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dc.contributor.authorSillence, KAen
dc.contributor.authorRoberts, LAen
dc.contributor.authorHollands, HJen
dc.contributor.authorThompson, HPen
dc.contributor.authorKiernan, Men
dc.contributor.authorMadgett, TEen
dc.contributor.authorWelch, CRen
dc.contributor.authorAvent, NDen

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.

dc.format.extent1399 - 1407en
dc.subjectBlood Specimen Collectionen
dc.subjectGenotyping Techniquesen
dc.subjectPolymerase Chain Reactionen
dc.subjectReal-Time Polymerase Chain Reactionen
dc.subjectRh-Hr Blood-Group Systemen
dc.subjectSensitivity and Specificityen
dc.subjectSex Determination Analysisen
dc.titleFetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR.en
dc.typeJournal Article
plymouth.journalClin Chemen
plymouth.organisational-group/Plymouth/00 Groups by role
plymouth.organisational-group/Plymouth/00 Groups by role/Academics
plymouth.organisational-group/Plymouth/00 Groups by role/Professional Services staff
plymouth.organisational-group/Plymouth/Faculty of Medicine and Dentistry
plymouth.organisational-group/Plymouth/Faculty of Medicine and Dentistry/Biomedical Research Group
plymouth.organisational-group/Plymouth/Faculty of Medicine and Dentistry/Biomedical Research Group/RC Reporting Group BRG
plymouth.organisational-group/Plymouth/Faculty of Medicine and Dentistry/School of Biomedical Sciences
plymouth.organisational-group/Plymouth/REF 2021 Researchers by UoA
plymouth.organisational-group/Plymouth/REF 2021 Researchers by UoA/UoA01 Clinical Medicine
plymouth.organisational-group/Plymouth/Research Groups
plymouth.organisational-group/Plymouth/Research Groups/Institute of Translational and Stratified Medicine (ITSMED)
plymouth.organisational-group/Plymouth/Research Groups/Institute of Translational and Stratified Medicine (ITSMED)/CBR
dc.publisher.placeUnited Statesen
dc.rights.embargoperiodNot knownen
rioxxterms.typeJournal Article/Reviewen

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