A Systematic Comparison of Alpha-Synuclein Seed Amplification Assays for Increasing Reproducibility

Seed amplification assays (SAAs) enable ultrasensitive detection of misfolded α-synuclein across biofluids and tissues. Yet, heterogeneity in protocols limits cross-study comparability and clinical translation. Here, we review α-synuclein SAA methods and their performance across various biological matrices. We catalog key variables (e.g., human recombinant α-synuclein variants, buffers, salts, beads, shaking, incubation temperature) and outline pre-analytical factors that shape outcomes. We propose a prioritized framework of key analytical and pre-analytical variables most likely to influence assay reproducibility and clinical interpretation. We also outline minimal reporting criteria and practical quality-control checkpoints, and recommend multicenter interlaboratory proficiency testing as essential steps to accelerate the robust clinical and research implementation of α-synuclein SAAs.