Thi-Thu-Trang Dong1, Akio Akagi2, Toshiaki Nonaka3, Takehiro Nakagaki4, Ban Mihara5, Masaki Takao6, Yasushi Iwasaki7, Noriyuki Nishida4, Katsuya Satoh8. 1. Department of Health Sciences, Unit of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Japan. 2. Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Aichi 480-1195, Japan. 3. Department of Health Sciences, Unit of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Japan; Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8501, Japan. 4. Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8501, Japan. 5. Department of Neurology, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Isesaki 372-0006, Japan. Electronic address: ban.mihara@mihara-ibbv.jp. 6. Department of Neurology, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Isesaki 372-0006, Japan; Department of Neurology International Medical Center, Saitama Medical University, Saitama 350-1298, Japan; Department of Clinical Laboratory National Center of Neurology and Psychiatry (NCNP), National Center Hospital, 4-1-1 Ogawa-higashi-cho, Kodaira, Tokyo 187-8551, Japan. 7. Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Aichi 480-1195, Japan. Electronic address: iwasaki@sc4.so-net.ne.jp. 8. Department of Health Sciences, Unit of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Japan. Electronic address: satoh-prion@nagasaki-u.ac.jp.
Abstract
BACKGROUND: The neuropathology of sporadic Creutzfeldt-Jakob disease (sCJD) is usually investigated using formalin-fixed and formic acid-treated brain tissue. However, formalin and formic acid treatment can interfere with immunostaining of abnormal prion protein. Therefore, there is a need for biochemical methods other than immunostaining to investigate abnormal prion protein in postmortem tissue. We developed RT-QuIC to quantitate the seeding activity (SD50) of sCJD brain tissue treated with formalin and formic acid. METHODS: We used endpoint RT-QuIC assays to analyze SD50 in formalin-fixed brain tissue from 19 sCJD patients (14 MM1 cases, 3 MM2-thalamic form [MM2T] cases and 2 MM2-cortical form [MM2C] cases) diagnosed according to Parchi's classification. We assessed SD50 in brains after incubation in formalin solution for over 1 month, and after treating formalin-fixed brain tissue with formic acid. We also examined how the SD50 values from formalin-fixed brain samples compared with neuropathological and immunohistochemical findings. RESULTS: The SD50 values of formalin-fixed brain samples from 14 MM1 cases, 2 MM2C cases, and 2 MM2T cases were 107.77±0.57/g tissue, 107.44±0.24/g tissue and 106.00±0.77/g tissue, respectively. The average SD50 value in MM1 unfixed brains decreased by 102.04 after formalin fixation for 1 month. In MM1 cases, after combined formalin and formic acid treatment, the SD50 value was reduced by approximately 105.16 compared with that of unfixed tissue. The SD50 values of formalin-fixed tissue showed a consistent pattern with the neuropathological findings in most brain regions examined. CONCLUSION: RT-QuIC enables the study of formalin-fixed brain tissue from sCJD patients that has not previously been amenable to analysis.
BACKGROUND: The neuropathology of sporadic Creutzfeldt-Jakob disease (sCJD) is usually investigated using formalin-fixed and formic acid-treated brain tissue. However, formalin and formic acid treatment can interfere with immunostaining of abnormal prion protein. Therefore, there is a need for biochemical methods other than immunostaining to investigate abnormal prion protein in postmortem tissue. We developed RT-QuIC to quantitate the seeding activity (SD50) of sCJD brain tissue treated with formalin and formic acid. METHODS: We used endpoint RT-QuIC assays to analyze SD50 in formalin-fixed brain tissue from 19 sCJD patients (14 MM1 cases, 3 MM2-thalamic form [MM2T] cases and 2 MM2-cortical form [MM2C] cases) diagnosed according to Parchi's classification. We assessed SD50 in brains after incubation in formalin solution for over 1 month, and after treating formalin-fixed brain tissue with formic acid. We also examined how the SD50 values from formalin-fixed brain samples compared with neuropathological and immunohistochemical findings. RESULTS: The SD50 values of formalin-fixed brain samples from 14 MM1 cases, 2 MM2C cases, and 2 MM2T cases were 107.77±0.57/g tissue, 107.44±0.24/g tissue and 106.00±0.77/g tissue, respectively. The average SD50 value in MM1 unfixed brains decreased by 102.04 after formalin fixation for 1 month. In MM1 cases, after combined formalin and formic acid treatment, the SD50 value was reduced by approximately 105.16 compared with that of unfixed tissue. The SD50 values of formalin-fixed tissue showed a consistent pattern with the neuropathological findings in most brain regions examined. CONCLUSION: RT-QuIC enables the study of formalin-fixed brain tissue from sCJD patients that has not previously been amenable to analysis.