Noninvasive fetal RHD genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal RHD genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal RHD genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the RHD gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the RHD genotyping. The RHD genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the RHD genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal RHD allele from the plasma of RhD-negative pregnant women.

Department of Blood Transfusion University Hospital and Palacky University Olomouc 775 20 Olomouc Czech Republic

Department of Medical Genetics University Hospital Olomouc and Faculty of Medicine and Dentistry Palacky University Olomouc 775 20 Olomouc Czech Republic

Department of Obstetrics and Gynecology University Hospital Olomouc and Faculty of Medicine and Dentistry Palacky University Olomouc 775 20 Olomouc Czech Republic

Institute of Biostatistics and Analyses Masaryk University 625 00 Brno Czech Republic

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Avent N.D., Reid M.E. The Rh blood group system: A review. Blood. 2000;95:375–387. doi: 10.1182/blood.V95.2.375. PubMed DOI

de Haas M., Finning K., Massey E., Roberts D.J. Anti-D prophylaxis: Past, present and future. Transfus. Med. 2014;24:1–7. doi: 10.1111/tme.12099. PubMed DOI

de Haas M., Thurik F.F., Koelewijn J.M., van der Schoot C.E. Haemolytic disease of the fetus and newborn. Vox Sang. 2015;109:99–113. doi: 10.1111/vox.12265. PubMed DOI

van der Schoot C.E., de Haas M., Clausen F.B. Genotyping to prevent Rh disease: Has the time come? Curr. Opin. Hematol. 2017;24:544–550. doi: 10.1097/MOH.0000000000000379. PubMed DOI

Kent J., Farrell A.-M., Soothill P. Routine administration of Anti-D: The ethical case for offering pregnant women fetal RHD genotyping and a review of policy and practice. BMC Pregnancy Childbirth. 2014;14:87. doi: 10.1186/1471-2393-14-87. PubMed DOI PMC

Kratochvílová T., Holusková I., Durdová V., Strašilová P., Ľubušký M. Klinický význam neinvazivního stanovení RHD a RHCE genotypu plodu v managementu těhotenství s rizikem rozvoje hemolytické nemoci plodu a novorozence [The clinical significance of the non-invasive fetal RHD and RHCE genotype assessment in the management of pregnancies at risk of hemolytic disease of the fetus and newborn] J. Postgrad. Med. 2016;18:362–369.

Innan H. A two-locus gene conversion model with selection and its application to the human RHCE and RHD genes. Proc. Natl. Acad. Sci. USA. 2003;100:8793–8798. doi: 10.1073/pnas.1031592100. PubMed DOI PMC

Palomaki G.E., Kloza E.M., Lambert-Messerlian G.M., Haddow J.E., Neveux L.M., Ehrich M., van den Boom D., Bombard A.T., Deciu C., Grody W.W., et al. DNA sequencing of maternal plasma to detect Down syndrome: An international clinical validation study. J. Genet. Med. 2011;13:913–920. doi: 10.1097/GIM.0b013e3182368a0e. PubMed DOI

de Haas M., Thurik F.F., van der Ploeg C.P.B., Veldhuisen B., Hirschberg H., Soussan A.A., Woortmeijer H., Abbink F., Page-Christiaens G.C.M.L., Scheffer P.G., et al. Sensitivity of fetal RHD screening for safe guidance of targeted anti-D immunoglobulin prophylaxis: Prospective cohort study of a nationwide programme in the Netherlands. BMJ. 2016;355 doi: 10.1097/01.ogx.0000513227.69777.ca. PubMed DOI PMC

Haimila K., Sulin K., Kuosmanen M., Sareneva I., Korhonen A., Natunen S., Tuimala J., Sainio S. Targeted antenatal anti-D prophylaxis program for RhD - negative pregnant women: Outcome of the first two years of a national program in Finland. Acta Obstet. Gynecol. Scand. 2017;96:1228–1233. doi: 10.1111/aogs.13191. PubMed DOI

