Fetal RHD Genotyping Research Articles

FOETAL RHESUS D AND SEX GENOTYPING FROM THE PLASMAS OF RH-NEGATIVE PREGNANT WOMEN IN DUHOK

ABSTRACT Background: This prospective study was done to evaluate the benefit of Real-time Polymerase Chain Reaction in the determination of RHD and SRY of the fetuses in the plasma of Rhesus D-negative enrolled ladies. Methods: Thirty-nine pregnant Rh-negative females with (RhD+) partners were registered in this study. Blood samples were taken from those participants whose pregnancy age was from 20 -28 weeks for the purposes of fetal RHD genotyping and DNA extraction/purification. Real-time PCR was used to determine the RH and sex genotype using particular RHD, SRY, and GLO gene primers and probes. Results: Out of the total 39 samples, 28/39(71.8 %) were positive for Rhesus D antigen and 11/39 (28.2 %) were negative. The results of prenatal fetal RHD genotyping based on exons 7, and 10 combined were concordant with postnatal RhD phenotyping in 94.9% of cases (37/39) with a sensitivity & specificity of 93.3 and 100% respectively, and precision was 100%. For the gene SYR, there was a concordance rate of 92.3% between the fetal sex genotype and the newborn sex with one false-positive (2.6%) and two false negatives (5.1%) results leading to a precision of 95.2%. Conclusion: The study demonstrated great accuracy of the Real-Time PCR technique through the usage of cffDNA for the genetic study of the fetal RHD and SYR genes and implicates the effectiveness of this procedure for predicting the necessity of Anti-D immunoprophylaxis in pregnant women whose fetuses have a peril of hemolytic anemia of the newborn due to RhD incompatibility between partners.

Evaluation of the clinical effect of a nationwide implementation of targeted routine antenatal anti-D prophylaxis in Denmark.

In 2010, Denmark was the first country to implement a targeted routine antenatal anti-D prophylaxis (tRAADP) program, offering fetal RHD genotyping to all nonimmunized D negative pregnant women. The program represented a shift from only postnatal prophylaxis to a combined antenatal and postnatal prophylaxis. This study aimed to evaluate the clinical effect of tRAADP in Denmark. This nationwide registry-based cohort study included all D negative women who gave birth between 2004-2020, identified through the National Medical Birth Register and the Departments of Clinical Immunology in Denmark. The clinical effect of tRAADP was assessed by comparing the incidence of new D immunization between 2004-2009 (non-tRAADP-cohort) and 2011-2018 (tRAADP-cohort). A total of 282 women were D immunized during pregnancy between 2004-2009 (non-tRAADP-cohort), and 167 between 2011-2018 (tRAADP-cohort). The incidence of new D immunization decreased from 0.46% (95% CI 0.41-0.52) in the non-tRAADP-cohort to 0.22% (95% CI 0.19-0.25) in the tRAADP-cohort. The risk reduction was statistically significant p < 0.001. Notably, in the tRAADP cohort 0.1% (95% CI 0.08-0.12) of new D immunizations occurred before the time of antenatal prophylaxis. tRAADP significantly reduced the incidence of new D immunization by more than half, thus demonstrating the expected effect. However, even with full adherence to the current program, some women with early fetomaternal hemorrhage (FMH) were still at risk. Future studies may evaluate the impact of administering an additional tRAADP dose earlier in the second trimester to prevent this.

Clinical Validation of a Prenatal Cell-Free DNA Screening Test for Fetal RHD in a Large U.S. Cohort.

