University of Plymouth PEARL https://pearl.plymouth.ac.uk Faculty of Health: Medicine, Dentistry and Human Sciences School of Biomedical Sciences 2015-11 Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR. Sillence, KA http://hdl.handle.net/10026.1/3798 10.1373/clinchem.2015.239137 Clin Chem All content in PEARL is protected by copyright law. Author manuscripts are made available in accordance with publisher policies. Please cite only the published version using the details provided on the item record or document. In the absence of an open licence (e.g. Creative Commons), permissions for further reuse of content should be sought from the publisher or author. Clinical Chemistry 61:11 Molecular Diagnostics and Genetics 1399–1407 (2015) Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR Kelly A. Sillence,1 Llinos A. Roberts,2 Heidi J. Hollands,2 Hannah P. Thompson,1 Michele Kiernan,1 Tracey E. Madgett,1 C. Ross Welch,2 and Neil D. Avent1* BACKGROUND: Noninvasive genotyping of fetal RHD inconclusive results, particularly when samples express (Rh blood group, D antigen) can prevent the unnecessary high levels of background maternal cell-free DNA. administration of prophylactic anti-D to women carry- © 2015 American Association for Clinical Chemistry ing RHD-negative fetuses. We evaluated laboratory methods for such genotyping. Diagnosis of fetal sex, RHD (Rh blood group, D anti- METHODS: Blood samples were collected in EDTA tubes gen)3 genotype, and chromosomal abnormalities can be and Streck® Cell-Free DNA™ blood collection tubes achieved only through analysis of fetal DNA. Initially, (Streck BCTs) from RHD-negative women (n ϭ 46). this could be achieved through invasive procedures such Using Y-specific and RHD-specific targets, we investi- as amniocentesis and chorionic villus sampling, quoted as gated variation in the cell-free fetal DNA (cffDNA) frac- having a 1% risk of miscarriage (1). Since the discovery tion and determined the sensitivity achieved for optimal 4 and suboptimal samples with a novel Droplet Digital™ of cell-free fetal DNA (cffDNA) in maternal plasma, PCR (ddPCR) platform compared with real-time quan- noninvasive prenatal testing is now a clinical reality (1– titative PCR (qPCR). 5). Fetal sex determination is offered in the clinic for families at risk of X-linked disorders, such as Duchenne RESULTS: The cffDNA fraction was significantly larger for muscular dystrophy (6). Determination of fetal sex is samples collected in Streck BCTs compared with samples especially beneficial in cases of congenital adrenal hyper- collected in EDTA tubes (P Ͻ 0.001). In samples expressing plasia, to allow therapy to be targeted to female fetuses optimal cffDNA fractions (Ն4%), both qPCR and digital only (7). Fetal aneuploidy detection requires accurate PCR (dPCR) showed 100% sensitivity for the TSPY1 quantification and presently can only be determined by (testis-specific protein, Y-linked 1) and RHD7 (RHD exon next-generation sequencing, which is too costly for rou- 7) assays. Although dPCR also had 100% sensitivity for tine testing (8). RHD5 (RHD exon 5), qPCR had reduced sensitivity (83%) The antigens of the Rh blood group system are for this target. For samples expressing suboptimal cffDNA coded for by 2 genes, RHD and RHCE (Rh blood group, fractions (Ͻ2%), dPCR achieved 100% sensitivity for all CcEe antigens), which are located on chromosome 1 assays, whereas qPCR achieved 100% sensitivity only for (p34–p36) (9). In white populations, most D-negative the TSPY1 (multicopy target) assay. phenotypes result from a complete RHD deletion (10). For D-negative individuals of African descent, only 18% CONCLUSIONS: qPCR was not found to be an effective are a result of RHD deletion. Instead, 66% and 15% of tool for RHD genotyping in suboptimal samples (Ͻ2% D-negative Africans have an inactive RHD gene cffDNA). However, when testing the same suboptimal (RHD⌿) or a hybrid gene (RHD-CE-DS or r’S), which do samples on the same day by dPCR, 100% sensitivity was not produce any RhD protein (10, 11). Many laborato- achieved for both fetal sex determination and RHD geno- ries currently provide noninvasive fetal RHD genotyping typing. Use of dPCR for identification of fetal specific for alloimmunized women as routine practice to manage markers can reduce the occurrence of false-negative and hemolytic disease of the fetus and newborn (HDFN) 1 School of Biomedical and Healthcare Sciences, Plymouth University, Plymouth Univer- naute RISC catalytic component 1 (formerly EIF2C1); TERT, telomerase reverse transcrip- sity Peninsula Schools of Medicine and Dentistry, Plymouth, UK; 2 Department of Fetal tase; ERV3–1, endogenous retrovirus group 3, member 1. Medicine, Plymouth Hospitals National Health Service Trust, Plymouth, UK. 4 Nonstandard abbreviations: cffDNA, cell-free fetal DNA; HDFN, hemolytic