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Objective We aimed to simplify our fetal RHD genotyping protocol by changing the method to attach Illumina’s sequencing adaptors to PCR products from the ligation-based method to a PCR-based method, and to improve its reliability and robustness by introducing unique molecular indexes, which allow us to count the numbers of DNA fragments used as PCR templates and to minimize the effects of PCR and sequencing errors. Results Both of the newly established protocols reduced time and cost compared with our conventional protocol. Removal of PCR duplicates using UMIs reduced the frequencies of erroneously mapped sequences reads likely generated by PCR and sequencing errors. The modified protocols will help us facilitate implementing fetal RHD genotyping for East Asian populations into clinical practice.

Purpose To evaluate the diagnostic accuracy of a commercially available test kit for noninvasive prenatal determination of the fetal RhD status (NIPT-RhD) with a focus on early gestation and multiple pregnancies. Methods The FetoGnost RhD assay (Ingenetix, Vienna, Austria) is routinely applied for clinical decision making either in woman with anti-D alloimmunization or to target the application of routine antenatal anti-D prophylaxis (RAADP) to women with a RhD positive fetus. Based on existing data in the laboratory information system the newborn’s serological RhD status was compared with NIPT RhD results. Results Since 2009 NIPT RhD was performed in 2968 pregnant women between weeks 5 + 6 and 40 + 0 of gestation (median 12 + 6) and conclusive results were obtained in 2888 (97.30%) cases. Diagnostic accuracy was calculated from those 2244 (77.70%) cases with the newborn’s serological RhD status reported. The sensitivity of the FetoGnost RhD assay was 99.93% (95% CI 99.61–99.99%) and the specificity was 99.61% (95% CI 98.86–99.87%). No false-positive or false-negative NIPT RhD result was observed in 203 multiple pregnancies. Conclusion NIPT RhD results are reliable when obtained with FetoGnost RhD assay. Targeted routine anti-D-prophylaxis can start as early as 11 + 0 weeks of gestation in singleton and multiple pregnancies.

Background and objective Routine antenatal anti-D prophylaxis (RAADP) to RhD-negative women is most often administered in gestational age (GA) 28-30 weeks with the next anti-D dose administered postpartum. The aim of this study was to analyse the proportion of RhD-negative women where RAADP is not detectable at term and in a pilot study to investigate whether RAADP administered in GA 28 and 38 results in detectable levels at term, post-term and post-delivery. Materials and methods In a retrospective analysis, 4280 RhD-negative women carrying an RHD positive fetus were included and the proportion with a negative antibody screen at delivery was determined. In the second part, 39 pregnancies were included prospectively, a second dose of RAADP was administered in GA 38 weeks, and anti-D was quantified before the second dose and then weekly for 5 weeks. Results In the retrospective analysis, 20·5% (856/4280) with RAADP administered in GA 28 were negative in routine antibody screening at delivery. In the small prospective study, 18% (7/39) had a negative antibody screen and 26% (10/39) had levels below 0·005 IU/ml, in the quantification assay, in GA 38. Anti-D prophylaxis administered in GA 38 showed detectable levels of anti-D up to 30 days post-delivery, with concentration at delivery 0·060 ± 0·034 IU/ml (mean ± SD). Conclusion Approximately 20% of the RhD-negative women show non-detectable levels of anti-D at term. A second dose of RAADP at GA 38 results in stable concentrations of anti-D at term, post-term and post-delivery, but with large interindividual variation.

Background: The WHO recommends that pre-transfusion testing should include ABO/RhD grouping followed by screen- ing for red blood cell (RBC) alloantibodies using the indirect antiglobulin test (IAT). However, in Uganda, current practice does not include RBC alloantibody screening. Objective: To assess the utility of ‘home-made’ reagent RBCs in alloantibody screening. Materials and methods: In a laboratory-based study, group O RhD positive volunteer donors were recruited and their extended phenotype performed for C, c, E, e, K, Fya, Fyb Jkb, S and s antigens. These ‘home-made’ reagent RBCs were preserved using Alsever’s solution and alloantibody detection tests performed. For quality assurance, repeat alloantibody screening of patients’ samples was done at Bloodworks Northwest Laboratory in Seattle, United States. Results: A total of 36 group O RhD positive individuals were recruited as reagent RBC donors (median age, 25 years; range, 21 – 58 years; male-to-female ratio, 1.6:1). Out of the 311 IATs performed, 32 (10.3%) were positive. Confirmatory IAT testing in the United States was in agreement with the findings in Uganda. Conclusion: Use of ‘home-made’ reagent RBCs during pre-transfusion testing in Uganda is feasible. We recommend the introduction of pre-transfusion IAT alloantibody screening in Uganda using ‘home-made’ reagent RBCs to improve trans- fusion safety. Keywords: Blood transfusion; ‘Home-made’ reagent RBCs; Pre-transfusion testing; RBC alloantibody screening; Uganda.

