AIMS: Formalin-fixed paraffin-embedded (FFPE) samples, routinely used in neuropathology, represent an invaluable resource for studying rare diseases like transmissible spongiform encephalopathies (TSE). Despite fixation-induced protein cross-linking, prion seeding activity can be effectively detected using the seeding amplification assays. In this study, we employed the second-generation real-time quaking-induced conversion (RT-QuIC) assay to analyse and quantify human prion seeding activity in FFPE brain tissues. METHODS: FFPE frontal brain tissues were deparaffinised in xylene, followed by rehydration through descending concentrations of ethanol. The prion seeding activity in tissue homogenates was assessed by RT-QuIC assay utilising short recombinant hamster prion protein (rHaPrP90-231) as a substrate. RESULTS: A total of 60 samples, including 30 cases of confirmed TSE, comprising both sporadic and genetic forms, as well as 30 non-TSE controls, were analysed. Prion seeding activity has been detected in all TSE samples except one sCJD (VV2) and one GSS (P102L) case, corresponding to an assay sensitivity of 93.3%. Conversely, we did not detect any RT-QuIC positivity in the control group, resulting in 100% specificity. The mean 50% prion seeding dose of FFPE sporadic TSE samples was 107.8/g of brain tissue. CONCLUSION: Our study emphasises high sensitivity and specificity of RT-QuIC assay for prion detection in archival human FFPE brain tissues and demonstrates its diagnostic reliability comparable to other tissue types even after years of storage. The applicability of FFPE samples in RT-QuIC assays facilitates retrospective diagnostics and provides logistical advantages for sample preservation and transportation.

• Keywords
• FFPE, RT‐QuIC, SAA, formalin‐fixed paraffin‐embedded, prion, prion disease, real‐time quaking‐induced conversion assay, seeding amplification assay,
• MeSH
• Tissue Fixation methods   MeSH
• Formaldehyde MeSH
• Humans MeSH
• Brain * pathology   metabolism   MeSH
• Prion Proteins MeSH
• Prion Diseases * pathology   diagnosis   metabolism   MeSH
• Prions * metabolism   MeSH
• Paraffin Embedding methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Formaldehyde MeSH
• Prion Proteins MeSH
• Prions * MeSH

Real-time quaking-induced conversion assay (RT-QuIC) exploits templating activity of pathogenic prion protein for ultrasensitive detection of prions. We have utilized second generation RT-QuIC assay to analyze matching post-mortem cerebrospinal fluid and skin samples of 38 prion disease patients and of 30 deceased neurological controls. The analysis of cerebrospinal fluid samples led to 100% sensitivity and 100% specificity, but some samples had to be diluted before the analysis to alleviate the effect of present RT-QuIC inhibitors. The analysis of the corresponding skin samples provided 89.5% sensitivity and 100% specificity. The median seeding dose present in the skin was one order of magnitude higher than in the cerebrospinal fluid, despite the overall fluorescent signal of the skin samples was comparatively lower. Our data support the use of post-mortem cerebrospinal fluid for confirmation of prion disease diagnosis and encourage further studies of the potential of skin biopsy samples for intra-vitam prion diseases´ diagnostics.

• MeSH
• Biological Assay MeSH
• Creutzfeldt-Jakob Syndrome * diagnosis   cerebrospinal fluid   MeSH
• Skin metabolism   MeSH
• Humans MeSH
• Prion Proteins MeSH
• Prion Diseases * diagnosis   MeSH
• Prions * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Prion Proteins MeSH
• Prions * MeSH