Enhanced quantitation of pathological α-synuclein in patient biospecimens by RT-QuIC seed amplification assays

Disease associated pathological aggregates of alpha-synuclein (αSynD) exhibit prion-like spreading in synucleinopathies such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Seed amplification assays (SAAs) such as real-time quaking-induced conversion (RT-QuIC) have shown high diagnostic sensitivity and specificity for detecting proteopathic αSynD seeds in a variety of biospecimens from PD and DLB patients. However, the extent to which relative proteopathic seed concentrations are useful as indices of a patient’s disease stage or prognosis remains unresolved. One feature of current SAAs that complicates attempts to correlate SAA results with patients’ clinical and other laboratory findings is their quantitative imprecision, which has typically been limited to discriminating large differences (e.g. 5–10 fold) in seed concentration. We used end-point dilution (ED) RT-QuIC assays to determine αSynD seed concentrations in patient biospecimens and tested the influence of various assay variables such as serial dilution factor, replicate number and data processing methods. The use of 2-fold versus 10-fold dilution factors and 12 versus 4 replicate reactions per dilution reduced ED-RT-QuIC assay error by as much as 70%. This enhanced assay format discriminated as little as 2-fold differences in αSynD seed concentration besides detecting ~2-16-fold seed reductions caused by inactivation treatments. In some scenarios, analysis of the data using Poisson and midSIN algorithms provided more consistent and statistically significant discrimination of different seed concentrations. We applied our improved assay strategies to multiple diagnostically relevant PD and DLB antemortem patient biospecimens, including cerebrospinal fluid, skin, and brushings of the olfactory mucosa. Using ED αSyn RT-QuIC as a model SAA, we show how to markedly improve the inter-assay reproducibility and quantitative accuracy. Enhanced quantitative SAA accuracy should facilitate assessments of pathological seeding activities as biomarkers in proteinopathy diagnostics and prognostics, as well as in patient cohort selection and assessments of pharmacodynamics and target engagement in drug trials.Author summary: Seed amplification assays (SAAs) such as RT-QuIC detect pathological α-Syn aggregates with prion-like self-propagating (seeding) activity from various tissue biospecimens of synucleinopathy patients. However, clinical applications of current SAAs to neurodegenerative diseases can be hampered by their quantitative imprecision in stratifying levels of pathological seeds as biomarkers. In this study, we tested the influence of various assay variables including dilution factor, number of replicates, and quantitation methods in end-point dilution RT-QuIC (ED RT-QuIC) using PD and DLB patient samples, specifically brain tissue, CSF, skin, and nasal brushings. Our study highlights how assay design can markedly improve seed quantification in clinical samples. Better proteopathic seed quantification should enable more precise evaluation of pathological seeding activity to support important clinical and research applications.