Use of cffDNA to avoid administration of anti-D to pregnant women when the fetus is RhD-negative: implementation in the NHS.

OBJECTIVE: To determine whether a policy of offering cffDNA testing to all RhD-negative women at about 16 weeks' gestation to avoid anti-D administration when the fetus is RhD-negative could be implemented successfully in the NHS without additional funding. DESIGN: Prospectively planned observational service implementation pilot and notes audit. SETTING: Three maternity services in the South West of England. POPULATION: All RhD-negative women in a 6-month period. METHODS: Prospective, intervention, cross-sectional observational study, using pre-intervention data as controls. MAIN OUTCOME MEASURES: Proportion of suitable women who offered and accepted the test. Accuracy of the cffDNA result as assessed by cord blood group result. Fall in anti-D doses administered. RESULTS: 529 samples were received; three were unsuitable. The results were reported as RhD-positive (n = 278), RhD-negative (n = 185) or inconclusive, treat as positive (n = 63). Cord blood results were available in 502 (95%) and the only incorrect result was one case of a false positive (cffDNA reported as positive, cord blood negative - and so given anti-D unnecessarily). The notes audit showed that women who declined this service were correctly managed and that anti-D was not given when the fetus was predicted to be RhD-negative. The total use of anti-D doses fell by about 29% which equated to about 35% of RhD-negative women not receiving anti-D in their pregnancy unnecessarily. CONCLUSIONS: We recommend this service is extended to all UK NHS services.

Anti-D prophylaxis: past, present and future.

The new British Committee for Standards in Haematology (BCSH) guidelines for the use of anti-D immunoglobulin in pregnancy provide a welcome clarification of the use of anti-D in ectopic pregnancy and after red cell salvage during caesarean section, of dosing with different preparations and distinguishing non-immune and immune anti-D. The routine use of anti-D prophylaxis (RAADP) to prevent Rhesus (Rh) D alloimmunisation during the third trimester is well established and requires careful and well-audited local implementation to achieve the maximum public health benefit. In the UK, such scrutiny may be provided by the reporting of failed anti-D prophylaxis at women who have produced an immune anti-D that is detectable for the first time in the current pregnancy through the voluntary Serious Hazards of Transfusion reporting scheme (SHOT). Application of fetal RHD genotyping would avoid giving anti-D to RhD negative women carrying an RhD negative fetus. RAADP is directed by fetal RHD genotyping in some countries in Northern Europe led by the Netherlands and Denmark. The economic case for RAADP directed by fetal RHD genotyping needs to be carefully evaluated and in England is under consideration by National Institute for Health and Clinical Excellence (NICE). Possible future developments include the use of monoclonal anti-D preparations, now in advanced clinical trials, and also testing the hypothesis that directed RAADP from early in the second trimester may further reduce anti-D immunisation.

Non-invasive prenatal determination of fetal sex: translating research into clinical practice.

The effectiveness and clinical utility of non-invasive prenatal diagnosis (NIPD) for fetal sex determination using cell-free fetal DNA (cffDNA) was assessed by undertaking a prospective national audit of UK testing. NIPD was performed using real-time polymerase chain reaction analysis of the DYS14 or SRY gene in cffDNA extracted from maternal plasma. All cases referred for fetal sex determination from 1 April 2006 to 31 March 2009 were ascertained from two laboratories offering the test. Fetal gender determined by NIPD was compared with that based on ultrasound, invasive test or phenotype at birth. Indication and rate of invasive testing was ascertained. In the first year, results were issued in 150/161 pregnancies tested. Of the 135 with outcome data, results were concordant in 130/135 [96.3% (95% CI 91.6-98.8%)]. Reporting criteria were changed and in the subsequent 511 pregnancies the concordancy rate increased to 401/403 [99.5% (95% CI 98.2-99.9%)]. Over the 3 years only 32.9% (174/528) underwent invasive testing. NIPD for fetal sex determination using cffDNA is highly accurate when performed in National Health Service laboratories if stringent reporting criteria are applied. Parents should be advised of the small risk of discordant results and possible need for repeat testing to resolve inconclusive results.

Early detection of cell-free fetal DNA in maternal plasma.

OBJECTIVES: We aimed to establish the earliest gestational age at which fetal DNA in maternal plasma could be detected and whether this was reliable at 12-13 weeks' gestation. STUDY DESIGN: A prospective observational cohort study of 32 pregnancies either after IVF or before prenatal diagnosis by CVS. Maternal blood was taken and RT-PCR was carried out to detect the multi-copy Y chromosome associated DSY14 gene. The end point was gender as assessed at delivery or on karyotype. RESULTS: Y signal was obtained as early as 14 days post conception (4 weeks' gestation) and has a good prediction rate by 12 weeks' gestation. CONCLUSION: Free fetal DNA allows very early prediction of fetal sex in some cases and could be useful for clinical use for X-linked conditions by the end of the first trimester.

Fetal RhD genotyping: a more efficient use of anti-D immunoglobulin.

