American Journal of Obstetrics and Gynecology (2005) 192, 666–9 www.ajog.org EDITORS’ CHOICE Fetal RhD genotyping by maternal serum analysis: A two-year experience Evelyne Gautier, MD,a Alexandra Benachi, MD,b Yves Giovangrandi, MD,c Pauline Ernault,a Martine Olivi,a Thierry Gaillon,a Jean-Marc Costaa,* Centre de Diagnostic Pre´natal, American Hospital of Paris, Neuilly, France,a Maternite´, Hoˆpital Necker-Enfants Malades, Paris, France,b and Maternite´, Hoˆpital Notre-Dame de Bon Secours, Paris, Francec Received for publication June 29, 2004; revised October 1, 2004; accepted October 28, 2004 KEY WORDS Fetal DNA Maternal serum RHD genotype Objective: The purpose of this study was to determine the accuracy of the none-invasive prenatal determination of polymerase chain reaction (PCR)-based fetal RhD genotyping. Study design: A prospective case series was undertaken on all RhD-negative pregnant women presenting for genetic counseling in our prenatal diagnosis center from January 2001 until December 2002. Results were compared with serologic RhD typing of the newborns. Results: Among the 285 pregnant women who participated in the study, fetal RhD status could be determined for 283 patients. In 2 cases, the RhD-negative phenotype of the mother was not the result of a complete RHD gene deletion, and therefore, the status of the fetus could not be determined. Neither false-negative nor false-positive results were observed. Conclusion: The present report demonstrates that a reliable fetal RHD genotype determination can be achieved with 100% accuracy. It is therefore possible to consider that such an assay could be systematically proposed to all RhD-negative pregnant women in order to more effectively utilize RhD prophylaxis. Ó 2005 Elsevier Inc. All rights reserved. The incidence of hemolytic disease of the fetus/ newborn has dropped dramatically (1 to 6 per 1000 live births) since 1968, when eﬀective prophylaxis by anti-D immunoglobulin injection to the mother became available. However, administration of blood derivatives is not devoid of risk, and every eﬀort should be made to improve techniques that could reduce the number of injections.1 Therefore, the new possibility of fetal RhD genotyping using polymerase chain reaction (PCR) is * Reprint requests: Jean-Marc Costa, Centre de Diagnostic Pre´natal, American Hospital of Paris, 63 bd Victor Hugo, 92200 Neuillysur-Seine, France. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ajog.2004.10.632 a signiﬁcant advance for clinical purposes. Fetal RhD genotype determination may also be useful in the management of RhD-negative sensitized women, but genotyping is also useful in RhD-negative pregnant women at risk for RhD immunization2 in order to avoid unnecessary administration of anti-D immunoglobulin in the case of an RhD-negative fetus. Although anecdotal diagnosis has been made using circulating fetal DNA (ie, myotonic dystrophy,3 achondroplasia4), most of the studies on the accuracy of fetal circulating DNA analysis have focused on both Y chromosome and RHD gene. In the past few years, some studies have reported a 100% accuracy in fetal sex determination in the ﬁrst trimester.5,6 This approach has Gautier et al 667 Real-time PCR for the RHD gene in maternal serum Figure Detection of the RHD gene in maternal serum using real-time PCR.Typical results observed from patient samples: one giving a positive result for fetal DNA (1), the second a negative one (2), and the third one corresponding to an RhDnegative woman but carrying the RHD gene in her genome (3). now modiﬁed the strategy of prenatal diagnosis in Xlinked disorders,7 as well as the management of pregnancies at risk for congenital adrenal hyperplasia.8 The use of cell-free DNA in maternal plasma or serum has also been achieved for noninvasive fetal RhD genotyping by several groups,9-13 but most often on a limited number of cases. More recently, 2 large studies14,15 have been conducted on 137 and 893 patients, respectively (most of them being alloimmunized), before implementation of routine clinical practice.14 In the present publication, we report the results of our 2-year experience in the noninvasive prenatal determination of fetal RhD genotyping routinely proposed in a prenatal diagnosis center. Material and methods Patients and samples Noninvasive prenatal determination by maternal serum analysis was systematically proposed to every RhDnegative pregnant woman presenting for genetic counseling in our prenatal diagnosis center from January 2001 to December 2002. Two hundred and eighty-ﬁve women elected to participate; all were of Caucasian origin but none were alloimmunized. After informed consent, 5 mL of blood were collected into Vacutainer SSTÒ tubes (Becton Dickinson, Meylan, France) before amniocentesis or CVS if indicated. Anti-D immunoglobulin was not provided to patients whose fetus tested to be RhD-negative. Immediately after clotting, serum was obtained by centrifugation for 10 minutes