Standardization non-invasive fetal RHD and SRY determination into clinical routine using a new multiplex RT-PCR assay for fetal cell-free DNA in pregnant women plasma: Results in clinical benefits and cost saving

• Published in: Clinica chimica acta 2012-02, Vol.413 (3), p.490-494
• Main Authors: Macher, Hada C., Noguerol, Pilar, Medrano-Campillo, Pablo, Garrido-Márquez, María R., Rubio-Calvo, Amalia, Carmona-González, Magdalena, Martin-Sánchez, Jesús, Pérez-Simón, José A., Guerrero, Juan M.
• Format: Article
• Language: English
• Subjects: Cell-Free System; Circulating DNA; Cost Savings - methods; DNA - blood; DNA - genetics; Female; Fetus - metabolism; Genotyping Techniques; Humans; Multiplex real-time PCR; Pregnancy; Real-Time Polymerase Chain Reaction - economics; Real-Time Polymerase Chain Reaction - standards; Reference Standards; Rh-Hr Blood-Group System - genetics; RHD genotyping; Sex-Determining Region Y Protein - genetics; SRY genotyping
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cca.2011.11.004

Among negative RhD mothers it is essential to know the fetal RhD status in order to avoid the possibility of hemolytic disease of the newborn. In this regard, the detection of fetal DNA in maternal plasma might become a new diagnostic tool. In the current study, we have evaluated the standardization of a Multiplex-PCR targeted towards two exons of the RHD and one SRY gene to monitor RhD negative women. The current study addresses questions concerning feasibility and applicability of this approach into the clinical practice. Both single and multiplex real-time PCRs targeting RHD exons 5 and 7 and SRY were applied for the detection of fetal-specific RHD sequences and sex in maternal plasma. A large cohort of 2127 women was studied between 10 and 28 weeks of pregnancy. 134 of them were used for single TaqMan PCR studies and 1993 were evaluated using Multiplex TaqMan PCR studies. All of them were serologically typed as RhD negative according to Spanish guidelines. Single and multiplex real-time PCR results were compared with postnatal serology and sex identification. There was a 100% concordance between results obtained with single and multiplex real-time PCR assays. At present, 1012 of the 1993 pregnant women studied gave birth and the results of RHD status obtained with the multiplex TaqMan PCR assay were confirmed postpartum by serological methods showing that sensitivity, specificity, and accuracy of the multiplex assay were 100, 98.6, and 99.3%, respectively. This procedure improved the speed of the assay, avoided over-treatment among RhD negative pregnant women bearing RhD negative fetus, and reduced the requirements for clinical and biological monitoring, resulting in a clinical benefit and cost saving. The routine determination of fetal RHD status and SRY in maternal plasma, using multiplex real-time PCR, is feasible. The use of multiplex real-time PCR allows improving the response of the laboratory, saving time and reagent costs, opening the door to a complete automatization of the process. ► New method to evaluate fetal RHD in the pregnant plasma using multiplex real time PCR. ► The method allows the automatization of non-invasive prenatal diagnosis of fetal RHD. ► The assay is 100% sensitive to detect RHD positive fetuses since week 10 of pregnancy.