To present a large U.S. clinical validation of a next-generation sequencing-based, noninvasive prenatal cell-free DNA test for fetal RHD. This clinical validation study assessed the performance of a commercially available, next-generation sequencing-based cell-free DNA test for fetal RHD status. Samples that passed quality metrics were included if the patient had a previously reported cell-free DNA result for fetal aneuploidy, maternal RhD-negative serology, newborn RhD serology, and maternal RHD deletion or RHD-CE-D hybrid(r's) genotype. Dizygotic twin pregnancies were excluded. Maternal and fetal RHD genotypes were evaluated with prospective cell-free DNA next-generation sequencing analysis. At the time of analysis, investigators were blinded to fetal RhD status. The cohort consisted of 655 pregnant patients with serologic results for RhD antigen. Patient demographics included a representative distribution of race and ethnicities in the RhD-negative U.S. population (74.0% White, 13.7% Hispanic, 7.0% Black, and 2.1% Asian). Cell-free DNA fetal RHD was not reported in two cases. There were zero false-negative cases; 356 of 356 fetuses were correctly identified as fetal RhD positive (sensitivity 100%, 95% CI, 98.9-100%). Of the 297 RhD-negative fetuses, 295 were correctly identified as RhD negative (specificity 99.3%, 95% CI, 97.6-99.8%). Of the fetuses with a negative RhD phenotype, the cell-free DNA test accurately identified three with the fetal RHD pseudogene (RHDΨ) genotype. Validation of this test in this large U.S. cohort of RhD-negative patients provides data on early and accurate noninvasive prenatal identification of fetal RHD genotype at 9 weeks of gestation or more. This test has the potential to assist patients and clinicians in the prevention and management of RhD alloimmunization.

Single-exon fetal RHD genotyping: a 31-month follow up in the obstetric population of Western Sweden.

The Rh blood group system is highly complex, polymorphic, and immunogenic. The presence of RHD gene variants in RhD negative pregnant women is a challenge in fetal RHD genotyping as it may influence the antenatal management of anti-D prophylaxis. The aim of this study was to determine the efficiency of a non-invasive single-exon approach in the obstetric population of Western Sweden in a 31-month follow up. The frequency and type of maternal RHD variants were explored and the relation to the ethnicity was elucidated. Discrepant results between fetal RHD genotyping and serological blood group typing of newborns were investigated and clarified. RHD exon 4 was analysed with quantitative real-time PCR technique in a total of 6,948 blood samples from RhD negative women in early pregnancy. All cases with suspected maternal RHD gene and discrepant results observed in newborn samples, were further investigated using both serological and molecular technologies. A total of 43 samples (0.6%) had inconclusive fetal genotyping result due the presence of a maternal RHD gene. These findings were in most cases (>66%) observed in pregnant women of non-European ancestry. Additionally, two novel RHD alleles were found. Seven discrepant results between fetal RHD genotype and serological RhD type of the newborns, were shown to be related to D antigen variants in newborns. Assay sensitivity was 99.95%, specificity 100%, and accuracy 99.97%. The single-exon approach for fetal RHD screening early in pregnancy is an appropriate choice in the population of Western Sweden, with a very low frequency of inconclusive results caused by the presence of maternal RHD gene variants. Due to the high sensitivity, specificity, and accuracy of the test, serological typing of neonates born to RhD negative women has no longer been performed at our laboratory since June 2023.

The use of free DNA for fetal RHD genotyping in the Rh negative pregnant patient—the time has come

Cell-free DNA (cfDNA) to determine the fetal RHD genotype from the maternal circulation was first described in 1993. High throughput assays using polymerase chain reaction technology were introduced in Europe and gained widespread acceptance in the management of the Rhesus alloimmunized pregnancy. The specificity and sensitivity of these assays approached 99%. As confidence was gained with these results, Scandinavian countries began to employ cfDNA for fetal RHD typing as an integral component of their introduction of antenatal Rhesus immune globulin (RhIG) in non-alloimmunized pregnancies. Since 40% of RhD-negative pregnant women will carry an RhD-negative fetus, doses of RhIG were conserved. Recently two U.S. companies have introduced cfDNA assays for RHD as part of their NIPT assays. Both utilize next generation sequencing and have developed methodologies to detect the aberrant RHD pseudogene and the hybrid RHD-CE-Ds genotype. In addition, excellent correlation studies with either neonatal genotyping or serology have been reported. The manufacturer of RhoGAM® has recently announced a national shortage. . Given the current availability of reliable cfDNA assays for determining the RHD status of the fetus, the time has come to implement this strategy to triage the antenatal use of Rhesus immune globulin in the U.S..

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 fetal blood group antigen genotyping.

Noninvasive fetal blood group antigen genotyping serves as a diagnostic tool to predict the risk of hemolytic disease of the fetus and newborn in pregnancies of immunized women. In addition, fetal RHD genotyping is used as an antenatal screening to guide targeted use of immunoglobulin prophylaxis in non-immunized RhD negative, pregnant women. Based on testing of cell-free DNA extracted from maternal plasma, these noninvasive assays demonstrate high performance accuracies. Consequently, noninvasive fetal blood group antigen genotyping has become standard care in transfusion medicine.

