Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay

Background Variant Creutzfeldt-Jakob disease (vCJD) has a pathogenesis dist inct from other forms of human prion disease: disease-related prion protein (PrPSc) is readily detectable in lymphoreticular tissues. Quantitation of risk of secondary transmission, and targeting of risk reduction strategies, is limited by lack of knowledge about relative prion titres in these and o ther peripheral tissues, the unknown prevalence of preclinical vCJD, and a transmission barrier which limits the sensitivity of bioassay. We aimed to improve immunoblotting methods for high sensitivity detection of PrPSc to i nvestigate the distribution of PrPSc in a range of vCJD tissues. Methods We obtained tissues at necropsy from four patients with neuropathol ogically confirmed vCJD and from individuals without neurological disease. Tissues were analysed by sodium phosphotungstic acid precipitation of PrPSc and western blotting using high sensitivity enhanced chemiluminescence. Findings We could reliably detect PrPSc in the equivalent of 50 nL 10% vCJD brain homogenate, with a maximum limit of detection equivalent to 5 nl. Pr PSc could be detected in tissue homogenates when present at concentrations 10(4)-10(5) fold lower than those reported in brain. Tonsil, spleen, and ly mph node were uniformly positive for PrPSc at concentrations in the range o f 0.1-15% of those found in brain: the highest concentrations were consiste ntly seen in tonsil. PrPSc was readily detected in the retina and proximal optic nerve of vCJD eye at levels of 2.5 and 25%, respectively of those fou nd in brain. Other peripheral tissues studied were negative for PrPSc with the exception of low concentrations in rectum, adrenal gland, and thymus fr om a single patient with vCJD. vCJD appendix and blood (Buffy coat fraction ) were negative for PrPSc at this level of assay sensitivity. Interpretation We have developed a highly sensitive immunoblot method for d etection of PrPSc in vCJD tissues that can be used to provide an upper limi t on PrPSc concentrations in peripheral tissues, including blood, to inform risk assessment models. Rectal and other gastrointestinal tissues should b e further investigated to assess risk of iatrogenic transmission via biopsy instruments. Ophthalmic surgical instruments used in procedures involving optic nerve and the posterior segment populations; of the eye, in particula r the retina, might represent a potential risk for iatrogenic transmission of vCJD. Tonsil is the tissue of choice for diagnostic biopsy and for popul ation screening of surgical tissues to assess prevalence of preclinical vCJ D infection within the UK and other populations.