Falsely Low Urinary Albumin Concentrations after Prolonged Frozen Storage of Urine Samples

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Abstract
Microalbuminuria, defined as a urinary albumin concentration (UAC) of 20–200 mg/L, is an early predictor of diabetic nephropathy (1)(2)(3)(4)(5)(6). In addition, microalbuminuria is a marker of cardiovascular morbidity and mortality, both in patients with diabetes mellitus and in the general population(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Consequently, there is great interest in screening for microalbuminuria in these groups. In cohort studies, urine samples are often kept frozen at −20 °C before analysis. Although some study results have indicated no effect of freezing on UAC(18)(19)(20)(21)(22)(23), other studies have found erroneously low values when samples were frozen at −20 °C(24)(25)(26)(27). Only a few studies have investigated the effect of longer storage periods, but these studies were small, and samples were in the macroalbuminuric range (>200 mg/L). We investigated the effects of storage at −20 °C for up to 24 months, mixing methods, and baseline UAC on samples in the normo- and microalbuminuric ranges. Urine samples were collected during the prospective PREVEND study in the general population initiated to investigate urinary albumin excretion as a predictor of renal and cardiovascular disease (28). The participants were asked to collect urine for two 24-h periods and to store it in two 3-L plastic containers at 7 °C and deliver it within 2 days to the clinic. Immediately after delivery, the urine sample volume was determined, and a portion of each of the 24-h samples was stored in 2-mL polypropylene aliquots at −20 °C for albumin assessment after freezing. For …