of the study is to research the epidemiological aspects of maternal alloimmunization against erythrocyte antigens of fetuses (AB0, Rhesus, Lewis, Kell, Duffy and others) and to identify the most common types of hemolytic disease of the newborn (HDN) in the West Herzegovina region.The 20-year retrospective epidemiological study includes all pregnant women who had been immunologically tested and newborn treated for HDN.The indirect antiglobulin (IAT) detected antibodies against antigens in 545 (1.8%) pregnant women of the 29 663 who were tested at the Department of Transfusion Medicine. During the 20-year-long study 310 (1.0%) newborn with HDN were treated. Our results indicate that 42% (230/545) of the pregnant women had AB0 immunization. The most common form of HDN is AB0 HDN 64% (199/310), whereas RhD HDN was treated in 19% (59/310) of the newborn infants. ETR was performed on 29 (19%) infants, 21 (72.4%) with AB0 HDN, and 7 (26%) with RhD HDN.This 20-year-long study concludes that, even though there has been significant progress in the prevention of immunization and proactive treatment of HDN, precautionary measures are still required as is the need for gynecologists and obstetricians to be active. The reasons for this are the non-existence of preventive measures for non-RhD immunization, the irregular immunological screening of RhD positive women in pregnancy in the region encompassed by the study in the past few years. The above raises new questions and recommends further research and monitoring of immunization and HDN treatment worldwide.ZIEL DIESER STUDIE: war die Untersuchung von epidemiologischen Aspekten mütterlicher Immunisierung gegen fetale Erythrozytenantigene(AB0, Rhesus, Lewis, Kell, Duffy und andere) und die Feststellung von häufigsten Ursachen der hämolytischen Erkrankung des Neugeborenen (MHN) in der Region der West-Herzegowina.20-jährige retrospektive epidemiologische Studie umfasste alle Schwangeren, die immunologische getestet sowie Neugeborene, die wegen MHN behandelt wurden.Durch den indirekten antiglobulin Test (IAT) wurden bei 545 (1,8%) Schwangeren Antikörper gegen Antigene nachgewiesen, von 29 663 Probanden, die an der Transfusionsanstalt UHC Mostar getestet wurden. Während der zwanzigjährigen Studie hatten 310 (1,0%) Neugeborene MHN. Unsere Ergebnisse zeigten, dass 42% (230/545) der Schwangeren die AB0-Immunisierung hatten. Die häufigste MHN bei dieser Forschung war die AB0 MHN 64% (199/310), während RhD MHN bei 19% (59/310) der Neugeborenen behandelt wurde.Die Blutaustauschtransfusion (ETR) erfolgte bei 29 (19%) Neugeborenen, 21 (72,4%) bei Neugeborenen mit AB0 MHNund 7 (26%) mit Rh D MHN.Obwohl es zu während dieser 20-jährigen Studie zu einem bedeutenden Fortschritt bei der Vorbeugung der Immunisierung und der proaktiven MHN-Behandlung kam, erfordert es weiterhin Vorsichtsmaßnahmen und die Aktivitäten der Gynäkologen und Neonatologen. Grund dafür ist das Nichtbestehen von Vorbeugungsmaßnahmen zur nicht-RhD Immunisierung (67% der Schwangeren hatten Nicht-RhD Antikörper), unregelmäßiges immunologisches Testen von RhD-positiven Schwangeren in den letzten Jahren im Untersuchungsgebiet. All dies eröffnet neue Fragen und Empfehlungen zur Erforschung und Beobachtung der Immunisierung sowie MHN-Behandlung auf der globalen immunologischen Szene.

