Noninvasive prenatal screening for RHD: the 1st national antenatal directed anti-D prophylaxis program – the Danish model or a guide to robust prediction of need of anti-D

Background As of January 1, 2010, Denmark implemented a national antenatal directed anti-D prophylaxis program. Prior to this, only postnatal prophylaxis directed by serology of cord blood was practiced. A preparatory work for modernization and optimization of fetal care was carried out by the National Board of Health with participation of leading obstetricians, specialists in fetal medicine, general practitioners, midwives, clinical immunologists and administrators. The promising results of non-invasive prenatal diagnosis based on cell free fetal DNA enabled an antenatal directed use of anti-D to those with a D positive fetus, thus eliminating approximately 40% of the antenatal use of anti-D. The complete program now includes an antenatal RHD screening in gestational week 25, and a postnatal serological RhD typing of cord blood, in both instances followed by administration of anti-D to those found positive. Aim The explicit aim of the RHD screening was to obtain the highest possible sensitivity to recommend anti-D in all cases where it might be indicated whereas specificity was of secondary priority. Methods Health services in Denmark are organized in five regions; accordingly screening has been implemented in every region, each using its own in-house screening methodology. However, all regions have implemented automated DNA purification and the assays are variations over a theme of multiple PCRs covering several exons. A control reaction for DNA purification is obligatory. However, a control for fetal DNA is not included. A working group with members from all regions has implemented a national scheme for quality control. Digital on-line delivery of results to general practitioners, midwives and obstetricians is implemented in most regions thus cutting down on cost and time. Results The continuation of postnatal serology has allowed a quality control of RHD screening. During the first six months of screening only two false negative results occurred out of 2312 tested (0·087%) suggesting a sensitivity of 99·9%. Due to the sacrifice of specificity some unnecessary use of anti-D occurred, 39 cases or 1·7%. Discussion The elimination of 38·3% of the use of anti-D, that is unnecessary and the related clinical contacts balances the expenses for the screening. An unforeseen benefit of the screening has emerged. The availability of the antenatal RHD results at the time of delivery has simplified logistics and preliminary clinical action is taken based on the antenatal result. Only in the very rare occasions of a (false negative) discrepancy between antenatal and postnatal results is communication needed between laboratory, clinician, and patient. The challenge will be to maintain the high standards in a possible future setting without serology serving as a benchmark for quality control.     

Noninvasive prenatal screening for RHD: the Stockholm study

The non-invasive technique to determine fetal RHD status opens the opportunity to change the antenatal screening and Rh-prophylaxis programs. During the period September 2009 to December 2011, we performed a study in the Stockholm area with approximately 27000 pregnancies per year. The study included routine cell free fetal DNA (cffDNA) RHD genotyping in early pregnancy followed by targeted RAADP in gestational week 29 to all RhD negative pregnant women carrying an RHD positive fetus. The new approach in our strategy, compared to previous studies, was that fetal RHD screening was done in early pregnancy at the first antenatal visit and based on a single-exon 4 assay. The implementation of this new screening program in a routine clinical setting is described. The final results of the study are still under analysis. The conclusion until now is that fetal RHD screening in early pregnancy is feasible and accurate with a high sensitivity and specificity, provided samples before gestational week eight were excluded.     

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.     

