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Letters |
1
Div. of Clin. Biochem., Women's College Hosp., Univ. of Toronto, 76 Grenville St., Toronto, Ontario M5S 1B2, Canada,
2
Dept. of Clin. Biochem., Univ. of Toronto
a Author for correspondence.
To the Editor:
"Microalbumin" (MA; albumin present in very small quantities) has assumed an increased role in the early diagnosis, assessment, and treatment of diabetic kidney disease (1)(2)(3)(4)(5). The measurement is reliably carried out by several methods, but the practical issue of MA stability during urine storage for extended periods remains controversial.
MA in urine stored at 4 °C is stable for at least 1 week and as long as 8 weeks (6)(7). Freezing the urine, however, has been reported by some to cause falsely low values (8)(9); others have reported no decrease in urines stored frozen for 4–6 months (6)(10), albeit with sporadic decreases in some urines (6). This is an important issue in cases when transport to a reference laboratory can take several days and storage at 4 °C is impractical. Townsend et al. have reported that the simple act of mixing the urine after thawing can prevent falsely low values for MA [11].
We measured MA by immunonephelometry with an Array 360 analyzer (Beckman Instruments, Brea, CA) calibrated once every 2 weeks as recommended by the manufacturer; no drift was detected over that period. Repeated assay of Beckman Level I (mean albumin 10.3 mg/L) control gave an interassay CV of 3.2% (n = 68). Fresh, unfrozen urines were centrifuged for 10 min at 3000g to remove debris. Frozen urines were stored uncentrifuged at -20 °C. After thawing, they were shaken (3–4 hand inversions) or not, centrifuged as above, and then analyzed. All determinations were carried out at room temperature.
Preliminary experiments showed that one or two freeze–thaw cycles resulted in the loss of MA concentration in most urine samples if they were not shaken after thawing. For another 23 samples, mixing after either a 1- or 3-week freeze resulted in all retaining the MA concentrations they had before freezing.
To confirm these results, we divided each of a subsequent 39 samples into three aliquots: The first aliquot from each sample (group 1) was used to measure MA in unfrozen urines; those in group 2 were frozen for a week, thawed, not mixed before centrifugation, and analyzed for MA; and those in group 3 were frozen for a week as in group 2, but were mixed before centrifuging and measuring MA. Most of the MA values in group 2 were falsely low: 77% (30 of 39) were at least 30% lower than the MA measured in the corresponding unfrozen aliquots—even after only one freeze–thaw cycle. For these 30 urines, the mean MA concentration was 7.3 mg/L, vs 11.8 mg/L for group 1, a 38% difference. Of the 39 samples in group 3, the MA concentration in 37 was the same as for the previously unfrozen aliquots (within the imprecision of the method, 3.2%); in the other two, the MA concentration decreased by ~50%: from 13.6 to 7.2 mg/L (patient 1) and from 11.3 to 6.2 mg/L (patient 2).
We rechecked in triplicate the original unfrozen urine from these two patients and found no difference from the original MA values reported for group 1. Similarly, reassay of the two samples in group 3 confirmed the decreased MA. Resuspending the latter samples by vigorous vortex-type mixing, recentrifuging, and reassaying again showed the same decreases in MA. We also resuspended the pellets from both samples in 1 mL of urine and measured these directly for MA. The MA concentration was not significantly higher in these resuspended specimens than in the original supernatants. We were thus unable to account for the "missing" MA.
Study of subsequent samples from these two patients along with three controls gave the following results. Although the MA in frozen urine from patient 1 did not return to the unfrozen sample values even with mixing (MA was still decreasedby 50%, as before), the MA results for patient 2, as well as for all of the controls, were, on mixing, the same as in the corresponding previously unfrozen samples (within experimental error). Adequate mixing seems essential to retain MA concentration after freezing and thawing. However, an unpredictable but permanent loss can occur in a few samples, for which we postulate that the antigen is distorted in such a way that the antibody may not detect the entire complement of MA present.
We conclude that in most cases (but not all) urine can be safely frozen for at least 3 weeks without alteration in MA, provided that, after thawing, the sample is well mixed (3–4 hand inversions) before centrifugation. Adequate mixing, or lack of it, may explain the reported discrepancies. As previously noted (6), MA is truly decreased in a small proportion of frozen urines, and restoration to the MA concentration in the unfrozen sample may not be possible, even with mixing. We suggest attaching a note to the MA result for a previously frozen specimen indicating that, where the clinical situation warrants, the result should be confirmed with an MA measurement on a fresh, unfrozen specimen.
References
The following articles in journals at HighWire Press have cited this article:
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  R. T. Gansevoort, J. Brinkman, S. J. L. Bakker, P. E. De Jong, and D. de Zeeuw Evaluation of Measures of Urinary Albumin Excretion Am. J. Epidemiol., October 15, 2006; 164(8): 725 - 727. [Abstract] [Full Text] [PDF]
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 J. W. Brinkman, D. de Zeeuw, J. J. Duker, R. T. Gansevoort, I. P. Kema, H. L. Hillege, P. E. de Jong, and S. J.L. Bakker Falsely Low Urinary Albumin Concentrations after Prolonged Frozen Storage of Urine Samples Clin. Chem., November 1, 2005; 51(11): 2181 - 2183. [Full Text] [PDF]
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I. S. Klasen, L. J.M. Reichert, C. M. de Kat Angelino, and J. F.M. Wetzels Quantitative Determination of Low and High Molecular Weight Proteins in Human Urine: Influence of Temperature and Storage Time Clin. Chem., March 1, 1999; 45(3): 430 - 432. [Full Text] [PDF]
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