Blood Test Increases Accuracy in Prenatal Testing

Laboratory methods have been evaluated for noninvasive genotyping of fetal RHD (Rh blood group, D antigen) that can prevent the unnecessary administration of prophylactic anti-D to women carrying RHD-negative fetuses. The test developed can be carried out on mothers at risk of X-linked genetic recessive diseases including hemophilia and Duchenne muscular dystrophy and mothers at risk of hemolytic disease of the new-born.

Scientists at the University of Plymouth (UK) recruited RHD-negative pregnant women (28 to 30 weeks' gestation), all of whom met inclusion criteria, from November 2013 to September 2014. Twenty-two maternal peripheral blood samples were collected in Ethylenediaminetetraacetic acid (EDTA) tubes and centrifuged at 1,600×g for 10 minutes at room temperature (samples 1–22). The plasma was carefully removed and transferred to a 15-mL tube. The plasma was then re-centrifuged at 16,000×g for 10 minutes.

Twenty-four maternal blood samples collected in Streck Cell-Free DNA blood collection tubes (Streck BCTs; La Vista, NE, USA) were centrifuged at 1,600×g for 15 minutes at room temperature (samples 23–46). Plasma was carefully removed, transferred to a 50-mL tube, and re-centrifuged at 2,500×g for 10 minutes. DNA was extracted from two 1-mL aliquots of plasma and quantified samples on the Qubit 2.0 Fluorometer (Thermo Fisher; Waltham, MA, USA). Using Y-specific and RHD-specific targets, the team investigated variation in the cell-free fetal DNA (cffDNA) fraction and determined the sensitivity achieved for optimal and suboptimal samples with a novel Droplet Digital polymerase chain reaction (ddPCR, Bio-Rad Laboratories; Hercules CA, USA) platform compared with real-time quantitative PCR (qPCR).

The cffDNA fraction was significantly larger for samples collected in Streck BCTs compared with samples collected in EDTA tubes. In samples expressing optimal cffDNA fractions greater than 4%, both qPCR and ddPCR showed 100% sensitivity for the testis-specific protein, Y-linked 1(TSPY1) and RHD exon 7 (RHD7) assays. Although ddPCR also had 100% sensitivity for RHD exon 5 (RHD5), qPCR had reduced sensitivity (83%) for this target. For samples expressing suboptimal cffDNA fractions, less than 2%, ddPCR achieved 100% sensitivity for all assays, whereas qPCR achieved 100% sensitivity only for the TSPY1 multicopy target assay.

Neil D. Avent, PhD, a professor and lead author of the study, said, “Although fetal blood grouping and sexing using maternal blood has been done for over a decade, this study proves a much more accurate and sensitive method of detecting fetal DNA. This offers great opportunities to detect other conditions using this technique, but is much cheaper than current noninvasive methods. The end is now in sight for the invasive techniques of amniocentesis and chorionic villus sampling.” The study was published in the November 2015 issue of the journal Clinical Chemistry.

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University of Plymouth 
Streck
Thermo Fisher