Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR.
BACKGROUND
Noninvasive genotyping of fetal RHD (Rh blood group, D antigen) can prevent the unnecessary administration of prophylactic anti-D to women carrying RHD-negative fetuses. We evaluated laboratory methods for such genotyping.


METHODS
Blood samples were collected in EDTA tubes and Streck® Cell-Free DNA™ blood collection tubes (Streck BCTs) from RHD-negative women (n = 46). Using Y-specific and RHD-specific targets, we 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™ PCR (ddPCR) platform compared with real-time quantitative PCR (qPCR).


RESULTS
The cffDNA fraction was significantly larger for samples collected in Streck BCTs compared with samples collected in EDTA tubes (P < 0.001). In samples expressing optimal cffDNA fractions (≥4%), both qPCR and digital PCR (dPCR) showed 100% sensitivity for the TSPY1 (testis-specific protein, Y-linked 1) and RHD7 (RHD exon 7) assays. Although dPCR also had 100% sensitivity for RHD5 (RHD exon 5), qPCR had reduced sensitivity (83%) for this target. For samples expressing suboptimal cffDNA fractions (<2%), dPCR achieved 100% sensitivity for all assays, whereas qPCR achieved 100% sensitivity only for the TSPY1 (multicopy target) assay.


CONCLUSIONS
qPCR was not found to be an effective tool for RHD genotyping in suboptimal samples (<2% cffDNA). However, when testing the same suboptimal samples on the same day by dPCR, 100% sensitivity was achieved for both fetal sex determination and RHD genotyping. Use of dPCR for identification of fetal specific markers can reduce the occurrence of false-negative and inconclusive results, particularly when samples express high levels of background maternal cell-free DNA.
School of Biomedical and Healthcare Sciences, Plymouth University, Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, UK;
Kelly A
KA
Sillence
Department of Fetal Medicine, Plymouth Hospitals National Health Service Trust, Plymouth, UK.
Llinos A
LA
Roberts
Department of Fetal Medicine, Plymouth Hospitals National Health Service Trust, Plymouth, UK.
Heidi J
HJ
Hollands
School of Biomedical and Healthcare Sciences, Plymouth University, Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, UK;
Hannah P
HP
Thompson
School of Biomedical and Healthcare Sciences, Plymouth University, Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, UK;
Michele
M
Kiernan
School of Biomedical and Healthcare Sciences, Plymouth University, Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, UK;
Tracey E
TE
Madgett
Department of Fetal Medicine, Plymouth Hospitals National Health Service Trust, Plymouth, UK.
C Ross
CR
Welch
School of Biomedical and Healthcare Sciences, Plymouth University, Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, UK; neil.avent@plymouth.ac.uk.
Neil D
ND
Avent
D001800:Blood Specimen Collection / Q000379:methods; D004247:DNA / Q000097:blood / Q000235:genetics*; D005260:Female; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012204:Rh-Hr Blood-Group System / Q000097:blood / Q000235:genetics*; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis / Q000379:methods*
D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; D004247:DNA