Hyland C.A., Millard G.M., O’Brien H., Schoeman E.M., Lopez G.H., McGowan E., Tremellen A., Puddephatt R., Gaerty K., Flower R.L., et al. Non-invasive fetal RHD genotyping for RhD negative women stratified into RHD gene deletion or variant groups: Comparative accuracy using two blood collection tube types. Pathology. 2017;49:757–764. doi: 10.1016/j.pathol.2017.08.010. PubMed DOI

Clausen F.B., Barrett A.N., Noninvasive Fetal RHD Genotyping EQA2017 Working Group Noninvasive fetal RHD genotyping to guide targeted anti-D prophylaxis-an external quality assessment workshop. Vox Sang. 2019;114:386–393. doi: 10.1111/vox.12768. PubMed DOI

Moezzi L., Keshavarz Z., Ranjbaran R., Aboualizadeh F., Behzad-Behbahani A., Abdullahi M., Ramezani A., Samsami A., Sharifzadeh S. Fetal RHD genotyping using real-time polymerase chain reaction analysis of cell-free fetal DNA in pregnancy of RhD negative women in South of Iran. Int. J. Fertil. Steril. 2016;10:62–70. doi: 10.22074/ijfs.2016.4770. PubMed DOI PMC

Wikman A.T., Tiblad E., Karlsson A., Olsson M.L., Westgren M., Reilly M. Noninvasive single-exon fetal RHD determination in a routine screening program in early pregnancy. Obstet. Gynecol. 2012;120:227–234. doi: 10.1097/AOG.0b013e31825d33d9. PubMed DOI

Clausen F.B., Christiansen M., Steffensen R., Jørgensen S., Nielsen C., Jakobsen M.A., Madsen R.D., Jensen K., Krog G.R., Rieneck K., et al. Report of the first nationally implemented clinical routine screening for fetal RHD in D- pregnant women to ascertain the requirement for antenatal RhD prophylaxis. Transfusion. 2012;52:752–758. doi: 10.1111/j.1537-2995.2011.03362.x. PubMed DOI

Böhmová J., Vodička R., Ľubušký M., Studničková M., Holusková I., Vrtěl R., Kratochvílová R., Frydrychová M., Krejčiříková E., Filipová H. Stanovení RHD genotypu plodu z plazmy periferní krve těhotné ženy a posouzení citlivosti nových diagnostických postupů pro zavedení do klinické praxe [RHD genotyping from cell-free fetal DNA circulating in pregnant women peripheral blood and sensitivity assessment of innovated diagnostic approaches for introduction into the clinical practice] Ces. Gynek. 2013;78:32–40. PubMed

Svobodová I., Pazourková E., Hořínek A., Novotná M., Calda P., Korabečná M. Performance of Droplet Digital PCR in non-invasive fetal RHD genotyping-comparison with a routine real-time PCR based approach. PLoS ONE. 2015;10:e0142572. doi: 10.1371/journal.pone.0142572. PubMed DOI PMC

Sillence K.A., Roberts L.A., Hollands H.J., Thompson H.P., Kiernan M., Madgett T.E., Welch C.R., Avent N.D. Fetal sex and RHD genotyping with digital PCR demonstrates greater sensitivity than real-time PCR. Clin. Chem. 2015;61:1399–1407. doi: 10.1373/clinchem.2015.239137. PubMed DOI

Kimura M., Sato C., Hara M., Ishihara O., Ikebuchi K. Noninvasive fetal RHD genotyping by maternal plasma with capillary electrophoresis. Transfusion. 2008;48:1156–1163. doi: 10.1111/j.1537-2995.2008.01681.x. PubMed DOI

Wienzek-Lischka S., Bachmann S., Fröhner V., Bein G. Potential of next-generation sequencing in noninvasive fetal molecular blood group genotyping. Transfus. Med. Hemother. 2020;47:14–22. doi: 10.1159/000505161. PubMed DOI PMC

Wienzek-Lischka S., Krautwurst A., Fröhner V., Hackstein H., Gattenlöhner S., Bräuninger A., Axt-Fliedner R., Degenhardt J., Deisting C., Santoso S., et al. Noninvasive fetal genotyping of human platelet antigen-1a using targeted massively parallel sequencing. Transfusion. 2015;55:1538–1544. doi: 10.1111/trf.13102. PubMed DOI

