Tear fluid as a novel specimen for detection of alpha-synuclein seeding activity in Parkinson’s disease

Parkinson's disease (PD) is a clinically heterogeneous neurodegenerative disorder characterized by intraneuronal accumulation of misfolded alpha-synuclein. Real-time quaking-induced conversion (RT-QuIC) detects seeding-competent alpha Syn species but commonly relies on invasive matrices. We evaluated tear fluid (TF) as a non-invasive biofluid for alpha Syn RT-QuIC in 44 PD patients and 32 matched healthy controls (HCs). ROC analyses were performed on participant-level readouts (mean of four technical replicates), and optimal cut-offs were selected using the Youden index before being applied to each technical replicate. At the subject level, the best trade-offs were observed at maximum fluorescence (MaxRFU) >= 2/4 (sensitivity 59.1%; specificity 65.6%), MedianRFU >= 3/4 (sensitivity 40.9%; specificity 78.1%), and area under fluorescence curve (AUFC) >= 3/4 (sensitivity 47.7%; specificity 71.9%). In a sensitivity analysis excluding RT-QuIC positive HCs with potential prodromal/risk features, specificity increased to 75% (MaxRFU), 86.2% (MedianRFU), and 79.3% (AUFC). Assay-characterization analyses, performed including all technical replicates, showed significantly higher fluorescence signals throughout the RT-QuIC amplification curve (p < 0.0001) with shorter lag phase, higher MedianRFU and MaxRFU, and larger AUFC values in PD than in HCs (all p < 0.05). Exploratory subgroup analyses suggested possible phenotype-related variability in test response. Our findings suggest that alpha Syn seeding activity may be detected in TF. Despite suboptimal sensitivity, non-invasiveness and promising specificity support further optimization of TF-based RT-QuIC as a diagnostic tool for synucleinopathies.