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Effect of Processing Delay and Storage Conditions on Urine Albumin-to-Creatinine Ratio.

Author(s): Herrington W, Illingworth N, Staplin N, Kumar A, Storey B, Hrusecka R, Judge P, Mahmood M, Parish S, Landray M, Haynes R, Baigent C, Hill M, Clark S

Publication: Clin J Am Soc Nephrol, 2016, Vol. 11(10), Page 1794-1801

PubMed ID: 27654930 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of preservation with boric acid; storage at -80, -40, -20, 4, 18 and 30˚C; and freeze-thaw cycling on levels of urinary albumin and creatinine and the uACR. Urine from 102 patients with kidney disease was collected into containers without additive and was transferred into containers with powdered boric acid or without preservative. Urine was aliquoted and analyzed within 2 h; after storage for 1, 2, 4, and 7 days at 4, 18, and 30°C; after up to 3 freeze-thaw cycles on consecutive days at -80˚C; and after 1 and 6 months of storage at -20, -40 and -80°C. Specimens were vortexed and centrifuged at 3000 x g for 10 min prior to analysis. Creatinine and albumin were determined using a clinical chemistry analyzer. At least 80 of the 102 specimens were used for each experimental protocol.

   Summary of Findings:

   Boric acid preservation resulted in an approximately 2% reduction in creatinine (-2.3% to -2.6%, P<0.001, all) and albumin (-1.6% to -2.4%, P<0.001, all) levels but because the percentage reductions were similar for both, the uACR was unaffected.  Generally, albumin levels were more affected by storage than creatinine and the changes increased with increasing storage temperature and duration, but the authors did not explore the significance of the changes. When stored without preservatives, uACR changed by more than 5% (the defined stability threshold) after 7 days at 30˚C, regardless of preservation, but the authors also state they were not stable after 4 days at 30˚C (4.2% change if preserved, 3.9% if not preserved). Regardless, uACR was stable in both preserved and unpreserved specimens stored for 7 days at 4˚C and in preserved specimens stored for 7 days at 18˚C.

   uACR was clearly stable in preserved urine specimens subjected to up to three freeze-thaw cycles or storage at -40 or -80˚C for up to 6 months and increased in unpreserved specimens by only 2.3% after 3 freeze-thaw cycles and decreased by 3.3% after 6 months at -40˚C. In contrast, the uACR declined 8.9% in preserved urine and 4.4% in unpreserved urine when stored at -20˚C for 1 month, a change that was mostly attributed to a decline in albumin as creatinine levels remained relatively unchanged. Importantly, the changes in uACR with one or two freeze-thaw cycles were larger in patients with macroalbuminuria than normo- or microalbuminuria (P=0.02 and P=0.03, respectively) and the changes in uACR during storage at -20˚C were larger in patients with normo- or microalbuminuria than macroalbuminuria (P=0.01 after 1 month and P<0.001 after 6 months).

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)
   • Other Preservative
   • Frozen

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   Protein	Clinical chemistry/auto analyzer
   Small molecule	Clinical chemistry/auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Biomarker level	Macroalbuminuria Normoalbuminuria Microalbuminuria
   Biospecimen Preservation	Type of fixation/preservation	Boric acid None (fresh) Frozen Refrigeration
   Storage	Freeze/thaw cycling	1 cycle 2 cycles 3 cycles
   Storage	Storage duration	0 days 1 day 2 days 4 days 7 days 1 month 6 months
   Storage	Storage temperature	-80˚C -40˚C -20˚C 4˚C 18˚C 30˚C

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