at 3000g, aliquoted and stored at 80(C until further processing if the assay was not performed on the same day. The mean gestational age was 15.2 weeks, ranging from 8 to 35. The procedure was similar to that previously described.5,13 Brieﬂy, as tracer for the DNA extraction and ampliﬁcation steps, a low amount (250 pg) of mouse DNA (Sigma, Grenoble, France) was added to each patient’s sample (400 mL of serum) immediately before DNA extraction. Total DNA was then extracted by the PCR Template Preparation Kit (Roche Biochemicals, Meylan, France), and the adsorbed DNA eluted with 50 mL of elution buﬀer, 10 mL of which was used per PCR reaction. Ampliﬁcation was carried out in a LightCyclerÒ instrument (Roche Biochemicals). PCR reactions were set up in a ﬁnal volume of 20 mL using the Fast DNA Master Hybridization Probes Kit (Roche Biochemicals), with 0.5 mmol/L of each primer, 0.25 mmol/L of each probe (Proligo, France) (Table), 1.25 units of uracil DNA glycosylase (UDG) (Biolabs, Saint-Quentin en Yvelines, France), 4.75 mmol/L of magnesium chloride. After an initial 1-minute incubation at 50(C, a ﬁrst denaturation step of 8 minutes at 95(C was followed by an ampliﬁcation performed for 50 cycles of denaturation (95(C, 10 seconds, ramping rate 20(C/second), annealing (56(C, 10 seconds, ramping rate 20(C/second), and extension (72(C, 20 seconds, ramping rate 2(C/second). Each sample was treated twice for DNA extraction, and the RHD assay was performed in duplicate on each DNA extract. Deﬁnitive results were considered only when the 4 PCR reactions were concordant. During each run, sera obtained from patients carrying an RhDpositive or an RhD-negative fetus were used as a positive and a negative control. The results were compared with those obtained later in pregnancy on amniotic ﬂuid cells and by RHD serology of the newborn. Results Among the 285 pregnant women tested for RHD gene presence in their serum, the status of the fetus could not be determined in 2 cases because the RhD-negative phenotype of the mother was not in relation with a complete RHD gene deletion. These 2 particular cases were easily identiﬁed using real-time PCR because the amount of RHD sequences detected in maternal serum was abnormally high (expressed by an unusual crossing point) to be from fetal origin (Figure). Because the maternal DNA represents the major part of DNA isolated from serum, it can be concluded that RHD sequences were present in the maternal genome. This hypothesis was conﬁrmed by analysis of DNA extracted from the maternal leukocytes. Fetal RhD genotype could be determined from all other 283 maternal sera. In 179 cases, RHD sequences had been detected in maternal serum, and the fetuses 668 Table Gautier et al Characteristics and sequences of primers and probes used in the PCR assay Gene target Primers sequences Probes sequences Human RHD exon 10 50 -GCCTGCATTTGTACGTGAGA-30 50 -CAAAGAGTGGCAGAGAAAGGA-30 30 FITC-TGACAGCAAAGTCTCCAATGTTCG 50 LCRed640-GCAGGCACTGGAGTCAGAGAAAA-30 Ph could therefore be considered as RhD-positive, while the others (n = 104) were RhD negative. For 11 patients, the result obtained by maternal serum analysis could not be conﬁrmed due to an early pregnancy termination in relation to fetal chromosomal abnormality (3 cases), or because the patients were lost to follow-up (8 cases). The results of the fetal RHD genotyping on maternal serum could be controlled for the other 272 patients either by analysis of amniotic ﬂuids for the presence of RHD gene (n = 209) and/or by serologic study of the newborn (n = 232). Results were in complete concordance, and neither false-negative nor false-positive results were observed; all sera from women carrying an RhD-positive fetus (n = 170) gave positive results for RHD gene detection, while all sera from women carrying an RhD-negative fetus (n = 102) gave negative results. Speciﬁcity and sensitivity of the assay were 100% (95% CI: 98-100). Comment Fetal RHD genotype can be determined with a high level of accuracy by analysis of fetal DNA circulating in maternal plasma and serum. It is therefore possible to consider that such an assay could be included in prenatal care of RhD-negative women.16 The results of a 2-year experience of such a clinical practice are reported herein. In 2 cases, the pregnant women did not carry a complete deletion of the RHD gene and a ﬁnal answer regarding the fetal RhD status could not be provided. Although a thorough analysis was not undertaken, theses patients were highly suspected of carrying a RHD pseudogene or variant. Except for these 2 pregnant women, the fetal RhD status could be addressed for the other 283 patients who had accepted the test (overall success rate 99.3%). The result was in complete concordance with that obtained either by analysis of amniotic ﬂuid for the presence of RHD gene (n = 209) and/or by serologic study of the newborn (n = 232). Therefore, the present report demonstrates that a reliable fetal RHD genotype