Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach

• Published in: PloS one Vol. 10; no. 11; p. e0142572
• Main Authors: Svobodová, Iveta, Pazourková, Eva, Hořínek, Aleš, Novotná, Michaela, Calda, Pavel, Korabečná, Marie
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 12.11.2015; Public Library of Science (PLoS)
• Subjects: Biology; Blood Specimen Collection - methods; Deoxyribonucleic acid; DNA; DNA - blood; DNA - genetics; DNA methylation; Drug dosages; Female; Fetus - metabolism; Fetuses; Genotype; Genotyping; Genotyping Techniques - methods; Gestational Age; Gynecology; Hospitals; Humans; Linearity; Low concentrations; Measurement methods; Medicine; Obstetrics; Plasma; Polymerase chain reaction; Polymerase Chain Reaction - methods; Pregnancy; Prenatal development; Prenatal Diagnosis - methods; Quantitative analysis; Real time; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; Rh-Hr Blood-Group System - genetics; Sensitivity analysis; Technology assessment; Workflow; Czech Republic
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0142572

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).