优化从脑脊液中分离无细胞 DNA 的方案

IF 4 2区 医学 Q1 MEDICAL LABORATORY TECHNOLOGY
Annals of Laboratory Medicine Pub Date : 2024-05-01 Epub Date: 2023-12-28 DOI:10.3343/alm.2023.0267
Ho Hyun Song, Hyeran Park, Doohwan Cho, Hae In Bang, Hyuk-Jin Oh, Jieun Kim
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引用次数: 0

摘要

目前还缺乏从脑脊液(CSF)中分离无细胞 DNA(cfDNA)的标准化方案。因此,我们建立了一个针对临床 CSF 标本进行优化的 cfDNA 分离方案,该方案整合了可接受的修改,并使用了由添加了无细胞肿瘤 DNA(ctDNA)参照物的残余 CSF 生成的人工 CSF。我们比较了体外诊断(IVD)认证的柱式分离(CB)和磁珠式分离(MB)的分离率。此外,我们还对这两种方法进行了改进,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,我们对参考ctDNA中的IDH1 R132C进行了液滴数字PCR(ddPCR)检测。与 CB 分离相比,MB 分离的产量更高(P
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Optimization of a Protocol for Isolating Cell-free DNA From Cerebrospinal Fluid.

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.

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来源期刊
Annals of Laboratory Medicine
Annals of Laboratory Medicine MEDICAL LABORATORY TECHNOLOGY-
CiteScore
8.30
自引率
12.20%
发文量
100
审稿时长
6-12 weeks
期刊介绍: Annals of Laboratory Medicine is the official journal of Korean Society for Laboratory Medicine. The journal title has been recently changed from the Korean Journal of Laboratory Medicine (ISSN, 1598-6535) from the January issue of 2012. The JCR 2017 Impact factor of Ann Lab Med was 1.916.
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