Non-invasive Fetal RHD Genotyping Research Articles

The determination of the fetal D status from maternal plasma for decision making on Rh prophylaxis is feasible

Noninvasive fetal RHD genotyping might become a valuable tool in decision making on antenatal Rh prophylaxis, which is currently in routine practice for all D- pregnancies in several countries. This study provides a large-scale validation study of this technology to address questions concerning feasibility and applicability of its introduction into clinical routine. Real-time polymerase chain reaction (PCR) targeting RHD Exons 5 and 7 was applied for the detection of fetal-specific RHD sequences in maternal plasma. A total of 1113 women in 6 to 32 weeks (median, Week 25) of pregnancy were recruited. All of them were serologically typed as D- according to current German guidelines. DNA was extracted via a spin-column method and a novel automated approach using magnetic tips. Real-time PCR results were compared with postnatal serology and discrepancies further elucidated by DNA sequencing from a newborn's buccal swab. Sensitivities of fetal RHD genotyping were 99.7 percent (spin columns) and 99.8 percent (magnetic tips), thus comparable with serology (99.5%). The detection of weak D variants was more reliable by real-time PCR. Specificities of fetal RHD genotyping were 99.2 percent (spin columns) and 98.1 percent (magnetic tips), which is lower than serology (>99.7%). Automation achieved significantly higher yields of cell-free fetal DNA. This prospective clinical trial revealed that routine determination of the fetal D status from maternal plasma is feasible. Noninvasive fetal RHD genotyping can be considered as sensitive as the traditional postnatal serologic assay.

Noninvasive fetal RHD genotyping by maternal plasma with capillary electrophoresis

Recently, a more accurate and reliable screening test has been investigated for noninvasive prenatal fetal RHD genotyping from D- women. The objective of this study was to perform the new method of noninvasive fetal RHD genotyping with maternal plasma from D- women by use of capillary electrophoresis. Blood samples were obtained from 8 D+ and 8 D- nonpregnant donors and mixed to make test plasma samples. DNA was extracted and the appropriate conditions relating to the initial sample volume as well as polymerase chain reaction cycle numbers were analyzed to detect the RHD gene with RHD exon 10 primer. Blood samples were also obtained from 13 D- pregnant women ranging from the 12th to 39th weeks of gestation. The presence of the RHD gene and Y-chromosome-specific STR (Y-STR) derived from the fetus was analyzed. The results were compared with the D status of newborns. In samples from 12 D- pregnant women, the RHD gene was detected. In one sample, the RHD gene was not detected but Y-STR loci were demonstrated in this sample, indicating a D- male baby. The results of fetal genotyping were all in concordance with the postpartum samples by serologic tests on D as well as with the sex of newborns. Capillary electrophoresis can be used for the determination of fetal RHD status in D- women. This diagnostic method is useful for the noninvasive prenatal diagnosis of the fetal RHD genotyping from D- women.

Noninvasive fetal RHD genotyping from maternal plasma : Use of a new developed Free DNA Fetal Kit RhD ®

Fetal RHD genotyping from maternal plasma was performed by real-time PCR amplification of exons 7 and 10 of the RHD gene and the amplified products were detected either with SYBR Green I dye according to our previously published method [Mol Diagn 8 (2004) 23–31] or with hydrolysis probes in a new Free DNA Fetal Kit RhD ®. Plasma specimen from 300 RhD-negative pregnant women (between 10 to 34 weeks of gestation) were analysed and validation of the results was ascertained either by RHD genotyping on amniotic cells or by blood typing of the neonate at birth. We found 100% concordant results when comparing the two methods. Two false-positive but no false-negative results were found. Thus, the sensitivity of the assay was 100% and the specificity superior than 99%. These data confirm the accuracy of fetal RHD genotyping on maternal plasma using the Free DNA Fetal Kit RhD ®, thus allowing to propose non invasive PCR-based fetal RHD genotyping for all RhD-negative pregnant women and to restrict the use of anti-D immunoglobulins only to those bearing an RhD-positive fetus.

Non-invasive fetal RHD genotyping by real-time polymerase chain reaction using plasma from D-negative Brazilian pregnant women

Non-invasive fetal RHD genotyping by real-time polymerase chain reaction using plasma from D-negative Brazilian pregnant women

Non-invasive Fetal RHD and RHCE Genotyping Using Real-time PCR Testing of Maternal Plasma in RhD-negative Pregnancies

We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation.

