An analysis of the impact of pre-analytical factors on the urine proteome: Sample processing time, temperature, and proteolysis.

PURPOSE: We have examined the impact of sample processing time delay, temperature, and the addition of protease inhibitors (PIs) on the urinary proteome and peptidome, an important aspect of biomarker studies. EXPERIMENTAL DESIGN: Ten urine samples from patients with varying pathologies were each divided and PIs added to one-half, with aliquots of each then processed and frozen immediately, or after a delay of 6 h at 4°C or room temperature (20-22°C), effectively yielding 60 samples in total. Samples were then analyzed by 2D-PAGE, SELDI-TOF-MS, and immunoassay. RESULTS: Interindividual variability in profiles was the dominant feature in all analyses. Minimal changes were observed by 2D-PAGE as a result of delay in processing, temperature, or PIs and no changes were seen in IgG, albumin, ß2 -microglobulin, or α1 -microglobulin measured by immunoassay. Analysis of peptides showed clustering of some samples by presence/absence of PIs but the extent was very patient-dependent with most samples showing minimal effects. CONCLUSIONS AND CLINICAL RELEVANCE: The extent of processing-induced changes and the benefit of PI addition are patient- and sample-dependent. A consistent processing methodology is essential within a study to avoid any confounding of the results.

A comparison of the analytical performance of five commercially available assays for neutrophil gelatinase-associated lipocalin using urine.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker for acute kidney injury that is beginning to be used in clinical practice in addition to research studies. The current study describes an independent validation and comparison of five commercially available NGAL assays, focusing on urine samples. This is an essential step in the translation of this marker to clinical use in terms of allowing valid inter-study comparison and generation of robust results. METHODS: Two CE (Conformité Européenne)-marked assays, the NGAL Test (BioPorto) on Siemens ADVIA(®) 1800 and the ARCHITECT Urine NGAL assay on i2000SR (Abbott Laboratories), and three research-use-only (RUO) ELISAs (R&D Systems, Hycult and BioPorto) were evaluated. Imprecision, parallelism, recovery, selectivity, limit of quantitation (LOQ), vulnerability to interference and hook effect were assessed and inter-assay agreement was determined using 68 urine samples from patients with various renal diseases and healthy controls. RESULTS: The Abbott and R&D Systems assays demonstrated satisfactory performance for all parameters tested. However for the other three assays evaluated, problems were identified with LOQ (BioPorto/ADVIA(®)), parallelism (BioPorto ELISA) or several parameters (Hycult). Between-method agreement varied with the Hycult assay in particular being markedly different and highlighting issues with standardization and form of NGAL measured. CONCLUSIONS: Variability exists between the five NGAL assays in terms of their performance and this should be taken into account when interpreting results from the various clinical or research studies measuring urinary NGAL.