OBJECTIVE To determine the occurrence of and risk factors for fetomaternal hemorrhage (FMH) among pregnant women at Korle Bu Teaching Hospital in Accra, Ghana. METHODS A prospective study of FMH among pregnant women without hemoglobinopathies in the second trimester attending prenatal care between October 2015 and May 2016 performed using the Kleihauer-Betke test. Volume of FMH was estimated; ABO and Rh blood groups of participants were determined. A data extraction form and structured questionnaire were used to collect demographic and clinical information, and data on risk factors. RESULTS Of 151 participants, 32 (21.2%) had FMH. Almost 18% (n=27) had FMH at baseline (16-24 weeks), 10% (10/100) at 28-32 weeks, and 11.1% (11/99) at 34-37 weeks of pregnancy. Volume of FMH was less than 30 mL in 30 (19.9%) women, whereas it was greater than 30 mL in 2 (1.3%) women. No identifiable patient-specific factors were associated with occurrence of FMH. CONCLUSION FMH is common among pregnant women in Ghana and can occur as early as 16 weeks, without identifiable risk factors. RhD negative women who may be pregnant with RhD positive fetuses should be screened early in pregnancy, not only at delivery, for occurrence of FMH.

Introduction Early transfusion reduces mortality in bleeding patients. In this setting, RhD-positive blood products might be transfused. This study determined the association between the RhD-alloimmunization rate and the number of RhD-positive products transfused. Methods RhD-negative patients between 13 and 50 years who were transfused with ≥1 RhD-positive red blood cell (RBC) or whole blood units between January 1, 2000 and December 31, 2019 in a healthcare network were identified. Study patients had to have had at least one antibody detection test performed ≥14 days after the index RhD-positive transfusion and not receive RhIg. Patients were stratified into groups that received 1, 2, 3-5, 6-10, 11-20, and >20 RhD-positive transfusions and the RhD-alloimmunization rate was determined for each group. Results There were 335 patients included; 52/335 (15.5%) were females. Overall, there were 117/335 (34.9%, CI: 29.8%-40.3%) recipients who became RhD-alloimmunized. There was no significant dosage effect in the RhD-alloimmunization rates as the exposure to RhD-positive units increased from one RhD-positive unit to more than 20 RhD-positive units (p = .270 for non-parametric trend test). In an exploratory analysis, patients who received 100% of their RhD-positive transfusions within 72 h of the index transfusion had a significantly higher rate of RhD-alloimmunization compared to those who were transfused over a longer period of time (42.3% vs. 21.4%, respectively; p = .001). Conclusion These results suggest that there may not be an increased RhD-alloimmunization risk with transfusing multiple RhD-positive units after one RhD-positive unit has been transfused. These findings need confirmation in larger studies.

Background Guidelines for laboratory assessment of fetomaternal haemorrhage (FMH) was published by the Australian and New Zealand Society of Blood Transfusion (ANZSBT) in 2002. However, data on adherence by practitioners and clinical outcomes are lacking. Aims The primary objective is to examine the follow-up testing and dosing of additional RhD immunoglobulin in RhD negative women who experienced large-volume FMH for whom additional intravenous RhD immunoglobulin was requested in Queensland, Australia. The secondary objectives are to examine the rate and risk factors of RhD alloimmunisation in these women. Materials and methods RhD negative women with FMH >6 mL for whom additional dose(s) of intravenous RhD immunoglobulin was requested through Australian Red Cross Lifeblood from February 2007 to February 2018 were identified. For each patient, the volume of FMH, methods and timing of FMH quantitation, dose of RhD immunoglobulin, maternal and cord blood groups were analysed against the corresponding antibody screen and identification. Results Following FMH >6 mL, only 15% and 11.5% of cases adhered to current ANZSBT guideline on follow-up testing and supplemental RhD immunoglobulin dosing respectively. Despite the provision of single supplemental RhD immunoglobulin at a ratio of 100 IU to 1 mL fetal red cells, the rate of RhD alloimmunisation in RhD negative women with RhD positive fetus or fetus of unknown RhD status following FMH >6 mL is at least 4%. Conclusions Poor compliance with guidelines for follow-up and management of large-volume FMH may contribute to increased risk of RhD alloimmunisation. Further analysis of data is warranted.