Orzińska A., Guz K., Mikula M., Kluska A., Balabas A., Ostrowski J., Uhrynowska M., Kopeć I., Debska M., Luterek K., et al. Prediction of fetal blood group and platelet antigens from maternal plasma using next-generation sequencing. Transfusion. 2020;60:884. doi: 10.1111/trf.15116. PubMed DOI

Vodička R., Vrtěl R., Scheinost O., Zapletalová J., Dušek L., Geierová M., Šantavý J. Refined quantitative fluorescent PCR of Y-chromosome DNA sequences mosaics in Turner’s syndrome patients--alternative to real-time PCR. J. Biochem. Biophys. Methods. 2004;60:151–162. doi: 10.1016/j.jbbm.2004.05.004. PubMed DOI

Vodička R., Vrtěl R., Procházka M., Šantavá A., Dušek L., Vrbická D., Singh R., Krejčiříková E., Schneiderová E., Šantavý J. Analýza volné fetální DNA v maternální plazme s vyuźitím STR lokusů [Analysis of free foetal DNA in maternal plasma using STR loci] Cas. Lek. Cesk. 2006;145:133–137. PubMed

Page K., Hava N., Ward B., Brown J., Guttery D.S., Ruangpratheep C., Blighe K., Sharma A., Walker R.A., Coombes R.C., et al. Detection of HER2 amplification in circulating free DNA in patients with breast cancer. Br. J. Cancer. 2011;104:1342–1348. doi: 10.1038/bjc.2011.89. PubMed DOI PMC

Sikora A., Zimmermann B.G., Rusterholz C., Birri D., Kolla V., Lapaire O., Hoesli I., Kiefer V., Jackson L., Hahn S. Detection of increased amounts of cell-free fetal DNA with short PCR amplicons. Clin. Chem. 2010;56:136–138. doi: 10.1373/clinchem.2009.132951. PubMed DOI

Nygren A.O.H., Dean J., Jensen T.J., Kruse S., Kwong W., van den Boom D., Ehrich W. Quantification of fetal DNA by use of methylation-based DNA discrimination. Clin. Chem. 2010;56:1627–1635. doi: 10.1373/clinchem.2010.146290. PubMed DOI

Fan H.C., Blumenfeld Y.J., Chitkara U., Hudgins L., Quake S.R. Analysis of the size distributions of fetal and maternal cell-free DNA by paired-end sequencing. Clin. Chem. 2010;56:1279–1286. doi: 10.1373/clinchem.2010.144188. PubMed DOI

Lun F.M.F., Chiu R.W.K., Chan K.C.A., Leung T.Y., Lau T.K., Lo Y.M.D. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin. Chem. 2008;54:1664–1672. doi: 10.1373/clinchem.2008.111385. PubMed DOI

Vodička R., Vrtěl R., Dušek L., Procházka M., Schneiderová E., Vrbická D., Krejčiříková E., Dhaifalah I., Šantavá A., Šantavý J. Refined fluorescent STR quantification of cell-free fetal DNA during pregnancy in physiological and Down syndrome fetuses. Prenat. Diagn. 2008;28:425–433. doi: 10.1002/pd.1996. PubMed DOI

Denis M.G., Knol A.-C., Théoleyre S., Vallée A., Dréno B. Efficient detection of BRAF mutation in plasma of patients after long-term storage of blood in cell-free DNA blood collection tubes. Clin. Chem. 2015;61:886–888. doi: 10.1373/clinchem.2015.238352. PubMed DOI

Toro P.V., Erlanger B., Beaver J.A., Cochran R.L., VanDenBerg D.A., Yakim E., Cravero K., Chu D., Zabransky D.J., Wong H.Y., et al. Comparison of cell stabilizing blood collection tubes for circulating plasma tumor DNA. Clin. Biochem. 2015;48:993–998. doi: 10.1016/j.clinbiochem.2015.07.097. PubMed DOI PMC

van Dessel L.F., Beije N., Helmijr J.C.A., Vitale S.R., Kraan J., Look M.P., de Wit R., Sleijfer S., Jansen M.P.H.M., Martens J.W.M., et al. Application of circulating tumor DNA in prospective clinical oncology trials-standardization of preanalytical conditions. Mol. Oncol. 2017;11:295–304. doi: 10.1002/1878-0261.12037. PubMed DOI PMC

