title: Stability and Reproducibility Underscore Utility of RT-QuIC for Diagnosis of Creutzfeldt-Jakob Disease.
abstract: Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein (PrP(Sc)). Recent studies applied the RT-QuIC methodology to cerebrospinal fluid (CSF) for diagnosing human prion diseases. However, to date, there has not been a formal multi-centre assessment of the reproducibility, validity and stability of RT-QuIC in this context, an indispensable step for establishment as a diagnostic test in clinical practice. In the present study, we analysed CSF from 110 prion disease patients and 400 control patients using the RT-QuIC method under various conditions. In addition, "blinded" ring trials between different participating sites were performed to estimate reproducibility. Using the previously established cut-off of 10,000 relative fluorescence units (rfu), we obtained a sensitivity of 85% and a specificity of 99%. The multi-centre inter-laboratory reproducibility of RT-QuIC revealed a Fleiss' kappa value of 0.83 (95% CI: 0.40-1.00) indicating an almost perfect agreement. Moreover, we investigated the impact of short-term CSF storage at different temperatures, long-term storage, repeated freezing and thawing cycles and the contamination of CSF with blood on the RT-QuIC seeding response. Our data indicated that the PrP(Sc) seed in CSF is stable to any type of storage condition but sensitive to contaminations with blood (>1250 erythrocytes/μL), which results in a false negative RT-QuIC response. Fresh blood-contaminated samples (3 days) can be rescued by removal of erythrocytes. The present study underlines the reproducibility and high stability of RT-QuIC across various CSF storage conditions with a remarkable sensitivity and specificity, suggesting RT-QuIC as an innovative and robust diagnostic method.
authors.affiliation: Department of Neurology, University Medical Center Goettingen and German Center for Neurodegenerative Diseases (DZNE)-site Goettingen, Robert-Koch Str. 40, 37075, Göttingen, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Cramm
authors.affiliation: Department of Neurology, University Medical Center Goettingen and German Center for Neurodegenerative Diseases (DZNE)-site Goettingen, Robert-Koch Str. 40, 37075, Göttingen, Germany. matthias.schmitz@med.uni-goettingen.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schmitz
authors.affiliation: Department of Neurology, University Medical Center Goettingen and German Center for Neurodegenerative Diseases (DZNE)-site Goettingen, Robert-Koch Str. 40, 37075, Göttingen, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Karch
authors.affiliation: Department of Prion Diseases, Slovak Medical University Bratislava, Limbová 14, 833-03, Bratislava, Slovakia.
authors.forename: Eva
authors.initials: E
authors.lastname: Mitrova
authors.affiliation: Thermo Fisher Scientific, Prionics AG, 8952, Schlieren, Switzerland.
authors.forename: Franziska
authors.initials: F
authors.lastname: Kuhn
authors.affiliation: Thermo Fisher Scientific, Prionics AG, 8952, Schlieren, Switzerland.
authors.forename: Bjoern
authors.initials: B
authors.lastname: Schroeder
authors.affiliation: Thermo Fisher Scientific, Prionics AG, 8952, Schlieren, Switzerland.
authors.forename: Alex
authors.initials: A
authors.lastname: Raeber
authors.affiliation: Department of Neurology, University Medical Center Goettingen and German Center for Neurodegenerative Diseases (DZNE)-site Goettingen, Robert-Koch Str. 40, 37075, Göttingen, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Varges
authors.affiliation: Ilsong Institute of Life Science, College of Medicine, Hallym University, Anyang, Republic of Korea.
authors.forename: Yong-Sun
authors.initials: YS
authors.lastname: Kim
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-12-4, Nagasaki, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Satoh
authors.affiliation: Department of Pathology, The University of Melbourne, Parkville, 3010, Australia.
authors.forename: Steven
authors.initials: S
authors.lastname: Collins
authors.affiliation: Department of Neurology, University Medical Center Goettingen and German Center for Neurodegenerative Diseases (DZNE)-site Goettingen, Robert-Koch Str. 40, 37075, Göttingen, Germany.
authors.forename: Inga
authors.initials: I
authors.lastname: Zerr
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001681:Biological Assay / Q000379:methods*; D007562:Creutzfeldt-Jakob Syndrome / Q000097:blood / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D011309:Preservation, Biological; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D055815:Young Adult; D016875:tau Proteins / Q000378:metabolism
chemical_list: D016875:tau Proteins
keywords: Cerebrospinal fluid; Creutzfeldt-Jakob disease; Diagnostic test; Prion protein; Real-time quaking-induced conversion
references.citation: Mol Med. 2000 Apr;6(4):291-302
references.pmid: 10949910
references.citation: Amyloid. 2000 Dec;7(4):245-58
references.pmid: 11132093
references.citation: Clin Lab Med. 2003 Mar;23(1):43-64
references.pmid: 12733424
references.citation: Ann Neurol. 1999 Aug;46(2):224-33
references.pmid: 10443888
references.citation: Cancer. 1950 Jan;3(1):32-5
references.pmid: 15405679
references.citation: Clin Chem. 2005 Jan;51(1):189-95
references.pmid: 15539465
references.citation: Mol Neurobiol. 2015 Feb;51(1):396-405
references.pmid: 24809690
references.citation: PLoS Pathog. 2010;6(12):e1001217
references.pmid: 21152012
references.citation: Nat Med. 2011 Feb;17(2):175-8
references.pmid: 21278748
references.citation: MBio. 2011;2(3):e00078-11
references.pmid: 21558432
references.citation: Ann Neurol. 2012 Aug;72(2):278-85
references.pmid: 22926858
references.citation: PLoS One. 2013;8(1):e54915
references.pmid: 23372790
references.citation: Hum Genet. 2005 Nov;118(2):166-74
references.pmid: 16187142