The paired skin and CSF samples from 29 patients with probable sCJD, gPrD or non-CJD were obtained from the tissue bank in the Center of Chinese CJD Surveillance System, including 12 sCJD, 1 T188K gCJD, 1 G114V gCJD, 1 D178N FFI, 9 non-CJD and 5 pending CJD cases. Among them, 4 patients provided two skin samples collected from different sites. The demography information of the patients, the clinical data, MRI and EEG data, the results of the Western blot for CSF 14-3-3 and PRNP sequencing were collected from the database of the Center of Chinese CJD Surveillance System.
In addition, 10% skin homogenates of neuropathologically-confirmed 10 sCJD patients and 5 non-CJD controls from the National Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University School of Medicine, Cleveland, Ohio, USA, were also enrolled in this study for validation of our skin RT-QuIC assay. All 5 non-CJD subjects were determined by neuropathological assays with the postmortem brains, which excluded the possibilities of sCJD or other human prion diseases but did not have the diagnosis for other neurological diseases.
All enrolled CSF samples were obtained by lumbar puncture and were free of blood contamination. Routine CSF biochemistry assays of those specimens, including cell count, glucose and total protein were all in the normal ranges.
Preparation of homogenates of skin
The sites for skin biopsies in this study included the skins behind the ears, inside the arms, inside the thighs, lower back, and/or abdomen. After disinfection with 75% alcohol, the patient received local anesthesia with subcutaneous injection of 1-2% lidocaine hydrochloride. A small piece of skin with a size of about 0.2 x 0.3 cm2 was taken with a disposable skin biopsy punch (Acupunch, Acuderm Inc, US), according to the manufacturer’s instruction. Usually, the biopsy skin specimen covers epidermis, dermis, and adipose tissues. 2% (w/v) of skin homogenate was prepared in lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 10 mM Tris, pH7.5) according to a previously described protocol[11, 12]. The homogenates were then stored at -80 ° C for further used.
The detail procedures of the RT-QuIC assay was described previously. Briefly RT-QuIC reaction contained 10 µg of rHaPrP90-231, 1X PBS, 170 mM NaCl, 1 mM EDTA, 0.01 mM ThT, 0.001% SDS, together with 15 µl CSF samples or 2 µl 10-2 to 10-4 diluted skin homogenates in a final volume of 100 µl. Each sample was assayed in triplicated or quadruplicated. The assay was conducted in a black 96-well, optical-bottomed plate (Nunc, 265301) on a BMG FLUOstar plate reader (BMG LABTECH). The main working conditions were fixed as follow: temperature, 55˚C; vibration speed, 700 rpm; vibration/incubation time, 60/60 sec; total reaction time, 60 h. ThT fluorescence (excitation wavelength, 450 nm; emission wavelength, 480nm) each reaction was automatically measured every 45 min and expressed as relative fluorescence units (rfu). The cutoff value was set as the mean value of the negative controls plus 10 times the standard deviation. A sample was considered to be positive when ≥2 wells revealed positive reaction curves. The positive control was 10-5 diluted the brain homogenate of the scrapie agent 263K-infected hamster, while the negative control was 10-5 diluted the brain homogenate of normal hamster.
Usages of the CSF and skin samples and relevant clinical information of the patients with different diseases in the Center of Chinese CJD Surveillance System has been approved by the Ethics Committee of the National Institute for Viral Disease Control and Prevention, China CDC, under the protocol of 2009ZX10004-101.