determination can be achieved with 100% accuracy. Although the presence of fetal DNA cannot be formally proven in maternal serum in case of RhD-negative fetus, a falsenegative result was never observed in our experience. This was the result of the use of a similar procedure successfully used for many years for fetal sex determination7 that includes a careful ultrasonographic control of the date of the pregnancy, the assay never being realized before 8 weeks. On the other hand, a false positive due to the transmission by the father of a silent RHD gene (women carrying such genes being easily detected by our assay) could not be excluded in the future. The patients were systematically informed about this without any medical risk possibility during genetic counseling. Among the studied women, 104 (36.8%) carried an RhD-negative fetus. Injection of anti-D immune globin was thus avoided, and pregnancy follow-up was therefore made easier, decreasing the patient’s anxiety. Determination of fetal RHD genotype using a noninvasive approach is an important challenge because it has crucial implication in the clinical management of sensitized RhD-negative pregnant women. Fetal DNA analysis in maternal serum oﬀers the opportunity to achieve this challenge. Attempts to identify the RhD-negative women who are at risk of sensitization contribute to a more eﬃcient prophylaxis of alloimmunization in obstetrics being targeted speciﬁcally at these women. Our data could be considered as part of a novel strategy in order to maintain and increase anti-D supplies, as well as (1) the use of minidose of anti-D in some circumstances (ie, ﬁrst trimester indications), (2) increased and accurate use of tests to assess feto-maternal hemorrhage, and (3) increase compliance with guidelines on the use of anti-D. Moreover, while eﬀorts are made to ensure that the blood used for the anti-D preparation is safe and free of human pathogens, it cannot be formally excluded that an infectious agent is present because of lack of availability or sensitivity of tests at present.17 The high accuracy rate achieved in the prediction of fetal D status has resulted in the implementation of a noninvasive fetal RHD genotyping service. Although this test is actually performed by a limited number of specialized laboratories in the world, increasing demand is anticipated in the near future. As a consequence, the actual ‘‘made-by-hand’’ assays must move towards more automated ones (until the availability of commercial tests) in order to ensure robustness and reliability. This major point was recently highlighted by an interlaboratory study.18 Therefore, time has come for an external quality control scheme. Finally, large-scale studies regarding the economic aspect of such routine oﬀer of fetal RhD genotyping are necessary. A consideration of cost-eﬀectiveness is particularly important because of the limited supply of anti-D Gautier et al available. These economic evaluations must be countrydependent because guidelines for anti-D use, if existing, may vary from one country to the other and from centers where no recommendations exist. Such a study is actually in progress in France. Attempts to predict other fetal blood groups (ie, Kell, Duﬀy) by maternal blood analysis is currently under investigation by some groups. However, the accurate, sensitive, and reliable determination of such ‘‘single base’’ modiﬁcations are diﬃcult to achieve using the actual methods caused by the presence of a high background of negative maternal DNA. Acknowledgments We are indebted to Dr Jocelyn McGinnis for reviewing this manuscript. References 1. Moise KJ Jr. Management of rhesus alloimmunization in pregnancy. Obstet Gynecol 2002;100:600-11. 2. American College of Obstetrics and Gynecology. ACOG practice bulletin. Prevention of RhD alloimmunization. Clinical management guidelines for obstetrician-gynecologists. Int J Gynaecol Obstet 1999;66:63-70. 3. Amicucci P, Gennarelli M, Novelli G, Dallapiccola B. Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem 2000;46:301-2. 4. Saito H, Sekizawa A, Morimoto T, Suzuki M, Yanaihara T. Prenatal DNA diagnosis of a single-gene disorder from maternal plasma. Lancet 2000;356:1170. 5. 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Large-scale pre-diagnosis study of fetal RHD genotyping by PCR on plasma DNA from RhD-negative pregnant women. Mol Diagn 2004;8:23-31. 16. Van Der Schoot CE, Tax GH, Rijnders RJ, De Haas M, Christiaens GC. Prenatal typing of Rh and Kell blood group system antigens: the edge of a watershed. Transfus Med Rev 2003;17:31-44. 17. Power JP, Davidson F, O’Riordan J, Simmonds P, Yap PL, Lawlor P. Hepatitis C infection from anti-D immunoglobulin. Lancet 1995;346:372-3. 18. Johnson KL, Dukes KA, Vidaver J, Leshane ES, Ramirez I, Weber WD, et al. Interlaboratory comparison of fetal male DNA detection from common maternal plasma by real-time PCR. Clin Chem 2004;50:516-21.
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