The most important application of blood group genotyping by molecular genetics is the prediction of fetal RhD phenotype in pregnant women with anti-D, in order to assess the risk of haemolytic disease of the fetus and newborn. This diagnostic test performed on cell-free fetal DNA in the maternal plasma, is now a routine procedure in some countries. High-throughput modifications of this form of fetal D-typing would be valuable for testing fetuses of all D-negative pregnant women to avoid unnecessary antenatal treatment with anti-D immunoglobulin in the 40% of D-negative pregnant women with a D-negative fetus. The results of trials in Bristol and Amsterdam suggest that such routine testing is feasible and accurate.

Detection of cell-free fetal DNA in maternal urine.

OBJECTIVE: To evaluate the presence of cell-free fetal DNA signals in maternal urine as a potential source of material for non-invasive prenatal diagnosis. STUDY DESIGN: Patients referred to the regional fetal medicine unit who underwent prenatal diagnosis by chorionic villus sampling (CVS) were asked to give blood and urine immediately before the procedure. Maternal blood and urine were centrifuged at 10,000 g for 10 min. Plasma (1 mL) and urine (1 mL) supernatant were transferred to a clean tube and centrifuged again. The plasma (0.8 mL) and urine (0.8 mL) supernatant were removed without disturbing the cell pellet and stored at - 80 degrees C. Following DNA extraction, each sample was tested for the presence of Y chromosome associated DYS14 gene using real-time polymerase chain reaction (PCR). The total amount (maternal and fetal) of DNA in each sample was estimated using a quantitative real-time PCR assay. RESULTS: Twenty patients were enrolled in the study. CVS was performed at a median gestational age of 13 weeks (range 11 + 5 - 14 + 1). There were 12 male and 8 female fetuses, as confirmed by karyotype. Y chromosome DNA was not detected in any of the 20 samples of maternal urine, including 12 of the 20 samples in which Y chromosome DNA was detected in maternal plasma (all of whom were subsequently confirmed to be carrying a male fetus). There was considerable variation in the amount of total free DNA detected in maternal urine. CONCLUSIONS: Cell-free fetal DNA either was not present or did not amplify in maternal urine.

Non-invasive first trimester determination of fetal gender: a new approach for prenatal diagnosis of haemophilia.

Ten carriers of haemophilia referred for prenatal diagnosis were offered first trimester non-invasive fetal gender determination by ultrasound and analysis of free fetal DNA (ffDNA) in maternal plasma in an attempt to reduce the need for an invasive diagnostic procedure in female pregnancies. Although repeat testing was required in three cases, fetal gender was determined correctly in all cases (four females, six males) at a median gestation of 12(+3) (11(+2) to 14(+1)) using both methods. In all cases of a female fetus, the mothers opted not to have invasive testing. Both methods provide a reliable option of avoiding invasive testing in female pregnancies.

Fetal blood group genotyping from DNA from maternal plasma: an important advance in the management and prevention of haemolytic disease of the fetus and newborn.

The cloning of blood group genes and subsequent identification of the molecular bases of blood group polymorphisms has made it possible to predict blood group phenotypes from DNA with a reasonable degree of accuracy. The major application of this technology, which has now become the standard of care, is the determination of a fetal RHD genotype in women with anti-D, to assess whether the fetus is at risk of haemolytic disease of the fetus and newborn (HDFN). Initially, the procurement of fetal DNA required the invasive procedures of amniocentesis or chorionic villus sampling. Since the discovery of fetal DNA in maternal plasma in 1997, the technology of detecting an RHD gene in this very small quantity of fetal DNA has developed rapidly, so that non-invasive fetal D typing can now be provided as a diagnostic service for D-negative pregnant women with anti-D. Within a few years, it is probable that fetuses of all D-negative pregnant women will be tested for RHD, to establish whether the mother requires antenatal anti-D immunoglobulin prophylaxis.

Fetal sex determination from maternal blood at 6 weeks of gestation when at risk for 21-hydroxylase deficiency.

BACKGROUND: 21-hydroxylase deficiency can lead to masculinization of female fetuses. Corticosteroid therapy may reduce these effects. When the fetus is male, this approach means that unnecessary treatment, with theoretic side effects, is given until the result of chorionic villus sampling (CVS), a procedure with known risks, is available. CASE: A woman was referred for prenatal assessment at 6 weeks' gestation because her first daughter had been born virilized from 21-hydroxylase deficiency. A real-time polymerase chain reaction assay was performed on maternal blood to detect the fetal Y chromosome-associated SRY gene. A positive signal for the SRY gene was observed. The assay was repeated a few days later, and the result was again consistent with a male fetus. CONCLUSION: Analysis of cell-free fetal deoxyribonucleic acid in maternal plasma for fetal sex determination might reduce the need for corticosteroid administration and CVS in women with fetuses at risk for 21-hydroxylase deficiency.

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

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSION: 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 determination of fetal blood group status.

