Seeding amplification assay with Universal Control Fluid: Standardized detection of α-synucleinopathies

Abstract

Seeding amplification assays, specifically the Real-Time Quaking-Induced Conversion method (RT-QuIC), have shown great diagnostic potential for α-synucleinopathies. Numerous research groups have demonstrated the method's high sensitivity and specificity using cerebrospinal fluid (CSF) samples and various RT-QuIC workflows. However, establishing a uniform and stably performing RT-QuIC protocol remains challenging. To address this, we established an RT-QuIC protocol with a Universal Control Fluid (UCF), which is simple to adopt, performs stably, and allows uniform preparation of both sample and control reactions. Firstly, we adapted and established a published 48-hour RT-QuIC protocol, including the in-house production of recombinant α-synuclein (rec α-syn), and evaluated its sensitivity and specificity through a blinded screening of an 81 CSF sample cohort consisting of Parkinson's disease (PD), dementia with Lewy bodies (DLB), Alzheimer's disease, motor neuron disease, multiple system atrophy, unidentified neurodegenerative diseases, and healthy controls. Additionally, we tested all CSF samples in three volumes to determine which volume provides the best diagnostic accuracy. The established RT-QuIC performs nearly equally well with 7 µL and 15 µL added CSF, resulting in 94% and 94.5% diagnostic accuracy, respectively. Secondly, we developed a UCF solution and tested its performance with the established RT-QuIC protocol. Results indicate that UCF, used in defined volume and concentration, standardizes the preparation of both sample and control reactions without compromising the assay's diagnostic accuracy and provides a stabilizing environment for the reactions, ensuring higher reproducibility. The established RT-QuIC protocol for pathologic α-synuclein detection in PD and DLB CSF samples is highly sensitive (92-96%) and specific (93-96%). Therefore, it is important that its adoption in clinical laboratories is uncomplicated and uniform. RT-QuIC with UCF simplifies, standardizes, and stabilizes the assay's performance and, thus, could be recommended as a standard protocol for accurate detection of α-synucleinopathies.

Fig 1. Kinetics of rec αSyn batches used in this study.

Fig 1 Kinetics of the 6 different in-house produced rec αSyn batches. Each batch was tested with 24 unseeded (dotted lines indicating mean RFU and 95% CI) and 24 seeded (continuous lines indicating mean RFU and 95% CI) reactions. Each batch is labeled with a letter (A to F) and color coded as listed in the figure legend.

Fig 2. Distribution of samples based on clinical diagnosis, added CSF volume and Area Under the Curve values.

Fig 2 Distribution of study cohort samples (from Table 1) based on their average Area Under the Curve value (AUC) and the volume of Cerebrospinal Fluid (CSF) added to the RT-QuIC reaction. Each sample group represents a different clinical diagnosis color coded as listed in the figure legend. DLB – Dementia with Lewy Bodies; PD - Parkinson’s Disease; MSA* - both Parkinsonian and Cerebellar Multiple System Atrophy samples, AD - Alzheimer’s Disease, MND – Motor Neuron Disease, and uNDD - unidentified neurodegenerative diseases; HC – healthy controls.

Fig 3. BLB BH seeded and unseeded 2xUCF and 4xUCF performance.

Fig 3 Distribution of 12 replicates based on their Area Under the Curve value (AUC) in unseeded (red) and DLB BH seeded (green) RT-QuIC reactions with different universal control fluid (UCF) volume and concentration combinations.

Fig 4. Linear correlation and kinetics of PD/DLB CSF dilutions in UCF and Area Under the Curve values.

Fig 4 a) Linear correlation between αSyn^D positive PD/DLB CSF sample dilutions in UCF and the AUC values; b) kinetics of the PD/DLB CSF samples diluted in UCF. Legend: The CSF/UCF ratio, ranging from 7 µL neat PD/DLB CSF to 7 µL neat UCF added to 93 µL master mix, is color coded as indicated in the figure legend and applies to both graph a) and b). UCF – Universal Control Fluid, AUC – Area Under the Curve value.

Fig 5. Comparison of diluted DLB BH with and without UCF.

Fig 5 Comparison of DLB BH serial 10-fold dilutions in 7 µL 4xUCF and in 2 µL water. a) Linear regression between DLB BH dilutions in UCF and in water, and relative fluorescence unit (RFU). The coefficient of determination (R²) and color code for the comparison of linear regressions is as indicated in the figure legend; b) kinetics of DLB BH dilutions in UCF; c) kinetics of DLB BH dilutions in water.

Fig 6. Comparison of diluted PFF with and without UCF. Fig 6 Comparison of serial 10-fold dilutions of pre-formed αSyn fibrils (dPFF) in 7 µL 4xUCF and in 2 µL water.

a) Linear regression between serial dilutions of pre-formed αSyn fibrils in UCF and in water, and relative fluorescence unit (RFU). The coefficient of determination (R²) and color code for the comparison of linear regressions is as indicated in the figure legend; b) kinetics of pre-formed αSyn fibrils dilutions with UCF; dilution factors and their color codes are indicated in the figure legend.