Ann Lab Med.  2024 May;44(3):294-298. 10.3343/alm.2023.0267.

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

• Song HH
• Park H
• Cho D
• Bang HI
• Oh HJ
• Kim J

• KMID: 2555708
• DOI: http://doi.org/10.3343/alm.2023.0267

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.

Keyword

Cell-free nucleic acids; Cerebrospinal fluid; Circulating tumor DNA; Droplet digital PCR; Vacuum

Figure

• 

  Fig. 1 Comparison of %yield and correlation between isolation and processing methods: elution, bead volume, and vacuum effects (A). Elution protocol for column-based isolation (50 µL twice, N=15; 100 µL once, N=14; *P<0.05) (B). %yield level in magnetic bead-based isolation with bead volume 50 µL vs. 75 µL vs. 100 µL (bead 50 µL, N=10, bead 75 µL, N=13; bead 100 µL, N=12, P<0.001) (C) %yield level in sample preparation with input poly(A) vs. non-poly(A) in magnetic bead-based isolation (input poly(A), N=26, non-poly(A), N=9, P>0.05) (D) %yield of column-based isolation and magnetic bead-based isolation (column-based, N=21; magnetic bead-based, N=20; P<0.0001) (E). Column-based isolation %yield level in before vs. after vacuum treatment (before, N=29; after, N=29; P<0.0001) (F). Magnetic bead-based isolation %yield level before vs. after vacuum treatment (before N=35; after N=35; P<0.0001) (G). Comparison of average size before vs. after vacuum concentration (ICC (2, 1)=0.78, 95% CI=0.19–0.91). Abbreviations: CB, column-based method; MB, magnetic bead-based method; ICC, intraclass coefficient correlation.

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  Fig. 2 Comparative evaluation of IDH1 R132C detection using ddPCR: column-based vs. magnetic bead-based isolation. (A) Column-based vs. magnetic bead-based isolation ddPCR event incubated with IDH1 R132C probe (column-based, N=26, magnetic bead-based, N=28, P<0.0001). (B) Variation allele frequency % of IDH1 R132C in column-based vs. magnetic bead-based isolation (column-based, N=26, magnetic bead-based, N=28, P=0.377).

Reference

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