disease of the * Address correspondence to this author at: School of Biomedical and Healthcare Sciences, fetus and newborn; qPCR, real-time quantitative PCR; dPCR, digital PCR; BCT, blood Plymouth University, Plymouth, UK PL4 8AA. E-mail [email protected]. collection tube; cfDNA, cell-free DNA; RHD5, Rh blood group, D antigen exon 5; RHD7, Received January 28, 2015; accepted August 24, 2015. Rh blood group, D antigen exon 7; FAM, carboxyfluorescein; HEX, hexachlorofluores- Previously published online at DOI: 10.1373/clinchem.2015.239137 cein; BLAST, Basic Local Alignment Search Tool; gDNA, genomic DNA; ddPCR, droplet © 2015 American Association for Clinical Chemistry digital PCR; Cq, quantification cycle; Ta, annealing temperature; NTC, no-template con- 3 Human genes: RHD, Rh blood group, D antigen; RHCE, Rh blood group, CcEe antigens; trol; 2D, 2-dimensional. SRY, sex-determining region Y; TSPY1, testis-specific protein, Y-linked 1; AGO1, argo- 1399 Table 1. Summary of amplicon location, length, and fluorescent label for each multiplex reaction. Multiplex Fluorescent reaction Amplicon Chromosome Gene Exon/intron reporter dye Length, bp Origin 1 Target Y SRY Exon FAM 137 Lo et al. (22) 2 Target Y TSPY1 Exon FAM 88 In-house 1 and 2 Reference X Xp22.3 Intron HEX 95 Fan et al. (23) 3 Target 1 RHD5 Exon (5) FAM 82 Finning et al. (24) 4 Target 1 RHD7 Exon (7) FAM 75 Finning et al. (24) 3 and 4 Reference 1 AGO1 Exon HEX 81 Fan et al. (23) (4, 12–14). Before 1970, HDFN was a major cause of SAMPLE PROCESSING fetal mortality (46/100 000 births in the UK alone) (15), Twenty-two maternal peripheral blood samples were col- but since the introduction of routine antenatal anti-D pro- lected in EDTA tubes (5–10 mL total blood volume) and phylaxis, incidence has decreased nearly 10-fold (16). Cur- centrifuged at 1600g for 10 min at room temperature rently, all RHD-negative women in the West are offered this (samples 1–22). The plasma was carefully removed and prophylaxis, which is costly, as it is produced from hyper- transferred to a 15-mL tube. The plasma was then recen- immunized male volunteers. Providing a noninvasive test trifuged at 16 000g for 10 min. All samples were pro- for fetal RHD genotyping allows administration to be tar- cessed within4hofcollection, and plasma aliquots (1 ϩ Ϫ geted to RHD-negative women who are known to be carry- mL) were stored at Ϫ80 °C. RHD and RHD human ing an RHD-positive fetus. This is now routine in the Neth- whole blood, collected in EDTA tubes (5 mL total blood erlands and Denmark with real-time quantitative PCR volume), was supplied by National Health Service Blood (qPCR) approaches (4, 17, 18). However, recent publica- and Transplant (Bristol, UK) as positive and negative tions have described the application of digital PCR (dPCR) controls, respectively. These samples were processed for the detection of low-level targets with improved preci- within 48–96 h by following the same double-spin pro- sion, resulting in reliable quantification well below the limit tocol described above. of quantification of qPCR (19, 20). Twenty-four maternal blood samples collected in The use of Streck® Cell-Free DNA™ blood collec- Streck BCTs (10–20 mL total blood volume) were cen- tion tubes (BCTs) instead of conventional EDTA tubes trifuged at 1600g for 15 min at room temperature (sam- has been shown to increase the proportion of cffDNA ples 23–46). Plasma was carefully removed, transferred (21). Streck BCTs contain proprietary cell-preserving re- to a 50-mL tube, and recentrifuged at 2500g for 10 min. agents, which prevent maternal cell lysis and conse- All samples were processed within 48 h of collection, and quently reduce the amount of maternal cell-free DNA plasma aliquots (1 mL) were stored at Ϫ80 °C. (cfDNA) released into the plasma. In this study, we com- pared the sensitivity of dPCR and qPCR for the nonin- DNA EXTRACTION vasive determination of fetal sex and RHD genotype for DNA was extracted from two 1-mL aliquots of plasma samples collected in both Streck BCTs and EDTA tubes. with the QIAamp Circulating Nucleic Acid kit (Qiagen) For technical reasons, some samples, despite being col- and QIAvac 24 Plus (Qiagen). The extraction process lected in the third trimester, expressed suboptimal followed the manufacturer’s protocol, and each sample cffDNA fractions (Ͻ2%). However, all samples were in- was eluted in 60 ␮L Buffer AVE [RNase-free water con- cluded to thoroughly test the capability of the dPCR taining 0.04% (wt/vol) sodium azide]. No DNase or assay against the current gold standard, qPCR. RNase treatment was used. After DNA extraction, we quantified samples on the Qubit® 2.0 Fluorometer (Life Materials and Methods Technologies) with the Qubit dsDNA HS assay kit (Life Technologies). Samples were stored at Ϫ20 °C as 60-␮L STUDY PARTICIPANTS aliquots for Յ4 weeks.