title: Urine stability studies for novel biomarkers of acute kidney injury.
abstract: BACKGROUND
  The study of novel urinary biomarkers of acute kidney injury has expanded exponentially. Effective interpretation of data and meaningful comparisons between studies require awareness of factors that can adversely affect measurement. We examined how variations in short-term storage and processing might affect the measurement of urine biomarkers.
  
  
  METHODS
  Cross-sectional prospective.
  
  
  METHODS
  Hospitalized patients from 2 sites: Yale New Haven Hospital (n=50) and University of California, San Francisco Medical Center (n=36).
  
  
  METHODS
  We tested the impact of 3 urine processing conditions on these biomarkers: (1) centrifugation and storage at 4°C for 48 hours before freezing at -80°C, (2) centrifugation and storage at 25°C for 48 hours before freezing at -80°C, and (3) uncentrifuged samples immediately frozen at -80°C.
  
  
  RESULTS
  Urine concentrations of 5 biomarkers: neutrophil gelatinase-associated lipocalin (NGAL), interleukin 18 (IL-18), kidney injury molecule 1 (KIM-1), liver-type fatty acid-binding protein (L-FABP), and cystatin C.
  
  
  METHODS
  We measured urine biomarkers by established enzyme-linked immunosorbent assay methods. Biomarker values were log-transformed, and agreement with a reference standard of immediate centrifugation and storage at -80°C was compared using concordance correlation coefficients (CCCs).
  
  
  RESULTS
  Neither storing samples at 4°C for 48 hours nor centrifugation had a significant effect on measured levels, with CCCs higher than 0.9 for all biomarkers tested. For samples stored at 25°C for 48 hours, excellent CCC values (>0.9) also were noted between the test sample and the reference standard for NGAL, cystatin C, L-FABP and KIM-1. However, the CCC for IL-18 between samples stored at 25°C for 48 hours and the reference standard was 0.81 (95% CI, 0.66-0.96).
  
  
  CONCLUSIONS
  No comparisons to fresh, unfrozen samples; no evaluation of the effect of protease inhibitors.
  
  
  CONCLUSIONS
  All candidate markers tested using the specified assays showed high stability with both short-term storage at 4°C and without centrifugation prior to freezing. For optimal fidelity, urine for IL-18 measurement should not be stored at 25°C before long-term storage or analysis.
authors.affiliation: Section of Nephrology, Department of Medicine, Yale University School of Medicine, New Haven, CT; Program of Applied Translational Research, Yale University School of Medicine, New Haven, CT. Electronic address: chirag.parikh@yale.edu.
authors.forename: Chirag R
authors.initials: CR
authors.lastname: Parikh
authors.affiliation: Section of Nephrology, Department of Medicine, Yale University School of Medicine, New Haven, CT; Program of Applied Translational Research, Yale University School of Medicine, New Haven, CT.
authors.forename: Isabel
authors.initials: I
authors.lastname: Butrymowicz
authors.affiliation: Section of Nephrology, Department of Medicine, Yale University School of Medicine, New Haven, CT; Program of Applied Translational Research, Yale University School of Medicine, New Haven, CT.
authors.forename: Angela
authors.initials: A
authors.lastname: Yu
authors.affiliation: Department of Public Health Sciences, Penn State College of Medicine, Hershey, PA.
authors.forename: Vernon M
authors.initials: VM
authors.lastname: Chinchilli
authors.affiliation: Division of Nephrology, Department of Medicine, University of California, San Francisco, CA.
authors.forename: Meyeon
authors.initials: M
authors.lastname: Park
authors.affiliation: Division of Nephrology, Department of Medicine, University of California, San Francisco, CA; Division of Research, Kaiser Permanente of Northern California, Oakland, CA.
authors.forename: Chi-Yuan
authors.initials: CY
authors.lastname: Hsu
authors.affiliation: Department of Medicine, Division of Nephrology, Penn State College of Medicine, Hershey, PA.
authors.forename: W Brian
authors.initials: WB
authors.lastname: Reeves
authors.affiliation: Nephrology and Hypertension, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
authors.forename: Prasad
authors.initials: P
authors.lastname: Devarajan
authors.affiliation: Division of Kidney, Urologic, and Hematologic Diseases, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD.
authors.forename: Paul L
authors.initials: PL
authors.lastname: Kimmel
authors.affiliation: Department of Medicine, Division of Nephrology and Hypertension, Vanderbilt University Medical Center, Nashville, TN.
authors.forename: Edward D
authors.initials: ED
authors.lastname: Siew
authors.affiliation: Division of Nephrology, Department of Medicine, University of California, San Francisco, CA.
authors.forename: Kathleen D
authors.initials: KD
authors.lastname: Liu
mesh_terms: D058186:Acute Kidney Injury / Q000652:urine*; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000652:urine*; D002498:Centrifugation; D003430:Cross-Sectional Studies; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005615:Freezing; D006801:Humans; D008297:Male; D011446:Prospective Studies; D059349:Urine Specimen Collection / Q000379:methods / Q000592:standards
chemical_list: D015415:Biomarkers
keywords: Proteins; acute kidney injury (AKI); acute renal failure (ARF); biospecimen handling; concordance; handling; protein stability; storage; urine biomarker