Optimization of a Protocol for Isolating Cell-free DNA from Cerebrospinal Fluid

Ann Lab Med. 2023 Dec 28. doi: 10.3343/alm.2023.0267. Online ahead of print.ABSTRACTA 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.PMID:38151854 | DOI:10.3343/alm.2023.0267
Source: Annals of Laboratory Medicine - Category: Laboratory Medicine Authors: Source Type: research