Cell-free fetal DNA in specimen from pregnant women is stable up to 5 days.

OBJECTIVE: Before noninvasive prenatal diagnosis on the fetal Rhesus D status (NIPD RhD) can be implemented on a mass-scale, it is crucial to define requirements regarding sample transport. The aim of this study was to determine the relation between the transport time of samples for NIPD and the concentration of fetal DNA in maternal plasma. METHOD: We analyzed qualitative and quantitative data obtained in a previous study performed with real-time PCR to determine the accuracy of NIPD RhD following two different DNA extraction protocols. The number of days from phlebotomy until freezing of plasma at the study site was recorded and defined as transport time. RESULTS: NIPD RhD results of 972 specimens were analyzed according to transport time, which varied from a few hours to a maximum of 8 days (median 2 days). No decrease of cell-free fetal DNA was observed in samples with less than 6 days transport time. There was a pivotal trend to higher cycle threshold values in samples with ≥ 6 days transport time compared with those with ≤ 5 days. CONCLUSION: Because only a few laboratories offer an NIPD RhD service, we suggest a maximal transport time of 5 days from phlebotomy until freezing at the testing laboratory.

High throughput non-invasive determination of foetal Rhesus D status using automated extraction of cell-free foetal DNA in maternal plasma and mass spectrometry.

PURPOSE: To examine the potential high throughput capability and efficiency of an automated DNA extraction system in combination with mass spectrometry for the non-invasive determination of the foetal Rhesus D status. METHODS: A total of 178 maternal plasma samples from RHD-negative pregnant women were examined, from which DNA was extracted using the automated Roche MagNA Pure system. Presence of the foetal RHD gene was detected by PCR for RHD exon 7 and subsequent analysis using the Sequenom MassArray mass spectrometric system. RESULTS: We determined that as little as 15 pg of RHD-positive genomic DNA could be detected in a background of 585 pg of RHD-negative genomic DNA. The analysis of the clinical samples yielded a sensitivity and specificity of 96.1 and 96.1%, respectively. CONCLUSION: Our study indicated that automated DNA extraction in combination with mass spectrometry permits the determination of foetal Rhesus D genotype with an accuracy comparable to the current approaches using real-time PCR.

The determination of the fetal D status from maternal plasma for decision making on Rh prophylaxis is feasible.

BACKGROUND: Noninvasive fetal RHD genotyping might become a valuable tool in decision making on antenatal Rh prophylaxis, which is currently in routine practice for all D- pregnancies in several countries. This study provides a large-scale validation study of this technology to address questions concerning feasibility and applicability of its introduction into clinical routine. STUDY DESIGN AND METHODS: Real-time polymerase chain reaction (PCR) targeting RHD Exons 5 and 7 was applied for the detection of fetal-specific RHD sequences in maternal plasma. A total of 1113 women in 6 to 32 weeks (median, Week 25) of pregnancy were recruited. All of them were serologically typed as D- according to current German guidelines. DNA was extracted via a spin-column method and a novel automated approach using magnetic tips. Real-time PCR results were compared with postnatal serology and discrepancies further elucidated by DNA sequencing from a newborn's buccal swab. RESULTS: Sensitivities of fetal RHD genotyping were 99.7 percent (spin columns) and 99.8 percent (magnetic tips), thus comparable with serology (99.5%). The detection of weak D variants was more reliable by real-time PCR. Specificities of fetal RHD genotyping were 99.2 percent (spin columns) and 98.1 percent (magnetic tips), which is lower than serology (>99.7%). Automation achieved significantly higher yields of cell-free fetal DNA. CONCLUSION: This prospective clinical trial revealed that routine determination of the fetal D status from maternal plasma is feasible. Noninvasive fetal RHD genotyping can be considered as sensitive as the traditional postnatal serologic assay.