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bioRxiv · 10.1101/2025.04.11.648439

Validation and pre-analytical considerations for processing cerebrospinal fluid samples on a high-throughput proximity extension assay platform

Hyde, M.·Cunningham, M.·Chai, X.·Hu, H. A.·Lu, C.·Vasanthakumar, A.
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Abstract

BackgroundAnalysis of cerebrospinal fluid (CSF) facilitates the understanding of brain-specific molecular changes that may associate with disease progression. Proximity extension assays (PEA) have been deployed in several CSF studies, however the validation of the assay and impact of freeze-thaw cycles on the protein signal has not been documented. We sought to (1) validate the assay on the PEA platform and (2) evaluate the effect of freeze-thaw cycles on the detectability of analytes on the PEA platform. ResultsWe have validated the PEA with Next Generation Sequencing (NGS) readout assay and report on the detectability and coefficient of variation observed in CSF samples. We have also evaluated proteomic signals with a minimum of 3 and a maximum of 9 freeze thaw cycles and detected very minimal change in signal with increasing cycle number. ConclusionOur study is the first to validate PEA using NGS readout platform with CSF samples. We report lower protein detection rates and higher variability in the expansion panels compared to the original 4 panels, with acceptable variation above detectability threshold. In addition, our work demonstrates that the proteomic signal is robust and continues to be stable across multiple freeze thaw cycles. This is highly impactful to the processing and analysis of clinical samples and facilitates the investigation of samples with variable pre-analytical conditions.

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Hyde, M., Cunningham, M., Chai, X., Hu, H. A., Lu, C., Vasanthakumar, A.. 2025-04-17. Validation and pre-analytical considerations for processing cerebrospinal fluid samples on a high-throughput proximity extension assay platform. https://doi.org/10.1101/2025.04.11.648439

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