Table 4. Selected NRPE sCJD patient plasma creatinine and urea levels. NRPE Patient ID Plasma Creatinine (52–114 mmol/L) Plasma Urea (2.9–7.7 mmol/L) Gross indication of renal function in sCJD patients was assessed by retrospective analysis of plasma creatinine and urea levels measured as part of the clinical assessment of sCJD patients. Normal reference ranges are indicated. Data obtained from patients during the clinical phase of disease and which have subsequently died and been confirmed post-mortem as definite sCJD are indicated. Those patients which are either still alive, or which have died but did not undergo post-mortem, are indicated as probable sCJD. Patients with plasma creatinine or urea levels out-with this expected range are indicated by an asterisk. Patients for which creatinine or urea measurements were not obtained are indicated (nd; not done) doi:10.1371/journal.pone.0003870.t004 high specificity and sensitivity of the ability of urinary a1-ACT measurement to discriminate sCJD status. In a ROC curve the true positive rate (Sensitivity) is plotted in function of the false positive rate (100-Specificity) for different cut-off points. Each point on the ROC plot represents a sensitivity/specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two distributions) has a ROC plot that passes through the upper left corner (100% sensitivity, 100% specificity). Therefore the closer the ROC plot is to the upper left corner, the higher the overall accuracy of the test. The a1-ACT ELISA assay predicted prion infection with excellent sensitivity and specificity. The highest degree of sensitivity and specificity was observed for urinary a1-ACT normalised to creatinine. Vastly increased excretion of a1-ACT observed by ELISA-based measurement, this was also confirmed by Western blot analysis. The enhanced excretion of a1-ACT in prion infections is surprising. In view of the unaltered plasma a1-ACT levels of sCJD patients, a1-ACT ‘‘spillover’’ from blood to urine is unlikely to underlie our observations. We therefore considered, and attempt- ed to control for, possible artifactual effects. First, we considered that increased a1-ACT excretion may result from glomerular dysfunction. However, normalization of data to either urinary creatinine or albumin, which would be increased in many renal 3.6 nd 6.7 nd 5.5 12* nd 4 nd 10.3* 7.4 3.6 6.1 5.5 3.6 nd nd nd 4.5 Prion Disease Biomarker diseases, did not affect the results. Secondly, urinary cystatin C, a marker of glomerular filtration rate [35], was unaltered in sCJD patients, suggesting that increased a1-ACT excretion does not result simply from compromised renal function. Thirdly, elevated a1-antichymotrypsin levels in sCJD urine are unlikely to result from proximal tubule defects, since urinary a1-microglobulin levels were not significantly different in urine of sCJD patients relative to respective controls. Fourthly, our retrospective analyses of sCJD patient plasma creatinine and urea indicate that renal insufficiency is not a typical feature of late-stage sCJD patients (Table 4). Finally, urinary a1-ACT levels were significantly higher in sCJD than (1) in post-kidney transplant subjects following diabetic nephropathy, focal segmental glomerulosclerosis, and reflux nephropathy, (2) in patients with bladder carcinoma, and (3) patients with pyogenic nephritis leading to bacteriuria. These aggregated data argue against increased levels of a1-ACT in urine from sCJD patients simply resulting from renal disease, or from generally compromised health. We considered that enhanced a1-ACT detection may result from sample collection or processing anomalies. We deem this unlikely for a number of reasons. Firstly, high urinary a1-ACT was diagnostic in sCJD patient collectives from each of three separate national reference centers. Secondly, no alterations of a1-ACT concentration in test urine were found when comparing voided vs. non-voided (P.0.05) urine, pre- vs. post-pellet urine (P.0.05), and urine samples collected in the morning or evening (P.0.05). Thirdly, the a1-ACT ELISA yielded similar results whether urines was stored immediately at 280uC or left at room temperature or 4uC overnight before testing (P.0.05). Therefore, none of the common sample collection and processing artifacts account for the reported results. Finally, elevated urinary a1-ACT was evident in a number of BSE-affected cattle, CWD-affected deer, and scrapie-affected sheep. In the latter, it was diagnostic at least 2 months prior to terminal disease. Enhanced a1-ACT excretion may prove useful for correlating responsiveness to experimental therapeutic regimens and perhaps for biochemical discrimination of vCJD from sCJD without invasive biopsies. Finally, considering its excellent specificity and good sensitivity, one might consider exploring the usefulness of urinary a1-ACT determinations as a means of deferring blood units from asymptomatic donors who may be harboring preclinical prion infections. Materials and Methods Prion inoculations Mice were maintained under specific pathogen-free conditions. Eight week-old inbred C57Bl/6 mice were inoculated intraperi- toneally with 100 ml (6 log LD50 units) of the Rocky Mountain laboratory (RML) scrapie strain (passage 5 in CD1 mice, hence called RML5) prepared as a 10% (w/v) clarified brain homoge- nate containing 5% BSA. The titer of this standard RML5 inoculum was 8.9 log LD50 g21 of brain tissue [36]. Age- and gender-matched control mice were inoculated intraperitoneally with 100 ml of a 10% (w/v) clarified brain homogenate from healthy CD1 mice. The incubation period of this model until onset of terminal disease was approximately 200–210 days post inoculation (dpi). Tissues from mice at 45, 100, 145 and 190 dpi stages of pathogenesis were used for real-time quantitative RT- PCR analyses. All manipulations were approved by the Animal Experimentation Committee of the Canton of Zu¨ rich. Tissue collection and RNA isolation Human occipital cortex tissue was provided via the Swiss Reference Center for Prion Diseases (NRPE) and was collected at 217 222 223 228 233 234 238 239 240 246 247 248 249 257 260 262 268 270 271 sCJD status Definite Definite Probable Definite Definite Definite Definite Definite Probable Definite Definite Definite Definite Probable Definite Definite Definite Probable Definite 58 113 124* 71 70 81 92 52 85 95 75 96 70 88 78 72 74 61 100 PLoS ONE | www.plosone.org 9 December 2008 | Volume 3 | Issue 12 | e3870