 days before assay.

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

“…The present study does not address the effect of prolonged storage beyond 8 months at −20°C. However, it has been reported that urine proteins are more severely affected, together with creatinine, after a longer period of 2 years of storage at −20°C[17]. These data, comparing specimen storage at −20°C to −70°C, are very similar to results reported by Manley et al .…”

“…These data confirm that the storage of urine specimens at −20°C results in a significant mean underestimation of albumin of 20%, that this effect is not consistent between specimens, with only the minority (36%) showing substantial underestimation, that all proteins are affected, but that creatinine is not affected at the duration of storage investigated[17]. The power of this study lies in the elimination of interassay variability from the estimation of the effects of storage.…”

“…These data, comparing specimen storage at −20°C to −70°C, are very similar to results reported by Manley et al . [17] of a comparison of fresh specimens to those stored at −20°C for 6 months. As storage at 4°C was not a practical option in the ORPS study, the question of how this compares with storage at −20°C or −70°C was not addressed.…”

“…Previous definitions and much of the data relating to the predictive power of microalbuminuria are based on specimens stored at −20°C[1–15]. Yet several studies have reported a significant underestimation of urine proteins after freezing at −20°C[16–17]. As a result, some authors recommend the use of fresh specimens (4°C for very short periods) or storage at −70°C[18–19] and these methods have been used in several recent studies[14–20].…”

“…Yet several studies have reported a significant underestimation of urine proteins after freezing at −20°C[16–17]. As a result, some authors recommend the use of fresh specimens (4°C for very short periods) or storage at −70°C[18–19] and these methods have been used in several recent studies[14–20]. Immediate analysis of fresh specimens (4°C) is impractical in large longitudinal studies and in the Oxford Regional Prospective Study (ORPS) of childhood diabetes it was opted to change from storage at −20°C to storage at −70°C[21] following these reports.…”