Prion Disease Biomarker Table 1. Plasma concentration of Cystatin C and a1-ACT in patients suffering from AD or sCJD. Patient age (years) Total protein (g/L) Cystatin C (mg/L) Cystatin C (ng/mg plasma protein) a1-ACT (mg/L) a1-ACT (mg/mg plasma protein) Control (n = 10) 70.90 (1.98) 65.79 (1.75) 1310.26 (91.64) 19.93 (1.23) 978.42 (61.30) 14.80 (0.67) AD (n = 10) 77.40 (2.12) 66.16 (2.40) 1394.46 (59.00) 21.15 (0.71) 931.08 (88.28) 14.15 (1.24) sCJD (n = 10) 69.00 (4.98) 64.31 (2.52) 1381.25 (108.51) 21.65 (1.68) 1000.59 (96.48) 15.86 (1.85) Plasma levels of cystatin C and a1-ACT. Cystatin C and a1-ACT levels were measured in plasma of sCJD patients, as well as in age and gender-matched collectives of healthy persons and AD patients by ELISA. Numbers in parenthesis represent s.e.m. doi:10.1371/journal.pone.0003870.t001 Table 2. Cystatin C and a1-ACT in patient cerebrospinal fluid. Patient age (years) Total protein (mg/L) Cystatin C (mg/L) Cystatin C (ng/mg CSF protein) a1-ACT (mg/L) a1-ACT (mg/mg CSF protein) Control A (n = 10) 66.20 (4.36) 871.58 (39.63) 3875.00 (362.90) 4471.19 (402.78) 7.67 (0.33) 8.90 (0.43) AD (n = 10) 67.44 (3.17) 771.94 (47.56) 3278.89 (345.50) 4275.30 (406.21) 7.17 (0.45) 9.30 (0.17) sCJD (n = 8) 66.50 (3.91) 917.14 (227.61) 3868.75 (333.85) 5275.04 (793.47) 11.86 (3.05) 12.98 (0.71)** Control B (n = 8) 37.25 (4.25) 500.87 (82.02) 4413.75 (642.03) 9781.25 (1254.68) 10.31 (2.86) 21.94 (3.85) vCJD (n = 10) 31.50 (2.44) 452.99 (61.08) 3113.00 (328.04) 8005.71 (1243.42) 6.97 (0.35) 17.41 (1.84) CSF levels of cystatin C and a1-ACT. Levels of cystatin C and a1-ACT were measured in patient CSF by ELISA and expressed as quantity per ml fluid or relative to total CSF protein. Values from sCJD patient CSF were compared to values obtained from CSF of both AD patients and healthy control groups of comparable age and gender distribution within groups (control group A). Since vCJD is typically prevalent in patients of a younger age, values for vCJD CSF were compared to those obtained from patients of comparable age (control group B). Numbers in parenthesis represent s.e.m. doi:10.1371/journal.pone.0003870.t002 determined by sandwich ELISA. Similarly to the CSF analysis reported above, urinary a1-ACT concentrations of sCJD patients were compared to those of AD patients and of control group A. We found dramatically elevated urinary a1-ACT in sCJD patients (Table 3, Figure 3). This was particularly evident when a1-ACT concentrations were normalized to albumin or to creatinine (p,0.001), but it reached significance even when raw concentra- tions were analyzed without any attempt at normalization (p,0.05). We considered several artifactual scenarios which may have led to the above observations. Since both cystatin C and a1-ACT are present in urine at concentrations far below those observed in Table 3. Cystatin C, a1-ACT and a1-microglobulin in the urine of patients. Control A (n = 10) AD (n = 9) sCJD (n = 29) Control B (n = 10) vCJD (n = 6) Control C (n = 13) Patient age (years) Cystatin C (mg/L) 73.50 (2.00) 43.18 (3.46) Cystatin C (mg/mg albumin) 6.21 (1.42) Cystatin C (mg/g Creatinine) 40.74 (6.44) a1-ACT (mg/L) a1-ACT (mg/mg albumin) a1-ACT (mg/g Creatinine) a1-microglobulin (mg/g Creatinine) a1-microglobulin (mg/g Creatinine) 131.21 (26.58) 888.73 (133.56) 13.17 (6.45) 3.11 (1.44) 80.20 (2.14) 46.80 (8.00) 5.67 (0.90) 40.59 (4.64) nd nd 67.52 (1.76) 74.88 (9.87) 4.18 (0.76) 91.18 (11.45) 53.86 (54.02) 4.40 (1.05) 23.50 (0.60) 62.78 (18.92) 7.00 (2.13) 79.13 (15.76) 5.83 (1.56) 0.44 (0.19) 28.67 (2.47) 120.29 (29.79) 15.00 (4.14) 80.28 (19.91) 52.69 (6.43) 69.86 (18.74) 7.52 (1.82) 70.70 (11.09) 37.33 (24.76) 3.23 (0.52)** nd nd 1008.68 (124.83) 913.01 (176.05) 16557.89 (3569.36)* 617.21 (26.11) 1431.98 (677.58) 2206.62 (849.88) 111.70 (21.56) 560.68 (84.62)** 78.10 (15.21) 122.83 (22.26) 213.93 (47.94)* 778.05 (108.90) 20200.2 (4503.22) ** 942.92 (84.48) 1959.31 (1534.83) 2125.57 (401.41) * Cystatin C and a1-ACT were measured in patient urine by ELISA and expressed as quantities per ml urine or relative to concentrations of albumin and creatinine. Values obtained from sCJD patient urine are directly comparable to values obtained from urine of AD patients and the healthy control group A consisting of patients of comparable age and gender distribution. Control group B represents a comparable control group for the vCJD measurements, consisting of patients initially suspected to have CJD but being later determined not to have CJD. Control group C consists of patients either pre-transplant with kidney disease, patients post-kidney transplant and patients with bladder carcinomas and nephritic inflammation. Numbers in parenthesis represent S.E.M. nd, not done. doi:10.1371/journal.pone.0003870.t003 PLoS ONE | www.plosone.org 4 December 2008 | Volume 3 | Issue 12 | e3870