Non-invasive fetal RHD and RHCE genotyping from maternal plasma in alloimmunized pregnancies

In this prospective study, we assessed the feasibility of fetal RH genotyping by analysis of DNA extracted from maternal plasma samples of alloimmunized pregnant women using real-time PCR and primers and probes targeted toward RHD (exon 7 and exon 10) and RHCE (intron 2 and exon 5) genes. We analysed 23 alloimmunized pregnant women (16 anti-D, 5 anti-D + C, 2 anti-E) at risk of haemolytic disease of the newborn (HDN) within 11th and 37th week of pregnancy and correlated the results with serological analysis of cord blood. Detection of the presence of the RHD gene, the C and/or E alleles of the RHCE gene in maternal plasma samples is highly accurate and enables implementation in a clinical diagnostic algorithm for following pregnancies at risk for HDN. The absence of RHD gene, the C and/or E alleles of RHCE gene in the current pregnancy excludes the risk of HDN caused by anti-D, anti-C and/or anti-E alloantibodies and the performance of invasive fetal-blood sampling.

Prediction of fetal D status from maternal plasma: introduction of a new noninvasive fetal RHD genotyping service.

Invasive procedures to obtain fetal DNA for prenatal blood grouping present a risk to the fetus. During pregnancy, cell-free fetal DNA is present in maternal blood. The detection of RHD sequences in maternal plasma has been used to predict fetal D status, based on the assumption that RHD is absent in D- genomes. Real-time PCR assays were designed to distinguish RHD from RHDpsi (possessed by the majority of D- black Africans). Plasma-derived DNA from 137 D- women was subjected to real-time PCR to detect fetal RHD and Y chromosome-associated SRY sequences. The accuracy of RHD genotyping from maternal plasma was investigated by comparing results with those obtained by conventional RHD genotyping from fetal tissue or serologic tests on the infant's RBCs. The quantity of fetal DNA in maternal plasma was investigated in 94 pregnancies. Fetal D status was predicted with 100-percent accuracy from maternal plasma. The number of copies of fetal DNA in maternal plasma was found to increase with gestation. Combination of the sensitivity of real-time PCR with an improved RHD typing assay to distinguish RHD from RHDpsi enables highly accurate prediction of fetal D status from maternal plasma. This has resulted in the implementation of a clinical noninvasive fetal RHD genotyping service.

Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma.

The ability to determine fetal RhD Status noninvasively is useful in the treatment of RhD-sensitized pregnant women whose partners are heterozygous for the RhD gene. The recent demonstration of fetal DNA in maternal plasma raises the possibility that fetal RhD genotyping may be possible with the use of maternal plasma. We studied 57 RhD-negative pregnant women and their singleton fetuses. DNA extracted from maternal plasma was analyzed for the RhD gene with a fluorescence-based polymerase-chain-reaction (PCR) test sensitive enough to detect the RhD gene in a single cell. Fetal RhD status was determined directly by serologic analysis of cord blood or PCR analysis of amniotic fluid. Among the 57 RhD-negative women, 12 were in their first trimester of pregnancy, 30 were in their second trimester, and 15 were in their third trimester. Thirty-nine fetuses were RhD-positive, and 18 were RhD-negative. In the samples obtained from women in their second or third trimester of pregnancy, the results of RhD PCR analysis of maternal plasma DNA were completely concordant with the results of serologic analysis. Among the maternal plasma samples collected in the first trimester, 2 contained no RhD DNA, but the fetuses were RhD-positive; the results in the other 10 samples were concordant (7 were RhD-positive, and 3 RhD-negative). Noninvasive fetal RhD genotyping can be performed rapidly and reliably with the use of maternal plasma beginning in the second trimester of pregnancy.

Fetal RhD genotyping in fetal cells flow sorted from maternal blood

OBJECTIVE: The aim of this study was to determine the accuracy of noninvasive fetal RhD genotyping by fetal cell isolation from maternal blood. STUDY DESIGN: Candidate fetal cells from 18 pregnant women (one twin gestation) were flow-sorted. Polymerase chain reaction amplification of a 261 bp fragment of the RhD gene was performed on sorted fetal cells. The presence of amplified product was considered predictive of the RhD-positive genotype in the fetus. RESULTS: Sixteen of the 19 fetal RhD genotypes were correctly predicted in fetal cells isolated from maternal blood (10 were Rh positive, 6 were Rh negative). In 3 cases no amplification products were detected in RhD-positive fetuses. The association between presence of the fragment and RhD-positive genotype was significant ( p = 0.003, Fisher's exact test). CONCLUSIONS: Noninvasive prenatal diagnosis of the fetal RhD genotype is feasible. Absence of amplification products in the reaction requires confirmation that fetal material is present. Improvements in fetal cell purity and yield should increase diagnostic accuracy, although the current protocol has a positive predictive value of 100% and a negative predictive value of 67%. (A M J O BSTET G YNECOL 1996;174:818-22.)