Regression analyses to determine the relationship between differences in standards and biomarkers over time.

<p>Scatter plots, based on data from 72 hematuric patients, plotting the differences between biomarker levels on recruitment and follow-up against the differences between protein levels on recruitment and follow-up for (A) IL-6, (B) IL-8 and (C) VEGF. The regression line and 95% confidence interval show significant associations (p<0.0001 for all biomarkers). Differences in biomarker levels across time were associated with differences in protein levels.</p

Relationship between osmolarity and creatinine.

<p>Triplicate levels of osmolarity and creatinine were measured in urine from 119 hematuric patients. There was a modest relationship between osmolarity and creatinine (R Square = 0.519).</p

Comparison of protein levels across final diagnostic categories.

<p>Urinary protein levels measured in 120 patients with hematuria were related to final diagnostic categories in (ANOVA; p = 0.022). Subsequently, we carried out a one way ANOVA with post-hoc Dunnett T3 analyses using log₁₀ transformed protein data. Higher protein levels were measured in urine from patients diagnosed with bladder cancer in comparison to those with no diagnosis (p = 0.073). There were no significant differences between the protein levels measured in patients with confounding pathologies and levels measured in the urines from bladder cancer patients (p = 0.621) or between patients with no diagnosis and patients with confounding pathologies (p = 0.316).</p

SDS PAGE on urine samples.

<p>SDS PAGE was carried out on urine from each patient. A dense band was frequently observed at approximately 64–66 kDa. This band represents albumin. Eight representative samples demonstrate the diverse relationship between this albumin band on the SDS PAGE and corresponding IL-8 levels measured in urine from the same patient sample. Corresponding IL-8 levels are illustrated in the 95% confidence limit error bar chart directly below each lane. The density of the albumin band was not always indicative of the IL-8 levels. Four patients had non-muscle invasive bladder cancer (NMI), one patient had muscle invasive bladder cancer (MI), two patients had no diagnosis (ND), and one patient had benign prostate enlargement.</p

Creatinine, Osmolarity and Protein distributions.

<p>Triplicate levels of the standards were measured in 120 hematuric patients and then averaged. (A) Osmolarity was normally distributed; (B) creatinine and (C) protein had skewed distributions.</p

Comparison between measured protein levels and protein dipstick analyses.

<p>Total protein levels (mg/ml) in urine were determined by Bradford assay A_{595 nm} (Hitachi U2800 spectrophotometer) using Bovine Serum Albumin as standard. Dipstick analyses were undertaken using Aution Sticks 10EA. Analyses were interpreted using PocketChem (Arkray factory, Inc. Japan). Protein levels were plotted against dipstick results with the Y –axis reference line indicating the usual lower limit of sensitivity for urine dipstick testing (0.25 mg/ml).</p

Paired t-test comparing standard levels measured on recruitment and at follow-up.

<p>Urine samples were obtained on two visits; one on recruitment and a second at follow-up (median = 11 (1 to 20 months)) from 72 patients who had presented with hematuria. The mean difference between log10 protein levels decreased over time (p = 0.097).</p

AUROC for IL-6, IL-8 and VEGF.

<p>The lowest area under receiver operating characteristic (AUROC) were determined after protein normalization as represented by the solid black curve which was always closest to the diagonal reference line i.e., IL-6 = 0.634 (0.523 to 0.745); IL-8 = 0.677 (0.570 to 0.784); and VEGF = 0.609 (0.501 to 0.716). The AUROCs for uncorrected biomarker levels (thick grey curve), and those standardized using osmolarity (dashed black curve) or creatinine (dashed grey curve) were very similar for individual biomarkers : (A) IL-6 = 0.693 (0.592 to 0.794), 0.683 (0.582 to 0.784) and 0.678 (0.578 to 0.779), respectively; (B) IL-8 = 0.706 (0.608 to 0.804), 0.701 (0.603 to 0.799) and 0.694 (0.592 to 0.795), respectively; and (C) VEGF = 0.705 (0.610 to 0.799), 0.687 (0.591 to 0.783) and 0.680 (0.583 to 0.777), respectively. Figures in brackets are 95% Confidence Intervals.</p

Pearson’s correlations between <i>NAMPT, ICAM1, SELL, FPR1, DEFA1-3</i> and <i>LL-37</i> genes expression.

<p>Only Significant correlations are shown (P≤5×10⁻³).</p><p>All genes expression were normalized to <i>POL2RA</i> mRNA levels.</p><p>r: Pearson’s correlation coefficient, P: P-value.</p

Characteristics of studied individuals.

<p>BMI: body mass index, BP: blood pressure, SBP: systolic blood pressure, DBP: diastolic blood pressure, MAF: minor allele frequency.</p