Abstract
Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein (PrPSc). 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 PrPSc 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-015-9133-2) contains supplementary material, which is available to authorized users.
Highlights
Transmissible spongiform encephalopathies or prion diseases are characterized by the aggregation and accumulation of misfolded scrapie prion protein (PrPSc) in brain tissue
Genetic CJD cases showed a sensitivity of 100 %, while analysis of Sporadic Creutzfeldt-Jakob disease (sCJD) and FFI cerebrospinal fluid (CSF) samples revealed a sensitivity of 80 and 57 %, respectively (Fig. 1c)
CSF sample 1 was from a patient originally clinically diagnosed as AD, while for sample 2, the diagnosis was still outstanding at the time of Real-time quaking-induced conversion (RT-QuIC) analysis
Summary
Transmissible spongiform encephalopathies or prion diseases are characterized by the aggregation and accumulation of misfolded scrapie prion protein (PrPSc) in brain tissue. They can occur spontaneously (sporadic) and be due to familial and iatrogenic causes. The adaptation of in vitro amplification systems for the detection of PrPSc in human cerebrospinal fluid (CSF) was an innovation for the pre-mortem diagnosis. RT-QuIC analysis uses recombinant prion protein (recPrP) as a substrate to amplify very small amounts of PrPSc seed in human CSF to detectable levels. Four previous studies demonstrated the diagnostic potential of RT-QuIC through amplification of PrPSc from human CSF, derived from sCJD and gCJD patients [4,5,6,7]
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