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Effect of frozen storage on urinary concentration of kidney damage markers.

Author(s): Nauta FL, Bakker SJ, Lambers Heerspink H, de Zeeuw D, van Oeveren W, Bilo H, de Jong PE, Gansevoort RT

Publication: Am J Kidney Dis, 2012, Vol. 59, Page 586-9

PubMed ID: 22206741 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effect of frozen storage temperature and duration, alkalinization, and addition of protease inhibitors on levels of urinary markers of kidney disease. Ninety-five urine specimens from patients with type I or type II diabetes were aliquoted for storage at -20°C or -80°C for one week, six months or a year. To test the effect of pH, specimen pH levels were adjusted to >8.0 with 1 mol/L sodium hydroxide before or after freezing. Protease inhibitor cocktails were added to some specimens before freezing. Specimens were thawed overnight, vortexed, centrifuged at 20,000x g, and analyzed.

   Summary of Findings:

   While levels of IgG, Kim-1, and H-FABP were significantly lower after one week at -80˚C than in fresh specimens (P<0.05, P<0.01, and P<0.05; respectively), they were not significantly different after six months at -80˚C. Levels of IgG4 and NAG were significantly lower after six months at -80˚C than in fresh specimens (P<0.01, both).  Compared to fresh specimens, specimens stored for one year at -80˚C had lower levels of KIM-1 (P<0.01), NAG (P<0.01), and NGAL (P<0.05) and higher levels of Cystatin C (P<0.01), but comparable levels of IgG4.  Alkalinization prior to storage at -80˚C for a year did not stabilize levels of KIM-1, NAG, CysC, or NGAL and resulted in significantly lower levels of NGAL, IgG, and IgG4 than when stored for a year without alkalinization (P<0.01, P<0.01, and P<0.05; respectively). Similarly, specimens that were alkalinized after one year of storage at -80˚C had significantly lower levels of IgG4, KIM-1, NAG, NGAL, and H-FABP than specimens that were not alkalinized (P<0.01, all), but the storage-mediated effect on CysC was partially attenuated. Similarly, addition of protease inhibitors did not stabilize any of the analytes during storage at -80˚C and resulted in significantly lower levels of IgG4, KIM-1, NAG, CysC, NGAL, and H-FABP than when stored at -80˚C (P<0.01, P<0.01, P<0.05, P<0.01, P<0.01, and P<0.01; respectively).

   Compared to fresh specimens, urine stored at -20˚C for one year had significantly lower levels of IgG4 (P<0.05), KIM-1 (P<0.01), NAG (P<0.01), CysC (P<0.01), and H-FABP (P<0.01). Further, levels of KIM-1, CysC, NGAL, and HFABP were lower after one year of storage at -20˚C than when stored at -80˚C (P<0.01, all). While most analytes declined further when specimens were alkalinized after one year at -20˚C, alkalinization partially attenuated the effects on CysC. 

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Diabetes Type 2
   • Diabetes Type 1

   Platform:

   Analyte	Technology Platform
   Protein	Immunoassay
   Glycoprotein	Immunoassay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	pH	> 8 < 8
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)
   Storage	Storage conditions	Alkalinized Untreated
   Storage	Storage temperature	-20˚C -80˚C
   Storage	Storage duration	0 days 1 week 6 months 1 year
   Analyte Extraction and Purification	Protease inhibitor	No protease inhibitor added Cocktail

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