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

Abstract

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.

DNA in extracellular vesicles: from evolution to its current application in health and disease

“…cfDNA- or EV-based diagnostic methods are very promising. cfDNA-based methods are already partially established in clinical areas such as perinatal diagnostics [ 83 ]. A recent proteomics profiling of 426 human samples from tissue explants, plasma, and other body fluids identified new EV biomarkers for pancreatic and lung cancer diagnosis and treatment [ 84 ].…”

Prädiktive molekulare Diagnostik beim Mammakarzinom

“… Zur DNA-Extraktion sollten erprobte Standardmethoden angewendet werden. Automatisierte Verfahren reduzieren die Variabilität, können jedoch die DNA-Ausbeute verringern [ 18 ]. Für die Next-Generation-Sequencing(NGS)-basierte Analyse von ctDNA müssen validierte Verfahren mit ausreichender Sensitivität zum Einsatz kommen.…”

“…Zur DNA-Extraktion sollten erprobte Standardmethoden angewendet werden. Automatisierte Verfahren reduzieren die Variabilität, können jedoch die DNA-Ausbeute verringern [ 18 ].…”

Circulating Tumor DNA Testing for Homology Recombination Repair Genes in Prostate Cancer: From the Lab to the Clinic

“…Automated cfDNA extraction has been less evaluated than manual approaches. Besides high throughput and low hands-on time, the use of automated methods reduces variability and the risk of sample-to-sample contamination, though they generate somewhat lower cfDNA yields than manual extraction [ 71 , 72 ]. Recently, results from a comparison study on four commercially available (semi-)automated cfDNA extraction protocols have been published [ 73 ].…”