Background and objectives: Fetal RHD genotyping of cell-free maternal plasma DNA from RhD negative pregnant women can be used to guide targeted antenatal and postnatal anti-D prophylaxis for the prevention of RhD immunization. To assure the quality of clinical testing, we conducted an external quality assessment workshop with the participation of 31 laboratories. Materials and methods: Aliquots of pooled maternal plasma from gestational week 25 were sent to each laboratory. One sample was fetal RHD positive, and a second sample was fetal RHD negative. A reporting scheme was supplied for data collection, including questions regarding the methodological setup, results and clinical recommendations. The samples were tested blindly. Results: Different methodological approaches were used; 29 laboratories used qPCR and two laboratories used ddPCR, employing a total of eight different combinations of RHD exon targets. Fetal RHD genotyping was performed with no false-negative and no false-positive results. One inconclusive result was reported for the RHD positive sample. All clinical conclusions were satisfactory. Conclusion: This external quality assessment workshop demonstrates that despite the different approaches taken to perform the clinical assays, fetal RHD genotyping is a reliable laboratory assay to guide targeted use of Rh prophylaxis in a clinical setting. (Less)

Background Previous studies suggest that RhD positive heterozygotes express better health status than RhD positive homozygotes and especially RhD negative subjects. This also applies to pregnant women. According to the Trivers-Willard hypothesis, women in better physical condition should have more sons. Aim To test the hypothesis that RhD positive heterozygous mothers have a male-skewed sex ratio. Study design Cross-sectional study. The data was analysed using Chi-Square test for all women, separately for RhD positive and RhD negative women, and separately for primiparous and multiparous women. The effects of maternal weight as a continuous predictor and the RhD phenotype of newborn as a categorical predictor of newborn sex were evaluated by the generalized linear model (GLZ) separately for RhD positive and RhD negative women using binomial distribution and logit link function. Outcome measures Clinical records comprised maternal weight before pregnancy, number of previous deliveries, sex of the newborn, maternal RhD phenotype, and RhD phenotype of the newborn. Subjects We analysed data from 5655 women who gave birth between 2008 and 2012 in General University Hospital in Prague. Results Secondary sex ratio was significantly higher (P = 0.028) in RhD positive mothers who had RhD negative newborns, i.e., in heterozygotes (SR = 1.23), than in RhD positive mothers who had RhD positive newborns, i.e., in a mixed population of heterozygotes and homozygotes (SR = 1.00), especially in primiparous women (P = 0.013; SR = 1.37 and 0.99 resp.). Conclusion The sex ratio at birth was significantly higher in RhD positive mothers who had RhD negative newborns than in RhD positive mothers who had RhD positive newborns.

Starting in 2013, blood donors must be tested at least using: (1) one monoclonal anti-D and one anti-CDE (alternatively full RhCcEe phenotyping), and (2) all RhD negative donors must be tested for RHD exons 5 and 10 plus one further exonic, or intronic RHD specificity, according to the guidelines of the Blood Transfusion Service of the Swiss Red Cross (BTS SRC). In 2012 an adequate stock of RHD screened donors was built. Of all 25,370 RhD negative Swiss donors tested in 2012, 20,015 tested at BTS Berne and 5355 at BTS Zürich, showed 120 (0.47%) RHD positivity. Thirty-seven (0.15%) had to be redefined as RhD positive. Routine molecular RHD screening is reliable, rapid and cost-effective and provides safer RBC units in Switzerland.