Fetal RHD Screening in RH1 Negative Pregnant Women: Experience in Switzerland.

RH1 incompatibility between mother and fetus can cause hemolytic disease of the fetus and newborn. In Switzerland, fetal RHD genotyping from maternal blood has been recommended from gestational age 18 onwards since the year 2020. This facilitates tailored administration of RH immunoglobulin (RHIG) only to RH1 negative women carrying a RH1 positive fetus. Data from 30 months of noninvasive fetal RHD screening is presented. Cell-free DNA was extracted from 7192 plasma samples using a commercial kit, followed by an in-house qPCR to detect RHD exons 5 and 7, in addition to an amplification control. Valid results were obtained from 7072 samples, with 4515 (64%) fetuses typed RHD positive and 2556 (36%) fetuses being RHD negative. A total of 120 samples led to inconclusive results due to the presence of maternal or fetal RHD variants (46%), followed by women being serologically RH1 positive (37%), and technical issues (17%). One sample was typed false positive, possibly due to contamination. No false negative results were observed. We show that unnecessary administration of RHIG can be avoided for more than one third of RH1 negative pregnant women in Switzerland. This reduces the risks of exposure to a blood-derived product and conserves this limited resource to women in actual need.

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.

Terminating Routine Cord Blood RhD Typing of the Newborns to Guide Postnatal Anti-D Immunoglobulin Prophylaxis Based on the Results of Fetal RHD Genotyping

Introduction: Targeted routine antenatal prophylaxis with anti-D immunoglobulin (Ig) only to RhD-negative pregnant women who carry RhD-positive fetuses (determined by fetal RHD genotyping) has reduced D-alloimmunization significantly when administered in addition to postnatal prophylaxis. Achieving high analysis sensitivity and few false-negative fetal RHD results will make RhD typing of the newborn redundant. Postnatal prophylaxis can then be given based on the result of fetal RHD genotyping. Terminating routine RhD typing of the newborns in cord blood will streamline maternity care. Accordingly, we compared the results of fetal RHD genotyping with RhD typing of the newborns. Methods: Fetal RHD genotyping was performed, and antenatal anti-D Ig was administered at gestational week 24 and 28, respectively. Data for 2017–2020 are reported. Results: Ten laboratories reported 18,536 fetal RHD genotypings, and 16,378 RhD typing results of newborns. We found 46 false-positive (0.28%) and seven false-negative (0.04%) results. Sensitivity of the assays was 99.93%, while specificity was 99.24%. Conclusion: Few false-negative results support the good analysis quality of fetal RHD genotyping. Routine cord blood RhD typing will therefore be discontinued nationwide and postnatal anti-D Ig will now be given based on the result of fetal RHD genotyping.

Prenatal screening service for fetal RHD genotyping to guide prophylaxis: the two-year experience of the Friuli Venezia Giulia region in Italy.

Fetal RHD genotyping of cell-free fetal DNA (cff-DNA) from RhD-negative pregnant women can be used to guide anti-D prophylaxis: the knowledge of fetal RhD type can direct and restrict the use of prenatal anti-D immunoglobulin exclusively to RhD-negative women carrying a RhD-positive fetus. Since November 2019 in the region of Friuli Venezia Giulia (Italy) a prenatal screening service has been offered to RhD-negative women at 22-24 weeks of gestation. The cff-DNA is extracted from a simple peripheral maternal blood sample to analyze the fetal RHD gene: the results are interpreted as RHD-positive fetus, RHD-negative fetus, or Inconclusive. The service is shared with all regional hospitals and tests are provided free of charge by the National Health System. Overall, 142 RhD-negative pregnant women were recruited in nearly 2 years. Fetal RHD genotyping was negative in 53 pregnancies and positive in 89 pregnancies. Thus, unnecessary treatment of pregnant women and exposure to a scarce plasma-derived medicinal product was avoided, by the use of a single blood sample, in 37.8% of cases, representing 100% of the RhD-negative women carrying a RhD-negative fetus in our cohort. The first Italian region-wide screening service for fetal RHD genotyping has been implemented for 2 years, despite the COVID-19 pandemic, in order to obtain the predicted fetal RhD phenotype before the 28th week of gestation, during which prenatal prophylaxis is usually administered. Giving prenatal anti-D immunoglobulin exclusively to RhD-negative women carrying a RhD-positive fetus reduces the overall use of anti-D immunoglobulin, which is becoming an ever more limited resource. The high sensitivity of the procedure provides evidence that the implementation of a diagnostic test in a reference laboratory guarantees the quality of the results, the concordance of reports and the sustainability of costs, representing an excellent guide to targeted use of prophylaxis.