Prenatal RHD genotyping in Croatia: preliminary results

ObjectivesCroatian Institute of Transfusion Medicine (CITM) implemented non-invasive fetal RHD genotyping as a request for targeted antenatal anti-D prophylaxis. The diagnostic performance of in-house RT-PCR method for fetal RHD genotyping and preliminary results are analyzed.Materials and methodsEvaluation included results of RHD genotyping for 205 RhD negative pregnant women, 12–36th week of gestation, whose samples were received in period between 2015 and 2020. QIAsymphony SP DSP Virus Midi Kit was used for cffDNA extraction on QIAsymphony SP platform (Qiagen, Germany). Fragments of RHD exons 7 and 10 and later exon 5 were RT-PCR amplified. As internal controls, amplification of SRY gene or RASSF1A fragment and β-actin genes digested with BsTUI were used.ResultsWe identified 70.72% (145/205) positive and 28.78% (59/205) negative fetal RHD genotypes. We had one inconclusive result (0.50%) due to the interference of maternal DNA with variant genotype RHD*09.02.00/01/*01N.01. When compared to newborns RhD phenotypes, no false negative and three false positive results (3/199, 1.50%) were observed. The test yielded 100% sensitivity and 95.08% specificity, while diagnostic accuracy was 98.48%. We were able to determine one case of fetal variant genotype RHD*04.04/*01N.01 inherited from the father. The negative and positive predictive test values were 100% and 97.86%, respectively.ConclusionAutomated cffDNA extraction and RT-PCR amplification of fetal RHD exons 5,7,10 and fragments of SRY, RASSF1A genes represents highly reliable system for determining fetal RHD status which enables targeted antenatal anti-D prophylaxis. To obtain high specificity of cffDNA extraction, strict and thoroughly cleaning procedures are required.     

Suivi immunohématologique des femmes enceintes : nouvelles recommandations

Despite the generalization of immunoprophylaxis by anti-RH immunoglobulins over 40 years, fetomaternal incompatibility due to RH1 antigen (RhD) is not completely eradicated, although perinatal consequences might be extremely serious. Additionally, allo-immunizations against other antigens, especially anti-RH4 (anti-c) and anti-KEL1 (anti-Kell), may cause severe haemolytic disease. Follow-up of allo-immunization during pregnancy and its prevention are therefore still a concern for all pregnant women. Immunohaematological tests used in antenatal patients are under practice for a long time. However, despite significant progress, it is clear that these tests provide only an indirect indication and will only help the obstetrician, in conjunction with over fetal parameters, to assess the severity of the haemolytic disease. Since almost two decades, fetal RHD genotyping became a reality, first using amniocytes, but more recently by analyzing fetal DNA present in the maternal plasma. RH prophylaxis concerns RH:-1 women, who are non-sensitized against RH1 antigen during and at the end of their pregnancy with a RH1 child. RH prophylaxis includes targeted prophylaxis after foetomaternal haemorrhage and now routine antenatal RH prophylaxis at 28 gestation weeks. Indications for RH prophylaxis and immunohaematological testing to assure an efficient therapeutic prevention have been summarized in France through specific recommendations of the National College of Gynecologists and Obstetricians.     

Blood grouping by molecular genetics

It is possible to predict most clinically important blood group phenotypes from genomic DNA. Clinical applications include the following: determining foetal blood group to assess risk of haemolytic disease of the foetus and newborn or whether the pregnant woman requires antenatal anti-D prophylaxis; testing multiply transfused and autoimmune haemolytic anaemia patients; defining D variants; detecting donors with very weak D antigens; testing donors and patients when appropriate antisera are not available; RHD zygosity testing; zygosity testing of panel cells; assisting in solving difficult serological problems; and, with the use of high-throughput technologies, extended blood grouping of donors. Over the last 7 years, ISBT Workshops have provided external quality assurance. Future technologies may involve matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and massively parallel (next generation) sequencing.     

A structural model for 30 Rh D epitopes based on serological and DNA sequence data from partial D phenotypes

Both cDNA RHD sequences and reactivity with monoclonal anti-D have been reported in a number of partial D phenotypes, where parts (some epitopes) of the normal D antigen are missing, and anti-D of restricted specificity may be made in response to challenge with normal D positive blood. This paper analyses these reports together and proposes a model for the structures which comprise the epitopes of the Rh D antigen. Some epitopes are proposed to be comprised of continuous peptide sequence within one extracellular loop, whereas others require interactions between two or the extracellular peptide loops.     

Early detection of RhD status in pregnancies at risk of hemolytic disease of the newborn

The aim of this work was to investigate the presence of the RHD gene in fetal cells obtained from amniotic fluid. We studied 65 samples of amniotic fluid, 11 from RhD-negative mothers sensitized with anit-D alloantibodies. The fetal origin of the DNA was confirmed with the analysis of 1 VNTR locus and 3 STR loci in DNA samples from amniotic fluid and maternal blood. The RHD genotyping was performed in non-contaminated samples (n=62) using a multiplex polymerase chain reaction strategy that yields three amplification products from RhD-positive phenotypes (intron 4 of both RHCE and RHD genes and exon 10 of the RHD gene) and 1 DNA fragment from RhD-negative phenotypes (intron 4 of the RHCE gene). We genotyped 54 RhD-positive fetuses (8 from RhD-negative sensitized mothers) and 8 RhD-negative fetuses (3 from RhD-negative sensitized mothers). The fetal DNA genotyping allows the diagnosis, from a single amniocentesis, of fetuses at real risk of hemolytic disease of the newborn. When the fetus is determined to be RhD-negative invasive procedures can be avoided. Received: 17 October 2001 / Accepted: 26 May 2002     

Effect of hypoxia during early organogenesis on cardiac activity and noradrenergic regulation in the postnatal period

Cardiac activity in rats during the postnatal period was studied in vitro and in vivo after exposure of rat pups to antenatal acute hypobaric hypoxia at the stage of organogenesis (day 9–10 of gestation). Cultured cardiomyocytes from rat pups exposed to antenatal hypoxia were characterized by increased rate of contractions and decreased reactivity to norepinephrine. Heart rate elevation, predominance of sympathetic influences on cardiac activity, and significant increase in norepinephrine concentration in the cerebral cortex were found in freely moving animals exposed to antenatal hypoxia. Our results indicate that hypoxia at the stage of organogenesis modulated cardiac activity during the postnatal period, which manifested at the level of effector structures in the heart and activity of regulatory systems. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 11, pp. 484–486, November, 2006     

Maternal antibody titration as a predictive parameter for fetal status and therapy effectiveness in pregnancies associated with alloimmune thrombocytopenia

Alloimmune thrombocytopenia is the most common cause of severe isolated fetal and neonatal thrombocytopenia. In view of the recurrence of thrombocytopenia in subsequent pregnancies with incompatible fetuses, antenatal management has been developed. Until recently the only possibility of assessing the fetal status both before and during therapy was to perform fetal blood samplings (FBS). In view of the risks involved in the procedure, FBS has been restricted and non-invasive strategies have been developed. The determination of maternal parameters predictive of severe fetal thrombocytopenia is crucial for tailored intervention. A first retrospective study was designed to analyse the predictive value of the maternal anti-HPA-1a antibody concentration. With this in view, we developed a quantitative method based on the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technique, which is the gold standard method for serological investigations in platelet immunology [ 1 ]. Maternal anti-HPA-1a antibody concentrations were determined at the same time as the FBS carried out as a part of the antenatal management prior to therapy. A statistically significant correlation was observed between the high antibody concentration [≥ 28 international units (IU)/ml] and severe fetal thrombocytopenia (< 50 × 10⁹/l; P = 0·0021 [ 2 ]). A larger retrospective study was subsequently performed to search for additional maternal predictive parameters during managed pregnancies, taking into account maternal anti-HPA-1a antibody concentrations and maternal genetic background (ABO blood group and HLA-DRB3 allele). We confirmed the predictive value of the maternal antibody concentration before 28 weeks of gestation (wg) and before treatment. The follow-up of the concentration during pregnancy allowed the measurement of the area under the curve, weighted by the weeks between the first and the last quantification. This new parameter was predictive of the therapy effectiveness: under 24 IU/ml/wg, a majority of women delivered a severely thrombocytopenic newborn (< 50 × 10⁹/l; P = 0·0153 [ 3 ]). In conclusion, our work gives new insights into maternal predictive parameters for fetal status and therapy effectiveness, allowing non-invasive strategies. Follow-up of the antibody concentration during pregnancy could help to predict the outcome of the pregnancy, so as to prevent severe hemorrhagic disorders in the neonate.     

The direct early diagnosis of cystic fibrosis by the detection of the deltaF508 CFTR gene mutation in a prematurely delivered boy

The suspicion of prenatal meconium ileus syndrome was raised in a pregnancy in a family with no history of cystic fibrosis because of significantly higher maternal serum alpha-fetoprotein in the 16th and 19th week of gestation, dispersed areas with increased echogenity in the fetal abdomen, slight fetal ascites in the 24th-25th weeks of gestation, decreased amniotic fluid gamma-glutamyltranspeptidase (GGT) activity and alpha-fetoprotein level in the 25th-26th weeks, and normal 46,XY karotype of the fetus. The detection of a homozygous deltaF508 cystic fibrosis transmembrane regulator (CFTR) gene mutation, by means of PCR from a small amount of white blood cells and urine sediment cells, substantiated the diagnosis of cystic fibrosis in a prematurely delivered boy in the 28th week of gestation. The repeated sweat test was unsuccessful. The autopsy examination confirmed the diagnosis of cystic fibrosis. Fetal meconium ileus syndrome was complicated by peritonitis and by formation of a meconium pseudocyst. Direct PCR typing improves postnatal diagnostic possibilities in the early neonatal period in prematurely delivered babies when the sweat test is difficult to perform.     

RHD positive among C/E+ and D-negative blood donors in Tunisia

Purpose of the studyThe aim of this study was to investigate RHD alleles among Tunisian blood donors with D-negative phenotype and positive for C and/or E antigen.Patients and methodsA total of 100 D-negative and C/E+ samples were analyzed by RHD genotyping using an initial test for RHD exon 10. In case of a positive reaction, further molecular investigations including real time quantitative PCR, allele specific PCR and nucleotide sequencing were done to elucidate the RHD involved mechanisms.ResultsSeventy-five percent of the studied samples lacked the RHD gene. Twenty-three percent carried the hybrid RHD-CE-D alleles (16 RHD-CE(3-7)-D, 5 RHD-CE(4-7)-D, 1 RHD-CE(4-8)-D, 1 RHD-CE(3-8)-D) and 2% were weak D (1 weak D type 1 and 1 weak D type 5).ConclusionOur study proved the high frequency of RHD gene among serologically D-negative samples, positive for C and/or E antigen. Thus achieving systematically RHCE phenotyping in all transfusion centers on the Tunisian territory and considering blood donated from D-negative C/E+ persons as D-positive will be recommended to reduce anti-D allo-immunization.     

Cell‐free fetal DNA and fetal blood group genotyping: non‐invasive prenatal testing

In transfusion medicine and clinical immunology, cell‐free fetal DNA (cffDNA) is analysed from maternal plasma of pregnant women to predict fetal blood groups with the purpose of (1) assessing the risk of haemolytic disease of the fetus and newborn (HDFN) in immunized women and (2) guiding targeted Rh prophylaxis in non‐immunized RhD‐negative women. National programmes for guiding prophylaxis are now implemented in around 6–7 European countries; assay accuracy is very high, with sensitivities of 99·9%. Sensitivity is challenged by low quantities of cffDNA, especially in early pregnancy. Specificity is challenged by the polymorphic Rh blood group system, where careful attention is needed to navigate among the many RHD variants that may complicate cffDNA analysis and interpretation of results, especially in populations with mixed ethnicities. However, fetal RHD testing is feasible when implemented with careful attention to these issues. The success of predicting fetal RhD and its successful clinical implementation should encourage widespread implementation. For blood groups that are determined by SNPs, such as KEL or Rhc, novel techniques such as next‐generation sequencing and droplet digital PCR are now providing accurate non‐invasive prediction of these fetal blood groups. Future work on non‐invasive prenatal testing of fetal blood groups determined by SNPs may consolidate the application for cell‐free DNA testing for such targets. At ISBT, the newly formed cfDNA subgroup of the Red Cell Immunogenetics and Blood Group Terminology Working Party will work to facilitate clinical applications, implementation and evaluation of cell‐free DNA testing.     

