A fixed brain seeded amplification assay to complement neuropathological prion disease diagnosis

Prion diseases are rare neurodegenerative disorders that share misfolding of the normal cellular prion protein into disease-causing isoforms known as "prions" as the critical pathophysiological event. Definite diagnosis can only be achieved through neuropathological confirmation. The neuropathological features of prion disease are well described; however, some molecular subtypes are typified by characteristic neuropathological features that are subtle or absent. Prion seeding assays have excellent specificity and have considerably improved premortem diagnostic accuracy but they have reduced sensitivity for some uncommon prion disease molecular subtypes. We developed a formalin-fixed, paraffin-embedded tissue-based prion seeding assay to serve as a complementary diagnostic tool for prion diseases. Fixed brain tissue was prepared through an optimized process involving careful defacing of tissue blocks prior to sampling and then stepwise deparaffinization and homogenization. Fixed tissue homogenates are then tested in an adapted version of a diagnostic cerebrospinal fluid (CSF) prion seeding assay, which utilizes full-length recombinant hamster prion protein as substrate. Two examples illustrate the utility of the assay by confirming prion seeding in fixed brain tissue from previously neuropathologically misdiagnosed obligate carriers of 2 different prion protein gene mutations. The importance of careful tissue sampling to rigorously maintain the diagnostic specificity of this assay is also highlighted.