Barrett A.N., Zimmermann B.G., Wang D., Holloway A., Chitty L.S. Implementing prenatal diagnosis based on cell-free fetal DNA: Accurate identification of factors affecting fetal DNA yield. PLoS ONE. 2011;6:e25202. doi: 10.1371/journal.pone.0025202. PubMed DOI PMC

Board R.E., Williams V.S., Knight L., Shaw J., Greystoke A., Ranson M., Dive C., Blackhall F.H., Hughes A. Isolation and extraction of circulating tumor DNA from patients with small cell lung cancer. Ann. N. Y. Acad. Sci. 2008;1137:98–107. doi: 10.1196/annals.1448.020. PubMed DOI

Kadam S.K., Farmen M., Brandt J.T. Quantitative measurement of cell-free plasma DNA and applications for detecting tumor genetic variation and promoter methylation in a clinical setting. J. Mol. Diagn. 2012;14:346–356. doi: 10.1016/j.jmoldx.2012.03.001. PubMed DOI

Norton S.E., Lechner J.M., Williams T., Fernando M.R. A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR. Clin. Biochem. 2013;46:1561–1565. doi: 10.1016/j.clinbiochem.2013.06.002. PubMed DOI

van Ginkel J.H., van den Broek D.A., van Kuik J., Linders D., de Weger R., Willems S.M., Huibers M.H.M. Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics. Cancer Med. 2017;6:2297–2307. doi: 10.1002/cam4.1184. PubMed DOI PMC

Liggett T., Melnikov A., Yi Q.-L., Replogle C., Brand R., Kaul K., Talamonti M., Abrams R.A., Levenson V. Differential methylation of cell-free circulating DNA among patients with pancreatic cancer versus chronic pancreatitis. Cancer. 2010;116:1674–1680. doi: 10.1002/cncr.24893. PubMed DOI

Christensen E., Nordentoft I., Vang S., Birkenkamp-Demtröder K., Jensen J.B., Agerbæk M., Pedersen J.S., Dyrskjøt L. Optimized targeted sequencing of cell-free plasma DNA from bladder cancer patients. Sci. Rep. 2018;8 doi: 10.1038/s41598-018-20282-8. PubMed DOI PMC

Wang B.G., Huang H.-Y., Chen Y.-C., Bristow R.E., Kassauei K., Cheng C.-C., Roden R., Sokoll L.J., Chan D.W., Shih E.-M. Increased plasma DNA integrity in cancer patients. Cancer Res. 2003;63:3966–3968. PubMed

Ren C.C., Miao X.H., Yang B., Zhao L., Sun R., Song W.Q. Methylation status of the fragile histidine triad and E-cadherin genes in plasma of cervical cancer patients. Int. J. Gynecol. Cancer. 2006;16:1862–1867. doi: 10.1111/j.1525-1438.2006.00669.x. PubMed DOI

Pinzani P., Salvianti F., Zaccara S., Massi D., De Giorgi V., Pazzagli M., Orlando C. Circulating cell-free DNA in plasma of melanoma patients: Qualitative and quantitative considerations. Clin. Chim. Acta. 2011;412:2141–2145. doi: 10.1016/j.cca.2011.07.027. PubMed DOI

Kamel A.M., Teama S., Fawzy A., El Deftar M. Plasma DNA integrity index as a potential molecular diagnostic marker for breast cancer. Tumour Biol. 2016;37:7565–7572. doi: 10.1007/s13277-015-4624-3. PubMed DOI

Leng S., Zheng J., Jin Y., Zhang H., Zhu Y., Wu J., Xu Y., Zhang P. Plasma cell-free DNA level and its integrity as biomarkers to distinguish non-small cell lung cancer from tuberculosis. Clin. Chim. Acta. 2018;477:160–165. doi: 10.1016/j.cca.2017.11.003. PubMed DOI