The frequency of red blood cell (RBC) alloimmunization in RhD positive pregnant women is not known in our population. We planned to determine its frequency and correlation with neonatal outcome. We included 1000 RhD positive pregnant women: 500 had 'normal pregnancy' (Group I) and another 500 had 'high risk pregnancy' (Group II). ABO and extended Rh phenotyping were done by tube technique, antibody screening and identification by gel technique. For alloimmunized women, the paternal and neonatal ABO and extended Rh typing were done. Neonatal direct antiglobulin test (DAT) was also done and their clinical outcome observed. The frequency of RBC alloimmunization was 0.7% (7/1000) and all these women were from group II (p = 0.015). The alloantibodies were anti-E (85.7%), anti-c (71.4%), anti-Cw (14.3%) and anti-S (14.3%). Also, 6 women had history of transfusion (p

To assess the impact of rheumatic heart disease (RHD) on child-reported health-related quality of life (HRQOL) in the context of a Ugandan school-based echocardiographic screening program. Echocardiography-based screening has emerged as a tool for the early detection of RHD, but little is known about its impact on those screened.Participants included 358 children from Gulu and 28 children from Kampala Uganda. The Pediatric Quality of Life Inventory Version 4.0 was used to assess HRQOL in 4 groups of children: Gulu prescreen, Gulu postscreen, Gulu previously linked to care, and Kampala previously linked to care. Children in the pre- and postscreen groups were selected from a single school before and after screening occurred and matched by age and sex. Children previously linked to care were recruited from previous screening studies.When the echocardiogram was normal, there was no difference in HRQOL in the prescreen and postscreen groups. In the postscreen group, identification of latent RHD resulted in lower physical (75.3 vs 68.3, P = .03) and emotional (71.7 vs 63.4, P .01) HRQOL, despite a lack of symptoms. The Kampala group had longer linkage to care (42 months vs 6 months, P .01) and demonstrated greater HRQOL scores compared with the Gulu-linked group (70.7 vs 77.8, P .01) and the combined Gulu cohort (77.8 vs 69.4, P = .02).Echocardiography-based screening for RHD does not diminish HRQOL in Ugandan children; rather, a diminished HRQOL score may be associated with being identified as RHD positive. Further investigation is needed to understand if longer linkage to care may ultimately normalize or improve HRQOL.

Rhesus-positive and Rhesus-negative persons differ in the presence-absence of highly immunogenic RhD protein on the erythrocyte membrane. The biological function of the RhD molecule is unknown. Its structure suggests that the molecular complex with RhD protein transports NH3 or CO2 molecules across the erythrocyte cell membrane. Some data indicate that RhD positive and RhD negative subjects differ in their tolerance to certain biological factors, including, Toxoplasma infection, aging and fatique. Present cross sectional study performed on 3,130 subjects) showed that Rhesus negative subjects differed in many indices of their health status, including incidences of many disorders. Rhesus negative subjects reported to have more frequent allergic, digestive, heart, hematological, immunity, mental health, and neurological problems. On the population level, a Rhesus-negativity-associated burden could be compensated for, for example, by the heterozygote advantage, but for Rhesus negative subjects this burden represents a serious problem.

This work aims to assess the value of a generalized molecular RHD screening strategy which could replace routine serological screening of weak D by indirect antiglobulin test. Three independent studies were performed at the two Blood Transfusion Services Berne and Zurich. Donors investigated were 652 RhD negative, but RhC and/or RhE positive, 17,391 mainly Rhccee, and 8200 with normal RhCcEe phenotype distribution. In study I single samples, in studies II and III minipools of 24 and 20 donor samples were tested, respectively. Among 26,243 phenotypically RhD negative blood donors, 65 carriers of RHD alleles were identified. Thirty-one of them were redefined as RhD positive.

Anti-RhD immunoglobulin was administered to RhD-negative women based on estimates of fetal bleed size obtained using a direct immunofluorescence flow cytometric technique employing a FITC-conjugated monoclonal human anti-D (BRAD 3). The effectiveness of the dose administered was assessed by (i) measuring the fraction of RhD-positive fetal cells in the maternal circulation at d0, and between d2 and d10 post RhD Ig administration, (ii) quantifying the amount of anti-D detectable in maternal plasma following RhD Ig injection in the perinatal period and (iii) assessing maternal serum for the presence of immune anti-D in follow-up samples taken 3 months to 3 years after delivery. Fifty-four women were assessed, 29 having fetal bleeds in excess of 4 mL. Follow-up samples were received from 20/29 mothers after RhD Ig administration; 43-99% and 69-99% of fetal cells had been cleared by d2/3 and d5/6, respectively, in 14/20 mothers, whereas less than 50% had been cleared in the remaining mothers. Long-term follow-up samples were obtained from eight of the 29 mothers (four with bleeds >/=20 mL, two with bleeds >95 mL): none had detectable anti-D in the serum 4 months to 3 years after delivery despite the persistence of up to 36% fetal RhD-positive cells in the maternal circulation six days after delivery.

