Formalin RT-QuIC Assay: A Novel Method to Detect Amyloid-Seeding Activity in Formalin-Fixed Brain Samples From Sporadic Creutzfeldt–Jakob Disease Patients

Background: Neuropathology in sporadic Creutzfeldt–Jakob disease (sCJD) brains is usually investigated using formalin-fixed brains and formic acid-treated brains. However, brain tissues prepared in these ways are not amenable to biochemical analyses. We therefore developed RT-QuIC to quantitate the seeding activity (SD50) of sCJD brains. Methods: We used endpoint RT-QuIC assays to analyse SD50 in formalin-fixed brain tissue from 19 sCJD patients (14 MM1 cases, 3 MM2T cases and 2 MM2C cases) according to Parchi’s classification. We estimated SD50 in brains after incubation in formalin solution for over 1 month, and checked SD50 after treating formalin-fixed brain samples with formic acid. We also compared the neuropathological findings with SD50 from formalin-fixed brain samples from MM1, MM2T, and MM2C patients. Findings: RT-QuIC enables the study of formalin-fixed brains in sCJD patients that have not previously been amenable to analysis. Interpretation: The SD50 values of formalin-fixed brain samples from fourteen MM1 cases, two MM2C cases, and two MM2T cases were 107.77±0.57/g tissue, 107.44±0.24/g tissue and 106.00±0.77/g tissue, respectively. The SD50 values decreased by 102.04 after formalin fixation for 1 month. After combined formalin and formic-acid treatment, the SD50 value was reduced by approximately 105.16 compare with native brain in MM1 cases. We identified some relationships between SD50 of formalin-fixed brain samples and neuropathological findings. Registration: The study was registered with the University Hospital Medical Information Network (ID: UMIN000003301) and (ID: UMIN000038398). Funding Statement: This study was financially supported by grants for scientific research from the Ministry of Health, Labour and Welfare of Japan (KSat: No. 14507303), the Research Committee of Prion Disease and Slow Virus Infection, Research on Policy Planning and Evaluation for Rare and Intractable Diseases, Health and Labour Sciences Research Grants, the Research Committee of Surveillance and Infection Control of Prion Disease, the Ministry of Health, Labour, and Welfare of Japan, and the Japan Agency for Medical Research and Development (AMED) (grant No. 18ek0109362h0001). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The protocol for investigation was approved by the Ethics Committee of Nagasaki University Hospital (ID: 10042823). The protocol was also granted ethical approval for the use of brain tissues by the Japan Surveillance Unit for human prion diseases.