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 bead-based (MB) isolation. Furthermore, we modified both methods, including pre- and post-elution 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.

中文翻译：

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从脑脊液中分离游离 DNA 的方案优化。

目前缺乏从脑脊液 (CSF) 中分离游离 DNA (cfDNA) 的标准化方案。因此，我们建立了针对临床 CSF 样本进行优化的 cfDNA 分离方案，整合了可接受的修改并使用由掺有参考无细胞肿瘤 DNA (ctDNA) 的残余 CSF 生成的人工 CSF。我们比较了体外诊断 (IVD) 认证的基于柱 (CB) 和基于磁珠 (MB) 的分离的分离率。此外，我们修改了两种方法，包括洗脱前和洗脱后步骤。为了确认 ctDNA 的完整性并量化分离后的变异等位基因频率，我们针对参考 ctDNA 中的IDH1 R132C 进行了液滴数字 PCR (ddPCR)。MB 分离的产量高于 CB 分离（P <0.0001），并且分离后真空增加了两种方法的最终浓度，对 cfDNA 完整性影响很小。我们的研究提供了一个在 IVD 测试和未来研究中最大化 CSF-ctDNA 浓度的方案。