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 measurement of urine biomarkers. STUDY DESIGN Cross-sectional, prospective. SETTING & PARTICIPANTS Hospitalized patients from two sites: Yale New Haven Hospital (n= 52) and University of California, San Francisco Medical Center (n=36) PREDICTORS We tested the impact of 3 urine processing conditions on these biomarkers: a) centrifugation and storage at 4°C for 48 hours before freezing at −80°C, b) centrifugation and storage at 25°C for 48 hours before freezing at −80°C, and c) uncentrifuged samples immediately frozen at −80°C. OUTCOMES Urine concentration of five 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 MEASUREMENTS We measured urine biomarkers by an established ELISA method. 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 above 0.9 for all biomarkers tested. For samples stored at 25°C for 48 hours, excellent CCC values (>0.9) were also 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). Limitations 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.

Biomarkers of Kidney Injury Among Nicaraguan Sugarcane Workers

“…Second, biomarker levels could potentially be affected by storage of biological specimens. Though NGAL and IL-18 levels do not seem to be greatly affected by short-term handling variations, 52 levels may decline over time in frozen samples. In one study, NAG was shown to decline 28-fold over one year while stored at −80°C, 53 although this finding has not been confirmed.…”

Method Validation for Preparing Urine Samples for Downstream Proteomic and Metabolomic Applications

“…33 It has recently been shown that post-centrifugation delays of up to 48 hours at either RT or 4°C do not have a significant impact on the levels of biomarkers of acute kidney injury (neutrophil gelatinase-associated lipocalin, interleukin-18, kidney injury molecule 1, liver-type fatty acid-binding protein). 34 In order to prevent degradation of more sensitive molecules, a post-centrifugation delay of less than 2 hours at 4°C, storage of urine supernatant at -80°C, and avoiding freeze/thaw cycles is recommended.…”

Assessment of Worldwide Acute Kidney Injury, Renal Angina and Epidemiology in Critically Ill Children (AWARE): A Prospective Study to Improve Diagnostic Precision

“…All urine samples will be kept in frozen form for 5 years at −80°C, establishing a ‘biorepository’ of urinary samples. Stability of urine for processing and analysis of urinary biomarkers after storage in this manner has been validated [21]. …”