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.

RHD exon 5, 7 and 10 targeted non-invasive prenatal screening of fetal Rhesus-D (RhD) in selected RhD negative pregnant women in Ethiopia.

BackgroundA majority of non-invasive prenatal screening studies determining fetal RhD status have been tested on Caucasian and Asian populations, but limited or no studies have been conducted on the Ethiopian population. In the current study, we carried non-invasive prenatal screening of fetal RHD genotype in selected RhD negative Ethiopian pregnant women.MethodsCell-free DNA was extracted from the plasma samples of 117 RhD pregnant women between 9 and 38 weeks of gestation. Fetal RHD genotypes were detected by targeting exons 5, 7 and 10 of the RHD gene by using real-time PCR assay. RHD genotypic results were confirmed by neonatal cord blood serology.ResultsFetal RHD genotyping was conclusive in all 117 subjects. RHD genotype was correctly predicted in 115 of 117 cases, thus the test yielded 98.3% accuracy (95%CI: 97.3–99.1%). Among 115 cases, 105 were genotyped as RHD positive and 12 were genotyped as RHD negative. The sensitivity and specificity of the test were 99.1% (95% CI: 94.8–99.9%) and 91.7% (95%CI: 61.5–99.7%) respectively. The negative and positive predictive values were 99.9% (95%CI: 99.2–99.9%) and 54.0% (95% CI: 15.2–88.4%) respectively. SRY genotyping results were in complete concordance with fetal sex.ConclusionMulti exon targeted non-invasive prenatal screening test for fetal RhD determination exhibited high accuracy and sensitivity. A confirmatory study with a bigger size of study subjects is warranted before enabling clinical implementation.

Following targeted routine antenatal anti-D prophylaxis, almost half of the pregnant women had undetectable anti-D prophylaxis at delivery.

In September 2016, a nationwide targeted routine antenatal anti-D prophylaxis program was implemented in Norway. The prophylaxis (anti-D immunoglobulin) aims to cover the whole third trimester and is administered in gestational week 28 to RhD-negative women who carry RhD-positive fetuses. However, in many women, antibody screening at delivery does not detect anti-D immunoglobulin. The goal of this study was to investigate the presumable role of dose and timing of antenatal anti-D immunoglobulin administration in non-detectable prophylaxis at the time of delivery. In this retrospective observational study, RhD-negative pregnant women who gave birth at Oslo University Hospital and Akershus University Hospital between January 2017 and December 2019 were analyzed. Women who received antenatal anti-D immunoglobulin (1500IU at Oslo University Hospital and 1250IU at Akershus University Hospital) when fetal RHD genotyping at gestational week 24 predicted an RhD-positive fetus were included if an antibody screen at delivery was available. Data from the blood bank, maternity information systems, and electronic patient records were used. Analysis of the 984 RhD-negative women at the two hospitals revealed that 45.4% had non-detectable anti-D at delivery. A significant difference between the two hospitals was observed: 40.5% at Oslo University Hospital (n=509) and 50.7% at Akershus University Hospital (n=475) (p=0.001). The proportion with non-detectable anti-D increased to 56.0 and 75.3%, respectively (p=0.008) in the group of women who gave birth 12 weeks after routine antenatal anti-D prophylaxis. Significantly fewer women had detectable anti-D at delivery when the lower anti-D immunoglobulin dose (1250IU) was administered antenatally. Multiple logistic regression indicated that the time interval between routine antenatal anti-D prophylaxis and delivery, in addition to anti-D dose, were significantly associated with detectable anti-D at delivery (p<0.001). We do not know which RhD-negative pregnant women, despite antenatal anti-D prophylaxis, are at risk of RhD alloimmunization, when antibody screening is negative at delivery. Administration of antenatal prophylaxis should probably be moved closer to delivery, since the risk of fetomaternal hemorrhage is higher during the last weeks of the third trimester.

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.