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

Abstract

Purpose To examine the potential high throughput capability and eYciency of an automated DNA extraction system in combination with mass spectrometry for the noninvasive 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 speciWcity 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 realtime PCR.

Future of Molecular Testing for Red Blood Cell Antigens

“…20 Using the Sequenom platform, a prenatal screening test for fetal RHD has been reported with claims of more than 99% sensitivity. 21 However, the current high instrument cots may limit its availability and use in the future.…”

The controversy about controls for fetal blood group genotyping by cell-free fetal DNA in maternal plasma

“…Using a panel of only eight indels, two independent laboratories in the United Kingdom found the approach not informative in more than 60% of cases, and this approach is no longer pursued by them [36]. Multiplexing more systems is more easily reached on a mass spectrometry platform [12,15,16]. Tynan et al [37 ] presented a universal multiplexed single nucleotide polymorphism (SNP) genotyping method on this platform.…”

Development and Validation of Multiplex Real-Time PCR Assay for Noninvasive Prenatal Assessment of Fetal RhD Status and Fetal Sex in Maternal Plasma

“…For the same reason, also the need of triplicate analysis for each sample has been stressed [21,22,23,24,25]. The use of an automated extractor ideally should help in reducing the risk of sample mishandling and cross-contamination, while also contributing to a reduction of costs [26,27,28,29,30,31]. …”