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Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains
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High Agreement Across Laboratories Between Different Alpha-Synuclein Seed Amplification Protocols.

Stefan Bräuer1,2, Maximilian Weber3, Christian Deuschle3

  • 1German Center for Neurodegenerative Diseases (DZNE), Dresden, Germany.

European Journal of Neurology
|April 16, 2025
PubMed
Summary

Seed amplification assays (SAA) for alpha-synuclein (aSYN) show high agreement across labs and protocols. Round-robin testing confirms SAA quality for detecting aSYN pathology in cerebrospinal fluid (CSF).

Keywords:
RT‐QuICalpha‐synucleinmethod validationproficiency testingring trial

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Area of Science:

  • Neuroscience
  • Biomarker Discovery
  • Assay Development

Background:

  • Seed amplification assays (SAA) detect alpha-synuclein (aSYN) pathology in cerebrospinal fluid (CSF), potentially before clinical symptoms.
  • Standardization of CSF-based SAA is crucial for clinical trials and research as these assays gain wider adoption.

Purpose of the Study:

  • To assess the inter-laboratory reproducibility of SAA for alpha-synuclein (aSYN) detection.
  • To compare SAA performance across different laboratories, recombinant substrates, and assay protocols.

Main Methods:

  • A cross-laboratory, cross-substrate, cross-protocol round-robin study was conducted.
  • Four research laboratories analyzed 38 cerebrospinal fluid (CSF) samples using SAA.
  • Three laboratories used a standardized protocol, while one used a different protocol; two distinct aSYN protein substrates were employed.

Main Results:

  • High qualitative agreement was observed, with 37 out of 38 samples yielding identical results in at least three of the four laboratories (Fleiss Kappa = 0.751).
  • Individual laboratory agreement with a reference laboratory exceeded 92%.
  • Agreement for the number of positive replicates was moderate (Fleiss Kappa = 0.45 for zero, 0.42 for four positive replicates).

Conclusions:

  • Qualitative SAA results demonstrate substantial agreement across different research laboratories, alpha-synuclein (aSYN) monomers, and assay protocols.
  • Observed inter-laboratory variations appear systematic, suggesting they reflect biologically relevant properties.
  • Round-robin testing is effective for evaluating and ensuring the quality and consistency of SAA across multiple laboratories.