Hu P., Liang D., Chen Y., Lin Y., Qiao F., Li H., Wang T., Peng C., Luo D., Liu H., et al. An enrichment method to increase cell-free fetal DNA fraction and significantly reduce false negatives and test failures for non-invasive prenatal screening: A feasibility study. J. Transl. Med. 2019;17 doi: 10.1186/s12967-019-1871-x. PubMed DOI PMC

Jain M., Balatsky A.V., Revina D.B., Samokhodskaya L.M. Direct comparison of QIAamp DSP Virus Kit and QIAamp Circulating Nucleic Acid Kit regarding cell-free fetal DNA isolation from maternal peripheral blood. Mol. Cell Probes. 2019;43:13–19. doi: 10.1016/j.mcp.2018.12.006. PubMed DOI

Wagner F.F., Gassner C., Müller T.H., Schönitzer D., Schunter F., Flegel W.A. Molecular basis of weak D phenotypes. Blood. 1999;93:385–393. doi: 10.1182/blood.V93.1.385. PubMed DOI

Flegel W.A. Molecular genetics and clinical applications for RH. Transfus. Apher. Sci. 2011;44:81–91. doi: 10.1016/j.transci.2010.12.013. PubMed DOI PMC

Colin Y., Chérif-Zahar B., Le van Kim C., Raynal V., Van Huffel V., Cartron J.P. Genetic basis of the RhD-positive and RhD - negative blood group polymorphism as determined by Southern analysis. Blood. 1991;78:2747–2752. doi: 10.1182/blood.V78.10.2747.2747. PubMed DOI

Wagner F.F., Flegel W.A. RHD gene deletion occurred in the Rhesus box. Blood. 2000;95:3662–3668. doi: 10.1182/blood.V95.12.3662. PubMed DOI

Pirelli K.J., Pietz B.C., Johnson S.T., Pinder H.L., Bellissimo D.B. Molecular determination of RHD zygosity: Predicting risk of hemolytic disease of the fetus and newborn related to anti-D. Prenat. Diagn. 2010;30:1207–1212. doi: 10.1002/pd.2652. PubMed DOI

Daniels G. Human Blood Groups. 2nd ed. Blackwell Science; Oxford, UK: 2002.

Singleton B.K., Green C.A., Avent N.D., Martin P.G., Smart E., Daka A., Narter-Olaga E.G., Hawthorne L.M., Daniels G. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in Africans with the Rh D-negative blood group phenotype. Blood. 2000;95:12–18. doi: 10.1182/blood.V95.1.12. PubMed DOI

Faas B.H., Beckers E.A., Wildoer P., Ligthart P.C., Overbeeke M.A., Zondervan H.A., von dem Borne A.E., van der Schoot C.E. Molecular background of vs. and weak C expression in blacks. Transfusion. 1997;37:38–44. doi: 10.1046/j.1537-2995.1997.37197176949.x. PubMed DOI

Daniels G.L., Faas B.H., Green C.A., Smart E., Maaskant-van Wijk P.A., Avent N.D., Zondervan H.A., von dem Borne A.E., van der Schoot C.E. The vs. and V blood group polymorphisms in Africans: A serologic and molecular analysis. Transfusion. 1998;38:951–958. doi: 10.1046/j.1537-2995.1998.381098440860.x. PubMed DOI

Takahashi K., Migita O., Sasaki A., Nasu M., Kawashima A., Sekizawa A., Sato T., Ito Y., Sago H., Okamoto A., et al. Amplicon Sequencing-Based Noninvasive Fetal Genotyping for RHD-Positive D Antigen-Negative Alleles. Clin. Chem. 2019;65:1307–1316. doi: 10.1373/clinchem.2019.307074. PubMed DOI

Hromadníková I., Veselá K., Benešová B., Nekovářová D., Dušková D., Vlk R., Špálová I., Gerychová R., Hakenová A., Rosenbaumová Z., et al. Non-invasive fetal RHD and RHCE genotyping from maternal plasma in alloimmunized pregnancies. Prenat. Diagn. 2005;25:1079–1083. doi: 10.1002/pd.1282. PubMed DOI

Risk Minimization of Hemolytic Disease of the Fetus and Newborn Using Droplet Digital PCR Method for Accurate Fetal Genotype Assessment of RHD, KEL, and RHCE from Cell-Free Fetal DNA of Maternal Plasma