BACKGROUND AND OBJECTIVES: The prenatal determination of fetal blood group status by molecular techniques has been used in the clinical management of alloimmunised pregnancies for seven years, in particular for the definition of fetal Rh D, c and E, K, Fya and Jka status. This has arisen in response to the definition of the molecular bases of human blood group polymorphism. MATERIALS AND METHODS: PCR-based amplification assays have been designed to define fetal blood group status, where the source of template DNA is normally derived from amniotic fluid or chorionic villus. Recently, non-invasive methods have been explored to obtain fetal DNA from maternal peripheral blood. RESULTS: PCR-based tests are now available to screen for all fetal medicine significant blood group antigens. The Rh system is the most complex, and assays to define Rh genotype have been modified in response to our increased understanding of the molecular biology of this blood group system. CONCLUSION: Prenatal diagnosis of fetal blood group status is now in widespread use in the clinical management of HDN. Non-invasive testing, if applied in the clinical setting may invoke a dramatic increase in the numbers of pregnancies that may be analysed prenatally.

Evidence of genetic diversity underlying Rh D-, weak D (Du), and partial D phenotypes as determined by multiplex polymerase chain reaction analysis of the RHD gene.

The human blood group Rh antigens are expressed by proteins encoded by a pair of highly homologous genes located at chromosome 1p34-36. One of the genes (RHCE) encodes Rh CcEe antigens, while the other (RHD) the D antigen. Point mutations in the RHCE gene generate the C/c and E/e polymorphisms, while it has been shown that an RHD gene deletion can generate the D-negative phenotype. We have analyzed intron 4 of the RHCE and RHD genes and have defined the site of an RHD-specific deletion located in this intron. Using a multiplex RHD typing assay, which combines a reverse polymerase chain reaction (PCR) primer, which straddles this RHD-specific sequence, and a pair of primers located in exon 10 of the RHD gene, we have analyzed 357 different genomic DNA samples derived from individuals expressing D+, D-, weak D, and partial D phenotypes. Of these, we have noted a significant discordance with our multiplex PCR assay in the D- phenotypes dCcee and dccEe (which have been previously described) and weak D phenotypes. Our results suggest that in five serologically D- individuals we have identified an apparently intact RHD gene. Sequence analysis of transcripts obtained from one of these individuals (of phenotype dCCee) illustrates the presence of full-length RHD transcripts, which have a point mutation at nucleotide 121 (C --> T), which generates an in-frame stop codon (Gln41Stop). Thus, we describe a different molecular basis for generating the D- phenotype to the complete RHD gene deletion described previously. We also show that there are discordances with serotype and the multiplex assay in weak D and partial D phenotypes, indicating that the underlying molecular basis can be heterogeneous. Existing Rh D PCR assays assume the complete absence of the RHD gene in D- phenotypes. We describe a different molecular basis for generating the D- phenotype to the complete RHD gene deletion described previously.

The serological profile and molecular basis of a new partial D phenotype, DHR.

BACKGROUND AND OBJECTIVES: The Rh D antigen comprises a mosaic of at least 30 epitopes expressed on a 30-kD non-glycosylated Rh D polypeptide. The equivalent Rh CeEe polypeptide expressing the Rh C/c and E/e antigens differs in only 36 of the 417 amino acid residues. Partial D individuals have been described who fail to express a number of D epitopes. MATERIALS AND METHODS: Serologic methods were applied with monoclonal anti-D to map epitopes on the red cells of a proposita aberrant D typing. Polymerase chain reaction (PCR) and DNA sequencing were also done. RESULTS: DNA sequence analysis derived by RT-PCR using total RNA isolated from peripheral blood of this person suggests two mechanisms for the genetic basis of this variants: one here gene conversion events result in the replacement of RHD gene exons with the equivalent RHCE exons; the second where point mutation in the RHD gene generates an amino acid substitution in the Rh D protein. CONCLUSIONS: We report here a new partial D, DHR, where a single point mutation (G to A at nucleotide 686) in exon 5 of the RHD gene results in a conservative amino acid substitution (Arg229Lys), in the predicted Rh D protein. This residue is localised on the fourth predicted exofacial loop of the Rh D polypeptide as determined by hydropathy analysis. This substitution results in the lack of epD 1, 2, 12 and 20 (30 epitope model) and indicates the involvement of loop 4, and in particular the requirement of Arg229, in the expression of these epitopes.

Molecular biology of partial D phenotypes.

We have examined all DVI variant phenotypes submitted to the workshop by a combination of RT-PCR, multiplex RHD PCR and immunoblotting with Rh antipeptide sera. Our findings suggest that all DVI phenotypes arise through hybrid RHD-RHCE-RHD genes. Genomic DNA derived from all DVI samples were shown to be RHD intron 4 negative when analysed with an RHD intron 4/exon 10 multiplex assay. We assume therefore that all DVI phenotypes involve gene conversion events involving at least exons 4 and 5 of the RHD gene. Analysis of a novel D and E variant phenotype individual (ISBT49) by RT-PCR has allowed the identification of a hybrid Rh gene composed of exons 1-4 RHD: 5 RHCE/D and 6-10 RHD. We propose that the partial D & E phenotype observed arises through D & E expression on the hybrid RHD-RHCE-RHD protein: as no transcripts encoding Rh E could be found.