title: Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach.
abstract: Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods' performance parameters-standard curve linearity, detection limit and measurement precision-were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438).
authors.affiliation: Institute for Biology and Medical Genetics, First Faculty of Medicine, Charles University in Prague and General Faculty Hospital in Prague, Czech Republic.
authors.forename: Iveta
authors.initials: I
authors.lastname: Svobodová
authors.affiliation: Department of Nephrology, First Faculty of Medicine, Charles University in Prague and General Faculty Hospital in Prague, Czech Republic.
authors.forename: Eva
authors.initials: E
authors.lastname: Pazourková
authors.affiliation: Institute for Biology and Medical Genetics, First Faculty of Medicine, Charles University in Prague and General Faculty Hospital in Prague, Czech Republic.
authors.forename: Aleš
authors.initials: A
authors.lastname: Hořínek
authors.affiliation: Department of Obstetrics and Gynecology, First Faculty of Medicine and General Faculty Hospital in Prague, Charles University in Prague, Czech Republic.
authors.forename: Michaela
authors.initials: M
authors.lastname: Novotná
authors.affiliation: Department of Obstetrics and Gynecology, First Faculty of Medicine and General Faculty Hospital in Prague, Charles University in Prague, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Calda
authors.affiliation: Institute for Biology and Medical Genetics, First Faculty of Medicine, Charles University in Prague and General Faculty Hospital in Prague, Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Korabečná
mesh_terms: D001800:Blood Specimen Collection / Q000379:methods; D004247:DNA / Q000097:blood / Q000235:genetics; D005260:Female; D005333:Fetus / Q000378:metabolism*; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D005865:Gestational Age; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012204:Rh-Hr Blood-Group System / Q000235:genetics*
chemical_list: D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; D004247:DNA