Qualitative and quantitative comparison of cell-free DNA and cell-free fetal DNA isolation by four (semi-)automated extraction methods: impact in two clinical applications: chimerism quantification and noninvasive prenatal diagnosis.

Autor: Pedini P; Department of Histocompatibility, Établissement Français du Sang PACA-Corse, 149 Bd Baille, 13005, Marseille, France. pascal.pedini@efs.sante.fr., Graiet H; Department of Histocompatibility, Établissement Français du Sang PACA-Corse, 149 Bd Baille, 13005, Marseille, France., Laget L; Department of Immunohematology, Établissement Français du Sang PACA-Corse, 149 Bd Baille, 13005, Marseille, France., Filosa L; Department of Immunohematology, Établissement Français du Sang PACA-Corse, 149 Bd Baille, 13005, Marseille, France., Chatron J; Department of Histocompatibility, Établissement Français du Sang PACA-Corse, 149 Bd Baille, 13005, Marseille, France., Cherouat N; Department of Histocompatibility, Établissement Français du Sang PACA-Corse, 149 Bd Baille, 13005, Marseille, France., Chiaroni J; Department of Immunohematology, Établissement Français du Sang PACA-Corse, 149 Bd Baille, 13005, Marseille, France.; UMR 7268, ADÉS Aix-Marseille Université/EFS, CNRS, 27 Bd Jean Moulin, 13385, Marseille Cedex 05, France., Hubert L; Department of Histocompatibility, Établissement Français du Sang PACA-Corse, 149 Bd Baille, 13005, Marseille, France., Frassati C; Department of Histocompatibility, Établissement Français du Sang PACA-Corse, 149 Bd Baille, 13005, Marseille, France., Picard C; Department of Histocompatibility, Établissement Français du Sang PACA-Corse, 149 Bd Baille, 13005, Marseille, France.; UMR 7268, ADÉS Aix-Marseille Université/EFS, CNRS, 27 Bd Jean Moulin, 13385, Marseille Cedex 05, France.
Jazyk: angličtina
Zdroj: Journal of translational medicine [J Transl Med] 2021 Jan 06; Vol. 19 (1), pp. 15. Date of Electronic Publication: 2021 Jan 06.
DOI: 10.1186/s12967-020-02671-8
Abstrakt: Background: Non-invasive molecular analysis of cell-free DNA (cfDNA) became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA (cffDNA) in noninvasive prenatal test. In this study, we compared the efficiencies of four (semi)-automated cfDNA isolation instruments using their respective isolation kit: MagNA Pure 24 (Roche®), IDEAL (IDSolution®), LABTurbo 24 (Taigen®) and Chemagic 360 (Perkin Elmer®). The cfDNA was isolated from 5 plasma samples and the Rhesus D (RhD)-cffDNA from 5 maternal plasmas. The cfDNA were quantified by digital droplet PCR (ddPCR), BIABooster system and QUBIT fluorometer. The cfDNA fragment size profiles were assessed by BIABooster system. Chimerism were quantified by home-made ddPCR and Devyser NGS kit. RhD-cffDNA in maternal plasma were detected between weeks 14 and 24 of amenorrhea using free DNA Fetal RHD Kit® (Biorad®).
Results: Statistical tests have shown differences in DNA yield depending on the isolation procedure and quantification method used. Magna Pure isolates smaller cfDNA fragment size than other extraction methods (90% ± 9% vs. 74% ± 8%; p = 0.009). Chimerism was only reliable from LABTurbo 24 extractions using the NGS but not with ddPCR whatever extraction methods. RhD-cffDNA were detected by all isolation methods, although IDEAL and LABTurbo 24 systems seemed more efficient.
Conclusions: This comparative study showed a dependency of cfDNA yield depending on isolation procedure and quantification method used. In total, these results suggest that the choice of pre-analytical isolation systems needs to be carefully validated in routine clinical practice.
Databáze: MEDLINE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje