Non-invasive Fetal RHD Genotyping Research Articles

Non-invasive fetal RHD genotyping in d- (RH:-1) patients with RHD genomic sequences: retrospective study over 11 years

Non-invasive fetal RHD genotyping in d- (RH:-1) patients with RHD genomic sequences: retrospective study over 11 years

Antenatal RHD screening to guide antenatal anti-D immunoprophylaxis in non-immunized D- pregnant women.

In pregnancy, D- pregnant women may be at risk of becoming immunized against D when carrying a D+ fetus, which may eventually lead to hemolytic disease of the fetus and newborn. Administrating antenatal and postnatal anti-D immunoglobulin prophylaxis decreases the risk of immunization substantially. Noninvasive fetal RHD genotyping, based on testing cell-free DNA extracted from maternal plasma, offers a reliable tool to predict the fetal RhD phenotype during pregnancy. Used as a screening program, antenatal RHD screening can guide the administration of antenatal prophylaxis in non-immunized D- pregnant women so that unnecessary prophylaxis is avoided in those women who carry a D- fetus. In Europe, antenatal RHD screening programs have been running since 2009, demonstrating high test accuracies and program feasibility. In this review, an overview is provided of current state-of-the-art antenatal RHD screening, which includes discussions on the rationale for its implementation, methodology, detection strategies, and test performance. The performance of antenatal RHD screening in a routine setting is characterized by high accuracy, with a high diagnostic sensitivity of ≥99.9 percent. The result of using antenatal RHD screening is that 97-99 percent of the women who carry a D- fetus avoid unnecessary prophylaxis. As such, this activity contributes to avoiding unnecessary treatment and saves valuable anti-D immunoglobulin, which has a shortage worldwide. The main challenges for a reliable noninvasive fetal RHD genotyping assay are low cell-free DNA levels, the genetics of the Rh blood group system, and choosing an appropriate detection strategy for an admixed population. In many parts of the world, however, the main challenge is to improve the basic care for D- pregnant women.

Noninvasive screening of fetal RHD genotype in Chinese pregnant women with serologic RhD-negative phenotype.

Noninvasive fetal RHD genotyping has been provided to nonimmunized RhD-negative pregnant women to guide anti-D prophylaxis. Among the Chinese, more than 30% of the RhD-negative phenotype is associated with variant RHD alleles, which would limit the accuracy of fetal RHD status prediction; thus, more targeting and proper programs need to be developed. Fluorescence quantitative polymerase chain reaction PCR (qPCR) or Sanger sequencing on all RHD exons was used to detect maternal RHD genotypes. For pregnant women with RHD*01N.01 or RHD*01N.03 alleles, the presence of RHD exons 5 and 10 in cell-free DNA was determined by qPCR. For pregnant women with the RHD(1227G>A) allele, high-throughput sequencing on exon 9 of the RHD gene and RHCE gene was used to predict fetal RhD phenotype. Among 65 cases of Chinese pregnant women with the serologic RhD-negative phenotype, three major genotypes were identified: RHD*01N.01/RHD*01N.01 (61.5%), RHD*01N.01/RHD(1227G>A) or RHD*01N.03/RHD(1227G>A) (20%), and RHD*01N.01/RHD*01N.03 (13.8%), along with three cases of minor genotypes (4.6%). For 43 pregnant women with the RHD*01N.01 or RHD*01N.03 alleles, qPCR on maternal cell-free DNA yielded a 98.5% (42/43) accuracy rate and 100% successful prediction rate. High-throughput sequencing was successfully used to predict fetal RhD phenotypes for 13 pregnant women with RHD(1227G>A). On the basis of maternal RHD genotyping, fetal genotyping through qPCR or high-throughput sequencing can improve the accuracy and success rate of prenatal fetal RhD phenotype prediction among Chinese pregnant women. It plays a potential role in guiding anti-D prophylaxis and pregnancy management in Chinese pregnant women.

Evaluation of an automated platform for non-invasive single-exon fetal RHD genotyping early in pregnancy.

RhD immunization is still the major cause of hemolytic disease of the fetus and newborn. Fetal RHD genotyping during pregnancy followed by tailored anti-D prophylaxis for pregnant RhD-negative women carrying an RHD-positive fetus to prevent RhD immunization is a well-established practice in many countries. This study aimed to validate a platform for high-throughput, non-invasive, single-exon, fetal RHD genotyping consisting of automated DNA extraction and PCR set-up, and a novel system for electronic data transfer to the real-time PCR instrument. We also investigated the effect of storage conditions of fresh or frozen samples on the outcome of the assay. Blood samples from 261 RhD-negative pregnant women collected in Gothenburg, Sweden, between November 2018 and April 2020 during gestation week 10-14 were either tested as fresh after storage for 0-7 days at room temperature or as thawed plasma samples previously separated and stored for up to 13 months at -80°C. Extraction of cell-free fetal DNA and PCR set-up were performed in a closed automated system. Fetal RHD genotyping was determined by real-time PCR amplification of the RHD gene exon 4. The outcome of RHD genotyping was compared with either the results obtained with serological RhD typing of newborns or with the results of RHD genotyping performed by other laboratories. No difference was observed in genotyping results when using fresh or frozen plasma during short- and long-term storage, revealing high stability of cell-free fetal DNA. The assay has shown high sensitivity (99.37%), specificity (100%), and accuracy (99.62%). These data confirm that the proposed platform for non-invasive, single-exon, RHD genotyping early in pregnancy is accurate and robust. Importantly, we demonstrated the stability of cell-free fetal DNA in fresh and frozen samples after short- and long-term storage.

Reaffirmed Guideline No. 343: Routine Non-Invasive Prenatal Prediction of Fetal RHD Genotype

<h2>ABSTRACT</h2><h3>Summary Statements</h3> <ul><li>1.Non-invasive antenatal determination of fetal RHD genotype by cellfree DNA is highly accurate with sensitivities above 99% and very few false-negative results (II-2).</li><li>2.While the risks of Rh immune globulin exposure are exceptionally low, it is no longer considered appropriate to treat all D-negative pregnant women with human plasma derivatives when there are no benefits to her or to the fetus in a substantial percentage of cases (II-2).</li><li>3.Implementation of non-invasive fetal RHD genotyping with targeted routine antenatal anti-D prophylaxis would enable up to 40% of D-negative women to avoid use of Rh immune globulin (II-2).</li><li>4.Fetal RHD genotyping is feasible as early as 10 weeks' gestation and earlier testing is preferable as it allows for targeted prophylaxis for women with sensitizing events prior to 28 weeks' gestation (II-2).</li><li>5.Implementation of non-invasive fetal RHD genotyping with selective prophylaxis requires interdisciplinary collaboration (clinical and laboratory) as well as the endorsement of provincial ministries of health (III).</li></ul> <h3>Recommendations</h3> <ul><li>1.The current optimal management of the D-negative pregnant woman is based on the prediction of the fetal D-blood group by cell-free DNA in maternal plasma with targeted antenatal anti-D prophylaxis. This approach should be adopted in Canada (II-2A).</li><li>2.While various algorithms of implementation of fetal RHD genotyping have been described, a model positioned in the first trimester appears to be most in alignment with the existing Canadian antenatal anti-D prophylaxis program and should be endorsed (II-2A).</li><li>3.While the risk of a false-negative result with RHD genotyping is very small and the benefits of knowing the fetal RHD status in terms of compliance with prophylaxis seem to outweigh the risks, the chance of immunization is not zero. Quality control at a laboratory and clinical level should be of utmost priority in program planning (II-3A).</li></ul>

Investigation of discrepancies obtained during 15years of non-invasive fetal RHD genotyping in apparent serologic RhD-negative pregnant women.

In some European countries, non-invasive fetal RHD genotyping is the first step of anti-D allo-immunized pregnant women management but presence of RHD variant alleles may interfere with the results accuracy. We developed an algorithm allowing solving discordant results (due to the presence of RHD variant) in fetal RHD genotyping assay. This study gathered the results of fetal RHD genotyping performed between 2006 and 2020 in the Medicine Laboratory of CHR Liège. Exons 4, 5 and 10 of the fetal RHD were profiled in maternal plasma using real time polymerase chain reaction (PCR). When the results were discrepant, maternal RHD variant was further explored by sequence-specific primer PCR on maternal buffy coat. A total of 11,630 pregnant women (mainly of both Caucasian and African origins) were tested during the study period and RHD variant alleles were detected in 247 women. The most frequent variant was RHD*08N.01 found in 66 women mainly of Black African origin. We identified 45 women with weak RHD variant type 1, 2 or 3. Women with weak RHD variant type 1, 2 or 3 can safely be considered as RhD positive in terms of RhIg prophylaxis and/or transfusion of blood components. Therefore, identification of RHD allele variants in women with discordant fetal RHD genotyping results contributes to save RhIg prophylaxis and RhD negative blood components.

Algorithm development and diagnostic accuracy testing for non-invasive foetal RHD genotyping: an Indian experience.

The discovery of the cell-free foetal DNA (cffDNA) circulating in the maternal plasma enabled prediction of foetal RHD thus eliminating the risks associated with invasive procedures. Non-invasive foetal RHD genotyping has now become the standard approach in developed countries for management of alloimmunised women and is also used for targeted antenatal prophylaxis in non-alloimmunised women. cffDNA was extracted from the plasma of 217 RhD negative pregnant women at a gestational age of 10-34 weeks. The foetal RHD genotype was determined by real-time polymerase chain reaction (real-time PCR) amplification of exons 4, 5 and 10 in duplicates. After an initial 54 samples, foetal typing was carried out with RHD exons 5 and 10 for the remaining samples. CCR5, SRY and RASSF1A genes were used as controls. Results were compared with cord blood serological typing at birth. Out of the 217 women, 193 were non-immunised and 24 were alloimmunised. A conclusive diagnosis was obtained in 203 samples. Diagnosis was inconclusive in 14 samples; of these, foetal RHD genotype could be resolved in six samples after maternal and paternal RHD genotyping. A 100% diagnostic accuracy, sensitivity and specificity were demonstrated in 209 women who had had a conclusive result. When the inconclusive samples were included, diagnostic accuracy and sensitivity were more than 95% and specificity was 78.95%. Anti-D is still the leading cause of haemolytic disease of the foetus and the newborn in India. There is, therefore, a need to establish and develop an algorithm for antenatal RhD negative women in India. The positive results of non-invasive foetal RHD genotyping, from the start of the 10th week of gestation using two RHD exons giving 100% diagnostic accuracy, show promise for routine diagnostic use to the benefit of the antenatal RhD negative Indian population.

Cost-effectiveness of noninvasive fetal RhD blood group genotyping in nonalloimmunized and alloimmunized pregnancies.

We sought to determine the cost-effectiveness of noninvasive fetal RhD blood group genotyping in nonalloimmunized and alloimmunized pregnancies in Canada. We developed two probabilistic state-transition (Markov) microsimulation models to compare fetal genotyping followed by targeted management versus usual care (i.e., universal Rh immunoglobulin [RhIG] prophylaxis in nonalloimmunized RhD-negative pregnancies, or universal intensive monitoring in alloimmunized pregnancies). The reference case considered a healthcare payer perspective and a 10-year time horizon. Sensitivity analysis examined assumptions related to test cost, paternal screening, subsequent pregnancies, other alloantibodies (e.g., K, Rh c/C/E), societal perspective, and lifetime horizon. Fetal genotyping in nonalloimmunized pregnancies (at per-sample test cost of C$247/US$311) was associated with a slightly higher probability of maternal alloimmunization (22 vs. 21 per 10,000) and a reduced number of RhIG injections (1.427 vs. 1.795) than usual care. It was more expensive (C$154/US$194, 95% Credible Interval [CrI]: C$139/US$175-C$169/US$213) and had little impact on QALYs (0.0007, 95%CrI: -0.01-0.01). These results were sensitive to the test cost (threshold achieved at C$88/US$111), and inclusion of paternal screening. Fetal genotyping in alloimmunized pregnancies (at test cost of C$328/US$413) was less expensive (-C$6280/US$7903, 95% CrI: -C$6325/US$7959 to -C$6229/US$7838) and more effective (0.19 QALYs, 95% CrI 0.17-0.20) than usual care. These cost savings remained robust in sensitivity analyses. Noninvasive fetal RhD genotyping saves resources and represents good value for the management of alloimmunized pregnancies. If the cost of genotyping is substantially decreased, the targeted intervention can become a viable option for nonalloimmunized pregnancies.

Noninvasive Prenatal Diagnosis of Fetal RHD Status Using Cell-free Fetal DNA in Maternal Plasma.

Background:The main cause of hemolytic disease of the fetus and newborn (HDFN) is the incompatibility of the RHD antigen between mother and fetus. Following the discovery of cell-free fetal DNA (cffDNA), noninvasive fetal RHD genotyping also became possible, which will help in the better management of immunized RHD negative mothers and in the targeted prenatal injection of Rho(D) immune globulin (RhIG). The objective of this study was to establish a reliable method with high accuracy to determine the fetal RHD genotype.Methods:The project was a prospective observational cohort study. After cell-free DNA (cfDNA) extraction from maternal plasma, fetal RHD genotyping was performed by duplex real-time polymerase chain reaction (PCR) and exons 5, 7, and 10 of the RHD gene were examined. SRY and RASSF1A genes were used as internal controls to confirm the presence of cffDNA in maternal plasma.Results:Out of 40 samples, 33 were RhD positive heterozygous mothers and 7 cases were RHD negative. In three cases where both the fetal RHD and SRY genotypes were negative, RASSF1A was amplified in cell-free DNA sample treated with the BstUI enzyme, and the presence of cffDNA was confirmed.Conclusion:The findings reveal that the strategy used in this study is reliable and it is possible to determine the fetal RHD status with high accuracy. The strategy can help targeted injection of RhIG and prevent unnecessary injection in RhD negative mothers who carry an RhD negative fetus.

Fetal RhD Genotyping using Cell Free Fetal DNA from Maternal Plasma in RhD Negative Women in Pakistan

Background: The Rhesus (Rh) blood group system is one of the most hemogenic and polymorphic system in the body. RhD negative pregnant women carrying RhD positive fetus are always at the risk of hemolytic disease of the fetus and newborn. Prenatal fetal RhD genotyping leads to appropriate management and targeted anti-D immunoprophylaxis in RhD-positive pregnancies only. Objective: To determine the accuracy and diagnostic feasibility of non-invasive fetal RhD genotyping by analysis of cell free fetal DNA based on real time polymerase chain reaction (RT-PCR). Materials and Methods: A total of 100 EDTA-blood samples from pregnant women between 10-32 weeks of gestation were collected. The cell free fetal DNA was extracted from maternal plasma followed by PCR analysis for SRY, RhD (exons 5,7,10) and RASSF1A genes using specific primers. Fetus was considered as RhD-positive when amplification of all targeted RhD exons were observed and in case, where no amplification for RhD exons was detected but Beta-globin gene and SRY gene was amplified, fetus were characterized as RhD-negative. When one or two RhD exons were amplified, the results were predicted to be inconclusive and RT-PCR assay with new extracted DNA samples was repeated. Results: Out of 100 pregnancies, none of the sample was found to be inconclusive for fetal RhD status. No false- positive and falsenegative results were registered. The sensitivity, specificity and diagnostic concordance was predicted to be 100%. Conclusion: The protocol we applied was highly accurate for non-invasive fetal RhD determination. We therefore recommend implementation of non-invasive RhD genotyping in prenatal screening. Keywords: Cff DNA; Fetal RhD genotyping; Maternal plasma

Recommendation for validation and quality assurance of non-invasive prenatal testing for foetal blood groups and implications for IVD risk classification according to EU regulations.

Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.

Prenatal RHD genotyping in Croatia: preliminary results.

Croatian 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. Evaluation 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. We 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. Automated 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.

Non-invasive fetal RHD genotyping: validation of the test and implementation of a screening program to guide anti-D prophylaxis

Non-invasive fetal RHD genotyping: validation of the test and implementation of a screening program to guide anti-D prophylaxis

Non-invasive fetal RHD genotyping

Immunization against RhD is the major cause of hemolytic disease of the fetus and newborn (HDFN), which causes fetal or neonatal death. The introduction of postnatal immune prophylaxis in the 1960s drastically reduced immunization incidents in pregnant, D-negative women. In several countries, antenatal prophylaxis is combined with postnatal prophylaxis to further minimize the immunization risk. Due to lack of knowledge of the fetal RhD type, antenatal prophylaxis is given to all D-negative women. In the European population, approximately 40% of pregnant women carry a D-negative fetus and are thus at no risk of immunization. Noninvasive fetal RhD genotyping enables antenatal prophylaxis to be targeted to only those women carrying a D-positive fetus to avoid unnecessary treatment. Based on an analysis of cell-free fetal DNA from the plasma of pregnant women, this approach has recently undergone technical improvements and rapid clinical implementation. As a screening assay, the sensitivity is >99.3% from a gestational age of approximately 10–11 weeks. In addition, fetal RhD genotyping is widely used to assess the risk of HDFN in anti-D immunized women.

Two Reliable Methodical Approaches for Non-Invasive RHD Genotyping of a Fetus from Maternal Plasma.

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.

Amplicon Sequencing-Based Noninvasive Fetal Genotyping for RHD-Positive D Antigen-Negative Alleles.

To avoid hemolytic disease of the fetus and newborn resulting from maternal alloantibodies against fetal Rh antigens, anti-D immunoglobulin is routinely administered to RhD-negative pregnant women in Japan. Fetal RHD genotyping using cell-free DNA may prevent unnecessary antibody administration; however, current PCR-based methods, which detect RHD deletion, do not address the higher rates of RHD-positive D antigen-negative alleles in nonwhite populations without additional inspections. We developed an amplicon-sequencing method that could estimate the type of paternally inherited fetal RHD allele from 4 major RHD alleles in the Japanese population: the D antigen-positive allele (RHD*01, 92.9%) and 3 D antigen-negative alleles (RHD*01N.01, 6.6%; RHD*01EL.01, 0.3%; RHD*01N.04, 0.1%) using cell-free DNA obtained from the blood plasma of pregnant women. The method correctly determined the fetal RhD type even when RhD-negative pregnant women possessed an RHD-positive D antigen-negative allele: RHD*01EL.01 or RHD*01N.04. This method is a reliable noninvasive fetal RHD genotyping method for Japanese and other East Asian populations. The genotyping principle of amplifying 2 different regions using the same primer pair and distinguishing them by their sequence difference during the subsequent mapping procedure is also theoretically applicable to RHD-positive D antigen-negative alleles prevalent in Africans. Therefore, this method offers an opportunity to consider targeted administration of anti-D immunoglobulin to RhD-negative pregnant women in East Asian and African countries and to increase the specificity of the fetal RHD genotyping implemented nationwide in several European countries.

Targeted Rhesus immunoglobulin for RhD-negative women undergoing an induced abortion: A clinical pilot study.

The aim of this clinical pilot study was to examine the accuracy of noninvasive fetal RHD genotyping in early pregnancy (8+0 to 11+6 weeks) and to clarify whether targeted administration of Rhesus immunoglobulin (RhIg) is possible for women undergoing an induced abortion such that unnecessary injections can be avoided. The study examines the correlation between gestational age and the amount of cell-free fetal DNA in maternal plasma, the fetal fraction of DNA and whether transportation time or body mass index affects these parameters. Fifty-two RhD-negative women undergoing a surgically induced abortion were included. A maternal blood sample was collected prior to the abortion and a tissue sample was collected from the placental part of the abortion material after the intervention. Fetal RhD type was determined by PCR analysis of cell-free fetal DNA extracted from maternal plasma and on DNA from the tissue sample, with the latter providing a reference standard. Copies of RHD/mL were determined on RHD-positive samples and the fetal fraction of DNA was calculated. We demonstrated complete concordance between results from plasma and tissue, with 31 RhD-positive and 21 RhD-negative samples, corresponding to 40% being RhD-negative, specificity 100% [95% confidence interval (CI) 88.8-100] and sensitivity 100% (95% CI 83.9-100). We found no significant correlation between gestational age and the amount or the fraction of cell-free fetal DNA in maternal plasma, nor did we find that transportation time or BMI significantly affected these factors in this setup. Fetal RHD genotyping can be accurately performed from the 8th week of gestation and unnecessary injections of RhIg can be avoided for women undergoing an induced abortion. A larger study is needed to determine a more accurate sensitivity for the analysis early in pregnancy.

A new biosensor for noninvasive determination of fetal RHD status in maternal blood of RhD negative pregnant women.

Prenatal detection of the fetal RHD status can be useful in the management of RhD incompatibility to identify fetuses at risk of hemolytic disease. Hemolytic disease causes morbidity and mortality of the fetus in the neonatal period. The routine use of antenatal and postnatal anti-D prophylaxis has reduced the incidence of hemolytic disease of the fetus and newborn. This study describe the detection of fetal RhD antigens in blood of RhD negative pregnant women using a nanopolymer coated electrochemical biosensor for medical diagnosis. Cell free fetal DNA in maternal plasma was also used to genotyping fetal RHD status using multiplex real-time PCR. Twenty-six RhD negative pregnant women in different gestational ages were included in the study. RhD positive fetal antibodies detected with a developed biosensor in maternal blood of RhD negative mothers. The electrochemical measurements were performed on a PalmSens potentiostat, and corundum ceramic based screen printed gold electrode combined with the reference Ag/AgCl electrode, and the auxiliary Au/Pd (98/2%) electrode. Fetal RHD genotyping performed using fluorescence-based multiplex real-time PCR exons 5 and 7 of the RHD gene. The fetal RHD status of 26 RhD negative cases were detected 21 as RhD positive and 5 as RhD negative with electrochemical biosensor. Fetal RHD status confirmed with extracted fetal DNA in maternal plasma using multiplex real-time PCR RHD genotyping and by serological test after delivery. The new method for fetal RhD detection in early pregnancy is useful and can be carry out rapidly in clinical diagnosis. Using automated biosensors are reproducible, quick and results can be generated within a few minutes compared to noninvasive fetal RHD genotyping from maternal plasma with real-time PCR-based techniques. We suggest the biosensor techniques could become an alternative part of fetal RHD genotyping from maternal plasma as a prenatal screening in the management of RhD incompatibility.

Usefulness of Non-Invasive Fetal RHD Genotyping towards Immunoprophylaxis Optimization

Introduction: Since anti-D immunoprophylaxis given to D-negative pregnant women is a blood product, blood donations have an impact on the availability of prophylactic doses. The Pan American Health Organization reported, in June 2017, that less than half of blood donors are volunteers in Latin America and the Caribbean. In these countries, guidelines for use of anti-D prophylaxis are still controversial. The aim of this study was to demonstrate the convenience of a simple and cost-effectivene non-invasive prenatal diagnostic assay for anti-D prophylaxis optimization in multiethnic populations. Methods: Cell-free fetal DNA from plasma samples of D-negative pregnant women were analyzed by real-time PCR for simultaneous amplification of sequences of exons 5 and 10 of the RHD gene. Fetal RHD genotype was determined in 111 pregnant women. Neonates' phenotype was determined 72 h after birth. Results: Genotyping predicted fetal phenotype with 100% accuracy. Prenatal diagnosis showed 78% RHD-positive and 22% RHD-negative neonates. Conclusion: We demonstrated that, beyond the large genetic variation of the Rh system and the numerous D variants present in multiethnic groups, non-invasive fetal RHD genotyping using two sequences of the gene can be enough for clinical application in an admixed population. This robust technique of simple implementation allows to determine fetal RHD in maternal blood with high sensitivity, specificity, and accuracy. The introduction of fetal RhD genotyping as part of an antenatal screening program constitutes a reliable manner to optimize anti-D prophylaxis; however, it has not been implemented so far in most American countries.

Noninvasive fetal RHD genotyping of RhD negative pregnant women for targeted anti-D therapy in Australia: A cost-effectiveness analysis.

To undertake a cost-effectiveness analysis of noninvasive fetal RHD genotyping to target pregnant women for antenatal anti-D prophylaxis therapy. A decision-analytic model was constructed to compare RHD testing and targeted anti-D prophylaxis, with current universal anti-D prophylaxis among pregnant women with RhD negative blood type. Model estimates were derived from national perinatal statistics, published literature, donor program records, and national cost sources. One-way sensitivity analyses addressed the uncertainty of variables on the main findings. The unit cost for RHD genotyping was estimated at AU$45.48 (US$31.84). The "mean cost per healthy baby" was AU$7495 (US$5247) for universal prophylaxis and AU$7471 (US$5230) for targeted prophylaxis. The findings were sensitive to the unit costs of anti-D 625IU (AU$59-AU$88) (US$41-US$62), the genetic test (AU$36-AU$55) (US$25-US$39), and packaging/transport costs of the samples for testing (AU$15-AU$40, US$11-US$28 per sample). With RHD genotyping, 13938 women would avoid antenatal anti-D prophylaxis at a total cost savings to the National Blood Authority of AU$2.1 million (US$1.5 million) per year. To the health system, net cost savings of AU$159701 (US$111791) per year (0.05%) were predicted for total health care costs. Given the vulnerable supply of donor plasma and other health concerns, RHD genotyping is an economically sound option for Australia.