Inter-assay and Inter-site Performance of Alpha-Synuclein Seed Amplification Assays in Synucleinopathies: A...

Area of Science:

• Neurology
• Biomarker Development
• Assay Validation

Background:

• The α-synuclein seed amplification assay (synSAA) is a key biomarker for synucleinopathies.
• Existing synSAA conditions vary in their ability to differentiate between Lewy body and glial inclusion diseases.
• Limited direct comparisons exist for cross-assay and inter-site synSAA performance.

Purpose of the Study:

• To compare two synSAA conditions (SPB-SAA and PIPES-SAA) across independent laboratories.
• To evaluate the diagnostic profiles of these synSAA conditions for differentiating synucleinopathies.
• To assess the reproducibility and replicability of synSAA assays.

Main Methods:

• Two synSAA conditions, sodium phosphate buffer (SPB) and piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), were tested in a 2x2 design.
• Each condition was performed at two independent laboratories using cerebrospinal fluid (CSF) from 60 participants.
• Samples included multiple system atrophy (MSA), Parkinson's disease/dementia with Lewy bodies (PD/DLB), and neurology controls.

Main Results:

• High inter-site concordance was observed for SPB-SAA (100%) and PIPES-SAA (95%).
• PIPES-SAA demonstrated sensitivity for MSA (75.0%-81.4%) with varying specificity (76.9%-92.3%).
• SPB-SAA showed 100% specificity for controls and PD/DLB but did not detect MSA; inter-assay concordance for MSA was low (31%).

Conclusions:

• Both synSAA conditions exhibit high reproducibility and replicability.
• PIPES-SAA detects synuclein seeds in MSA, while SPB-SAA is specific for PD/DLB.
• Assay selection should be context-dependent, and larger multicenter validation is needed for clinical application.