Standardization of Diagnostic Biomarker Concentrations in Urine: The Hematuria Caveat

PLoS One. 2012;7(12):e53354. doi: 10.1371/journal.pone.0053354. Epub 2012 Dec 31.


Sensitive and specific urinary biomarkers can improve patient outcomes in many diseases through informing early diagnosis. Unfortunately, to date, the accuracy and translation of diagnostic urinary biomarkers into clinical practice has been disappointing. We believe this may be due to inappropriate standardization of diagnostic urinary biomarkers. Our objective was therefore to characterize the effects of standardizing urinary levels of IL-6, IL-8, and VEGF using the commonly applied standards namely urinary creatinine, osmolarity and protein. First, we report results based on the biomarker levels measured in 120 hematuric patients, 80 with pathologically confirmed bladder cancer, 27 with confounding pathologies and 13 in whom no underlying cause for their hematuria was identified, designated "no diagnosis". Protein levels were related to final diagnostic categories (p = 0.022, ANOVA). Osmolarity (mean = 529 mOsm; median = 528 mOsm) was normally distributed, while creatinine (mean = 10163 µmol/l, median = 9350 µmol/l) and protein (0.3297, 0.1155 mg/ml) distributions were not. When we compared AUROCs for IL-6, IL-8 and VEGF levels, we found that protein standardized levels consistently resulted in the lowest AUROCs. The latter suggests that protein standardization attenuates the "true" differences in biomarker levels across controls and bladder cancer samples. Second, in 72 hematuric patients; 48 bladder cancer and 24 controls, in whom urine samples had been collected on recruitment and at follow-up (median = 11 (1 to 20 months)), we demonstrate that protein levels were approximately 24% lower at follow-up (Bland Altman plots). There was an association between differences in individual biomarkers and differences in protein levels over time, particularly in control patients. Collectively, our findings identify caveats intrinsic to the common practice of protein standardization in biomarker discovery studies conducted on urine, particularly in patients with hematuria.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Biomarkers / urine
  • Carcinoma, Transitional Cell / diagnosis*
  • Carcinoma, Transitional Cell / pathology
  • Carcinoma, Transitional Cell / urine
  • Case-Control Studies
  • Creatinine / urine
  • Hematuria / urine*
  • Humans
  • Interleukin-6 / urine*
  • Interleukin-8 / urine*
  • Male
  • Middle Aged
  • Reference Standards
  • Sensitivity and Specificity
  • Urinary Bladder Neoplasms / diagnosis*
  • Urinary Bladder Neoplasms / pathology
  • Urinary Bladder Neoplasms / urine
  • Vascular Endothelial Growth Factor A / urine*


  • Biomarkers
  • Interleukin-6
  • Interleukin-8
  • Vascular Endothelial Growth Factor A
  • Creatinine

Grant support

Randox Laboratories undertook laboratory analyses on anonymised patient samples. Randox played no role in data analyses which were undertaken by MS, FES and KW (Queen’s University Belfast). Randox employees contributed to the preparation of the manuscript but were not involved in the decision to publish which was taken by Queen’s University Belfast. The funders, Randox Laboratories Ltd., played no role in the design of the case control study and were not involved in collection of patient data and samples.