Morphometry of the kidney in rat pups from uninephrectomized mothers

Background: The present study was designed to explore whether maternal renal dysfunction affects fetal kidney development and if the effects are lasting during the postnatal development. Methods: Kidneys of 1-day-old and 6-week-old pups from mothers which were uninephrectomized on day 5 of gestation were studied. Results: One day after birth, both the number of immature glomeruli and average volume of mature glomeruli of the neonates from uninephrectomized mothers were significantly larger than those from sham-operated mothers. Six weeks after birth, no significant differences in parameters of the kidney were observed between the pups from uninephrectomized and those from sham-operated mothers. Furthermore, blood urea nitrogen (BUN) concentration in adult female rats was determined at various days after uninephrectomy. BUN concentration in uninephrectomized rats was significantly higher than that in sham-operated ones. Conclusions: These results suggest that the fetal kidney development is accelerated by the elevated BUN level following maternal uninephrectomy and that the renotropic activity does not last during the postnatal developmental period. © 1994 Wiley-Liss, Inc.     

Die extralobare Sequestration einer fetalen Lunge

The extralobar sequestration is a rare pulmonary malformation. An accurate antenatal evaluation is required for a timely therapy and subsequently a good outcome. Here an unusual case of extralobar pulmonary sequestration in a male human fetus is reported. Antenatal ultrasound at 28th week of gestation has revealed a fetal hydrothorax in coexistence with pulmonary hypoplasia and an isolated pulmonary structure. Authors summarise their postnatal findings with special reference to the pathogenesis of an accessory lung. The aim of this report is to define the association of clinical, gross, and histological features of this rare congential malformation in order to improve the antenatal diagnosis. This case indicates that an extralobar pulmonary sequester is not connected to the tracheobronchial tree, and that the arterial as well as the venous blood supply is realised by aberrant systemic vessels. Moreover, histologically revealed dilatations of the normally differentiated terminal airways within the sequester suggest that hyperechogenity can not be a reliable diagnostic criterion. For the accurate assessment of a pulmonary sequestration a detailed antenatal evaluation of both, the arterial and the venous blood supply is essential.     

Non-invasive antenatal RHD typing.

The existence of cell free fetal DNA, derived from apoptotic syncytiotrophoblast, in the maternal circulation has opened new possibilities of non-invasive prenatal diagnosis. Although still some technical problems exists, especially the lack of a generic positive control on the presence of fetal DNA and the aspecific amplification of background maternal DNA, non-invasive prenatal RHD typing has been successfully introduced in several laboratories, especially in Europe. The diagnostic accuracy reaches>99%. In the Netherlands PCR guided administration of antenatal anti-D prophylaxis is cost-effective and nearby. In this review the main characteristics and applications of cell free fetal DNA are discussed, with an emphasis on prenatal RHD genotyping.     

The Crawford variant as a cause of RhD typing discrepancies in blood banks: A case report

We present the case of a 55-year-old Colombian male who showed a discrepancy in the serological typing of the RhD antigen in his first platelet donation. The discrepancy persisted after a serological investigation with multiple Anti-D monoclonal reagents (IgG and IgM) under different conditions (22 °C and 37 °C, saline, and LISS/Coombs). Furthermore, partial RhD typing was performed, obtaining negative results with a commercially available panel of six Anti-D reagents. Molecular analysis showed a homozygous deletion of RHD and heterozygosity for the Crawford variant (RHCE*ce, RHCE*ceCF), with a predicted phenotype of C–, c+, E–, e+, Vs+, V+. Following the investigation of this case, this man has made 14 platelet donations showing variable reactivity, with agglutinations ranging from – to 2+. Since Crawford red blood cells express some RhD antigen epitopes, they could cause alloimmunization in RhD negative receptors. Likewise, Anti-D alloantibodies have been documented in Crawford variant carriers. Therefore, it is recommended that carriers of this variant be classified as RhD positive if they are blood donors and RhD negative if they are transfusion recipients. Also, in pregnant women carrying a Crawford variant, Anti-D immunoprophylaxis is recommended.     