Background Echocardiographic screening for detection of latent RHD has shown potential as a strategy to decrease the burden of disease. However, further research is needed to determine optimal implementation strategies. RHD results from a complex interplay between environment and host susceptibility. Family members share both and relatives of children with latent RHD may represent a high-risk group. The objective of this study was to use echocardiographic family screening to determine the relative risk of RHD among first-degree relatives of children with latent RHD compared to the risk in first-degree relatives of healthy peers. Methodology/Principal Findings Previous school-based screening data were used to identify RHD positive children and RHD negative peers. All first-degree relatives ≥ 5 years were invited for echocardiography screening (2012 World Heart Federation Criteria). Sixty RHD positive cases (30 borderline/30 definite RHD) and 67 RHD negative cases were recruited. A total of 455/667 (68%) family members were screened. Definite RHD was more common in childhood siblings of RHD positive compared to RHD negative (p = 0.05). Children with any RHD were 4.5 times as likely to have a sibling with definite RHD, a risk that increased to 5.6 times when considering only cases with definite RHD. Mothers of RHD positive and RHD negative cases had an unexpectedly high rate of latent RHD (9.3%). Conclusions/Significance Siblings of RHD positive cases with RHD are more likely to have definite RHD and the relative risk is highest if the index case has definite RHD. Future screening programs should consider implementation of sibling screening following detection of an RHD positive child. Larger screening studies of adults are needed, as data on prevalence of latent RHD outside of childhood are sparse. Future studies should prioritize implementation research to answer questions of how RHD screening can best be integrated into existing healthcare structures, ensuring practical and sustainable screening programs., Author Summary Rheumatic heart disease (RHD) affects at least 33 million people, most of who live in low-resource environments. RHD is a cumulative process and there exists a latent period between early valve damage and presentation with symptoms. Echocardiographic screening (ultrasound of the heart) has proven highly sensitive for latent RHD detection, but implementation research is needed to effectively develop sustainable public health strategies. Critical to this research is determining whom to screen. As family members have both a shared environment and shared genetic susceptibility, they may represent a high-risk group that could be targeted once a case of RHD is identified. We conducted an echocardiographic family screening study to determine the risk of RHD in families with and without an RHD positive child and found that siblings of children with latent RHD are more likely to have latent RHD themselves. Our data suggest that siblings may represent a particularly high-risk group that could be targeted for echocardiographic screening. Future studies are needed to answer questions of how RHD screening can best be integrated into existing healthcare structures, ensuring practical and sustainable RHD screening programs.

A 65-year-old woman, blood group A RhD positive, who had completed her first course of induction chemotherapy for acute myeloid leukaemia was transfused with apheresis platelets over a number of days. On three occasions she received group O RhD positive units, which had been screened and found not to contain high-titre anti-A,B isoagglutinins. Following the third unit, she developed a haemolytic transfusion reaction and died soon thereafter. This has led to change in policy of the supplying centre in testing for high-titre anti-A,B isoagglutinins. Blood group O apheresis platelets and fresh-frozen plasma units are now labelled as high titre with a cut-off of 1/50 as compared to the previous cut-off of 1/100 for anti-A,B isoagglutinins. A universal approach to testing donations for high-titre anti-A,B isoagglutinins, better compliance of guidelines and monitoring of patients is necessary.

To the Editor: The RhD status of transfusion recipients and donors is routinely matched for red-cell transfusion. This worldwide practice is due to the potent immunogenicity of RhD. In East Asians, the frequency of RhD-negative status is only about 0.3%, which sharply limits the supply of RhD-negative blood. However, approximately 30% of RhD-negative persons carry an RhD variant, termed “Asia type” DEL.1 Beginning in 2008, my colleagues and I organized a collaborative group of 10 laboratories, located in 10 cities in northern, central, and southern China. This group retrospectively evaluated 104 RhD-negative pregnant women with anti-D alloantibodies from 2005 through . . .