Annals of Laboratory Medicine

Korean Society for Laboratory Medicine (대한진단검사의학회)
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Optimization of a Protocol for Isolating Cell-free DNA From Cerebrospinal Fluid

  • Ho Hyun Song (Department of Interdisciplinary Program in Biomedical Science, Graduate School, Soonchunhyang University) ;
  • Hyeran Park (Department of Neurosurgery, Soonchunhyang University Seoul Hospital) ;
  • Doohwan Cho (Department of Laboratory Medicine, Soonchunhyang University Seoul Hospital) ;
  • Hae In Bang (Department of Laboratory Medicine, Soonchunhyang University Seoul Hospital) ;
  • Hyuk-Jin Oh (Department of Neurosurgery, Soonchunhyang University Cheonan Hospital) ;
  • Jieun Kim (Department of Laboratory Medicine, Soonchunhyang University Seoul Hospital)
  • Received : 2023.07.10
  • Accepted : 2023.12.08
  • Published : 2024.05.01
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Abstract

A standardized protocol for the isolation of cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) is lacking. Therefore, we established a cfDNA isolation protocol optimized for clinical CSF specimens, integrating acceptable modifications and using artificial CSF generated from remnant CSF spiked with reference cell-free tumor DNA (ctDNA). We compared the isolation yields of in vitro diagnostic (IVD)-certified column-based (CB) and magnetic beadbased (MB) isolation. Furthermore, we modified both methods, including pre- and postelution steps. To confirm ctDNA integrity and quantify the variant allele frequency after isolation, we performed droplet digital PCR (ddPCR) targeting IDH1 R132C in the reference ctDNA. MB isolation had a higher yield than CB isolation (P <0.0001), and post-isolation vacuum increased the final concentration in both methods, with little effect on cfDNA integrity. Our study provides a protocol to maximize CSF-ctDNA concentrations in IVD testing and future studies.

Keywords

Acknowledgement

We thank Suyeon Park at the Department of Biostatistics, Soonchunhyang University Seoul Hospital, Korea for statistical consultation.

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