Maturation of the extracellular material of the semilunar heart valves in the mouse

Summary The developmental changes of collagen and mucopolysaccharides in the semilunar valves of the mouse were studied during the embryonic, fetal and postnatal period. The valvular collagen was investigated using Van Gieson and Sirius red-polarization microscopy methods. These procedures showed that the establishment of the fibrosa layer of the cusps does not occur until the second week of the postnatal period.The nature and distribution of the valvular mucopolysaccharides were investigated by staining with Alcian blue at specific pH values and at various critical electrolyte concentrations, with the appropiate enzymatic controls using Streptomyces and testicular hyaluronidase. The results show that hyaluronate and chondroitin 4- and 6-sulphate are the major components during the embryonic and fetal period. In the older fetal stages and during the postnatal period the relative amount of hyaluronate decreases, while chondroitin sulphate increases.It is concluded from this study that the maturation of the valves occurs over a long period of the postnatal life.     

Blood group molecular genotyping

DNA-based typing methods of red cell antigens are applied in several fields. Genotyping is used to clarify problems in patient serology. It is also increasingly applied for routine mass-scale typing of blood donors for minor red cell antigens and screening for donors with rare blood types. An advance in this field is the non-invasive fetal RHD diagnostics in pregnancies of D-negative women to determine the fetal RHD. Reference centres for immunohaematology commonly use molecular methods to clarify problems, discrepancies or unusual results in patient serology. It is often applied in individuals with variant RhD expression to detect the contributing weak D or partial D types. Genotyping is also used to identify the individual ABO and RhD blood group in patients after mismatched transfusion. In some patients with positive direct antiglobulin test (DAT) it is necessary to type for antigens by DNA techniques. Genotyping is also helpful when reagents are not available or only weakly reactive, or to confirm weakly expressed antigens. Blood establishments are constantly challenged with the blood supply for patients carrying irregular red cell antibodies. A high number of donors have to be typed to find compatible blood units. Increasingly, DNA-based methods are used alongside standard serological typing. The advantages of molecular methods are the wider range of different antigens available for typing and that some DNA methods are less expensive than phenotyping. Extensive donor antigen typing leads to short response times, from request to issuing blood units. Genotyping is also used for identifying donors with extremely weak RhD expression (DEL) to prevent mistyping as RhD-negative. Non-invasive fetal typing of RHD from maternal blood in pregnancies of D-negative women is implemented in many Caucasian blood establishments. It allows that D-negative fetuses can be identified at early stage. In these cases, no anti-D immunoglobulin is required, which provides a more efficient use of this human blood derivate. Furthermore, in anti-D alloimmunized women, the assay allows to accurately identify fetuses at risk for haemolytic disease.     

Foetomaternal erythrocyte incompatibilities: from immunohaematologic surveillance of pregnant women to haemolytic disease of the newborn]

Despite the generalization of prevention measures against foetomaternal alloimmunization with anti-D immunoprophylaxis since 1970s, retrospectively 30 years later, its complications (new-born child's severe haemolytic disease, foetal death by anemia or nuclear icterus by bilirubin encephalopathy) have not disappeared. At the same time, alloimmunizations against antigens other than D increase with no possible prevention. As part of the set up in France of regional files analysing and making an inventory of serious foetomaternal incompatibilities requiring in utero or neonatal transfusion, we felt the need to synthesize current data, biological profiles (early screening of erythrocytic alloimmunization and its follow up during pregnancy, father's immunohaematologic status, evaluation of in utero immune haemolysis and impact of new non invasive techniques of diagnosis-RH1 foetal genotypage from ADN foetal of RH1--mothers' maternal plasma), clinical and paraclinical data (evaluation of foetal haemolysis by echography, recording of foetal movements and foetal cardiac rhythm), therapeutic indicators (in utero foetal transfusions or exsanguinotransfusions, neo and postnatal transfusions or exsanguinotransfusions, induced premature labour, newborn's intensive continue phototherapy and Rhesus immunoprophylaxis) in order to enable medical and paramedical professionals to carry out the specific supervision of pregnancies with foetomaternal incompatibility, the in utero, neo- and postnatal treatment of child and the efficient therapeutic prevention of anti-D alloimmunization, in a cooperative way.