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Introduction <p>The α-synuclein seed amplification assay (synSAA) is a biomarker test for synucleinopathies. Different synSAA conditions have different properties, and some of these conditions enable differentiation between diseases associated with Lewy bodies versus those associated with glial inclusions. Direct cross-assay and inter-site synSAA comparisons evaluating these features remain limited.</p> Methods <p>Two synSAA conditions (identified here as sodium phosphate buffer (SPB) and piperazine-<i>N</i>,<i>N</i>′-bis(2-ethanesulfonic acid) (PIPES) SAA conditions) were tested in a controlled 2 × 2 design, each performed at two independent laboratories. Cerebrospinal fluid (CSF) from 60 participants was analyzed, comprising 29 multiple system atrophy (MSA) samples and 31 additional CSF samples including 16 Parkinson’s disease/dementia with Lewy bodies (PD/DLB) cases previously screened using SPB-SAA conditions, and 15 neurology ward controls, which were used as analytical controls.</p> Results <p>Inter-site concordance was high for SPB-SAA (100%) and PIPES-SAA (95%, 0.913 Fleiss’ kappa). Compared to clinical diagnosis, sensitivity for detecting synSAA+ MSA cases was 75.0% for PIPES-SAA_A and 81.4% for PIPES-SAA_C, with specificities of 76.9% and 92.3%, respectively. SPB-SAA did not detect MSA at either of the two sites and showed 100% specificity. Inter-assay concordance was high for PD/DLB (88%) and controls (86%), but low for MSA (31%), reflecting the different sensitivity in MSA cases. The overall inter-assay concordance yielded a Fleiss’ kappa of 0.23.</p> Conclusion <p>Both synSAA conditions exhibit high reproducibility and replicability across laboratories yet differ in their diagnostic profiles: PIPES-SAA conditions enable detection of synuclein seeds in MSA, while SPB-SAA conditions showed high specificity for PD/DLB but not detect synuclein seeds in MSA. These findings support context-dependent assay selection and underscore the need for larger multicenter validation of analytical tools intended for clinical use.</p>

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Inter-assay and Inter-site Performance of Alpha-Synuclein Seed Amplification Assays in Synucleinopathies: A Multicenter 2 × 2 Protocol Comparison

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月27日

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  • Roger Plaza-Clar,
  • Maximilian Weber,
  • Vaibhavi Kadam,
  • Christian Deuschle,
  • Yihua Ma,
  • Carly M. Farris,
  • Luis Concha-Marambio,
  • David Mengel,
  • Marcello Rossi,
  • Angela Mammana,
  • Piero Parchi,
  • Matthis Synofzik,
  • Kathrin Brockmann,
  • Thomas Gasser

摘要

Introduction

The α-synuclein seed amplification assay (synSAA) is a biomarker test for synucleinopathies. Different synSAA conditions have different properties, and some of these conditions enable differentiation between diseases associated with Lewy bodies versus those associated with glial inclusions. Direct cross-assay and inter-site synSAA comparisons evaluating these features remain limited.

Methods

Two synSAA conditions (identified here as sodium phosphate buffer (SPB) and piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) SAA conditions) were tested in a controlled 2 × 2 design, each performed at two independent laboratories. Cerebrospinal fluid (CSF) from 60 participants was analyzed, comprising 29 multiple system atrophy (MSA) samples and 31 additional CSF samples including 16 Parkinson’s disease/dementia with Lewy bodies (PD/DLB) cases previously screened using SPB-SAA conditions, and 15 neurology ward controls, which were used as analytical controls.

Results

Inter-site concordance was high for SPB-SAA (100%) and PIPES-SAA (95%, 0.913 Fleiss’ kappa). Compared to clinical diagnosis, sensitivity for detecting synSAA+ MSA cases was 75.0% for PIPES-SAA_A and 81.4% for PIPES-SAA_C, with specificities of 76.9% and 92.3%, respectively. SPB-SAA did not detect MSA at either of the two sites and showed 100% specificity. Inter-assay concordance was high for PD/DLB (88%) and controls (86%), but low for MSA (31%), reflecting the different sensitivity in MSA cases. The overall inter-assay concordance yielded a Fleiss’ kappa of 0.23.

Conclusion

Both synSAA conditions exhibit high reproducibility and replicability across laboratories yet differ in their diagnostic profiles: PIPES-SAA conditions enable detection of synuclein seeds in MSA, while SPB-SAA conditions showed high specificity for PD/DLB but not detect synuclein seeds in MSA. These findings support context-dependent assay selection and underscore the need for larger multicenter validation of analytical tools intended for clinical use.