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Seeding amplification assay with Universal Control Fluid: Standardized detection of α-synucleinopathies
Remarh Bsoul1, Anja H Simonsen2, Kristian S Frederiksen2,3
1Danish Reference Center for Prion Diseases, Department of Pathology, Copenhagen University Hospital, Copenhagen, Denmark.
Plos One
|June 25, 2025
Summary
Real-Time Quaking-Induced Conversion (RT-QuIC) assays show promise for diagnosing alpha-synucleinopathies. A new protocol using Universal Control Fluid (UCF) standardizes and stabilizes RT-QuIC, improving accuracy for detecting Parkinson's disease and dementia with Lewy bodies.
Area of Science:
- Neuroscience
- Biochemistry
- Medical Diagnostics
Background:
- Seeding amplification assays, particularly Real-Time Quaking-Induced Conversion (RT-QuIC), offer high sensitivity and specificity for diagnosing alpha-synucleinopathies using cerebrospinal fluid (CSF).
- Standardization and stable performance of RT-QuIC protocols across different laboratories remain a significant challenge, hindering widespread clinical adoption.
Purpose of the Study:
- To establish a simplified, standardized, and stably performing RT-QuIC protocol for the detection of pathological alpha-synuclein in CSF.
- To evaluate the diagnostic accuracy of the established RT-QuIC protocol using a blinded cohort of CSF samples.
- To assess the impact of a novel Universal Control Fluid (UCF) on assay standardization, stability, and diagnostic performance.
Main Methods:
- Adapted and established a 48-hour RT-QuIC protocol, including in-house production of recombinant alpha-synuclein.
- Evaluated sensitivity and specificity using a blinded screening of 81 CSF samples from patients with various neurodegenerative diseases and healthy controls.
- Tested CSF samples in three different volumes (7 µL and 15 µL) and assessed the performance of a newly developed Universal Control Fluid (UCF).
Main Results:
- The established RT-QuIC protocol demonstrated high diagnostic accuracy (94% with 7 µL and 94.5% with 15 µL CSF).
- The Universal Control Fluid (UCF) standardized reaction preparation and stabilized the assay without compromising diagnostic accuracy, enhancing reproducibility.
- The optimized RT-QuIC protocol achieved high sensitivity (92-96%) and specificity (93-96%) for detecting pathological alpha-synuclein in Parkinson's disease and dementia with Lewy bodies CSF samples.
Conclusions:
- The developed RT-QuIC protocol, incorporating Universal Control Fluid (UCF), simplifies, standardizes, and stabilizes the assay for pathological alpha-synuclein detection.
- This standardized protocol demonstrates excellent diagnostic performance for alpha-synucleinopathies, including Parkinson's disease and dementia with Lewy bodies.
- The RT-QuIC with UCF approach is recommended for adoption in clinical laboratories to ensure uncomplicated and uniform implementation for accurate diagnosis.
