Feasibility for non-invasive prenatal fetal blood group and platelet genotyping by massively parallel sequencing: A single test system for multiple atypical red cell, platelet and quality control markers.

Non-invasive prenatal tests (NIPT) to predict fetal red cell or platelet antigen status for alloimmunised women are provided for select antigens. This study reports on massively parallel sequencing (MPS) using a red cell and platelet probe panel targeting multiple nucleotide variants, plus individual identification single nucleotide polymorphisms (IISNPs). Maternal blood samples were provided from 33 alloimmunised cases, including seven with two red cell antibodies. Cell-free and genomic DNA was sequenced using targeted MPS and bioinformatically analysed using low-frequency variant detection. The resulting maternal genomic DNA allele frequency was subtracted from the cell-free DNA counterpart. Outcomes were matched against validated phenotyping/genotyping methods, where available. A 2.5% subtractive allele frequency threshold was set after comparing MPS predictions for K, RhC/c, RhE/e and Fya /Fyb against expected outcomes. This threshold was used for subsequent predictions, including HPA-15a, Jka /Jkb , Kpa /Kpb and Lua . MPS outcomes were 97.2% concordant with validated methods; one RhC case was discordantly negative and lacked IISNPs. IISNPs were informative for 30/33 cases as controls. NIPT MPS is feasible for fetal blood group genotyping and covers multiple blood groups and control targets in a single test. Noting caution for the Rh system, this has the potential to provide a personalised service for alloimmunised women.

A cold case of hemolytic disease of the fetus and newborn resolved by genomic sequencing and population studies to define a new antigen in the Rh system.

BACKGROUND: We report an obstetric case involving an RhD-positive woman who had developed a red blood cell (RBC) antibody that was not detected until after delivery of a newborn, who presented with a positive direct antiglobulin test result. Immunohematology studies suggested that the maternal antibody was directed against a low-prevalence antigen on the paternal and newborn RBCs. RESULTS: Comprehensive blood group profiling by targeted exome sequencing revealed a novel nonsynonymous single nucleotide variant (SNV) RHCE c.486C>G (GenBank MZ326705) on the RHCE*Ce allele, for both the father and newborn. A subsequent genomic-based study to profile blood groups in an Indigenous Australian population revealed the same SNV in 2 of 247 individuals. Serology testing showed that the maternal antibody reacted specifically with RBCs from these two individuals. DISCUSSION: The maternal antibody was directed against a novel antigen in the Rh blood group system arising from an RHCE c.486C>G variant on the RHCE*Ce allele linked to RHD*01. The variant predicts a p.Asn162Lys change on the RhCE protein and has been registered as the 56th antigen in the Rh system, ISBT RH 004063. CONCLUSION: This antibody was of clinical significance, resulting in a mild to moderate hemolytic disease of the fetus and newborn (HDFN). In the past, the cause of such HDFN cases may have remained unresolved. Genomic sequencing combined with population studies now assists in resolving such cases. Further population studies have potential to inform the need to design population-specific red cell antibody typing panels for antibody screening in the Australian population.

Recurrent pregnancy loss in a patient with anti-Rh17.

BACKGROUND: Rh is one of the most important blood group systems in transfusion medicine. The two homologous genes RHD and RHCE are located on chromosome 1p36.11 and encode for RhD and RhCE proteins, respectively. Complex genetic polymorphisms result in a variety of antigenic expression of D, C, E, c, and e. Here, we describe a case of a young female with D-- who developed anti-Rh17 secondary to blood transfusion and had signs of haemolytic disease of the fetus and fetal death in five consecutive pregnancies. CASE DESCRIPTION: EDTA-whole blood samples were collected from the patient, husband and eight siblings for blood grouping, phenotyping, and red cell antibody screening. Extracted DNA was genotyped by SNP-microarray and massively parallel sequencing (MPS) with targeted blood group exome sequencing. Copy number variation analysis was performed to identify structural variants in the RHD and RHCE. Routine phenotyping showed all family members were D+. The patient's red blood cells were C-E-c-e-, Rh17- and Rh46- and had anti-Rh17 and anti-e antibodies. MPS showed the patient carried a wildtype RHD sequence and homozygous for RHCE (1)-D (2-9)-CE (10) hybrid gene predicted to express a D-- phenotype. CONCLUSIONS: Our patient had a rare D-- phenotype and confirmed to have RHCE/RHD hybrid gene with replacement of 2-9 exons of RHCE by RHD sequences. Unfortunately, our patient developed anti-Rh17 and anti-e antibodies due to blood transfusion and suffered fetal demise in her very first pregnancy. The adverse outcomes could have been prevented by active prenatal management.

Impact of transcription factors KLF1 and GATA1 on red blood cell antigen expression: a review.

KLF transcription factor 1 (KLF1) and GATA binding protein 1 (GATA1) are transcription factors (TFs) that initiate and regulate transcription of the genes involved in erythropoiesis. These TFs possess DNA-binding domains that recognize specific nucleotide sequences in genes, to which they bind and regulate transcription. Variants in the genes that encode either KLF1 or GATA1 can result in a range of hematologic phenotypes-from benign to severe forms of thrombocytopenia and anemia; they can also weaken the expression of blood group antigens. The Lutheran (LU) blood group system is susceptible to TF gene variations, particularly KLF1 variants. Individuals heterozygous for KLF1 gene variants show reduced Lutheran antigens on red blood cells that are not usually detected by routine hemagglutination methods. This reduced antigen expression is referred to as the In(Lu) phenotype. For accurate blood typing, it is important to distinguish between the In(Lu) phenotype, which has very weak antigen expression, and the true Lunull phenotype, which has no antigen expression. The International Society of Blood Transfusion blood group allele database registers KLF1 and GATA1 variants associated with modified Lutheran expression. Here, we review KLF1 and recent novel gene variants defined through investigating blood group phenotype and genotype discrepancies or, for one report, investigating cases with unexplained chronic anemia. In addition, we include a review of the GATA1 TF, including a case report describing the second GATA1 variant associated with a serologic Lu(a-b-) phenotype. Finally, we review both past and recent reports on variations in the DNA sequence motifs on the blood group genes that disrupt the binding of the GATA1 TF and either remove or reduce erythroid antigen expression. This review highlights the diversity and complexity of the transcription process itself and the need to consider these factors as an added component for accurate blood group phenotyping.

Hemolytic disease of the fetus and newborn caused by anti-sD antibody in a GP.Mur/Mur Thai mother and review of the prevalence of sD in Thai blood donors.

BACKGROUND: Low-prevalence antigen sD (MNS23) is encoded by GYPB c.173C > G. Hemolytic disease of the fetus and newborn (HDFN) due to anti-sD is rare. A mother delivered a newborn whose red blood cells (RBCs) were DAT-positive and was later diagnosed with HDFN. Serum from the mother was incompatible with the father's RBCs and was used to screen 184 Thai blood donors. This study aimed to investigate the cause of HDFN in a Thai family and determine the prevalence of sD in Thai blood donors. MATERIALS AND METHODS: Three family members and four blood donors were investigated in the study. Massively Parallel Sequencing (MPS) was used for genotyping. Standard hemagglutination techniques were used in titration studies, phenotyping, and enzyme/chemical studies. Anti-s, anti-Mia , anti-JENU, and anti-sD reagents were used in serological investigations. RESULTS: The mother was GYP*Mur/Mur. The father and the four donors were GYPB*s/sD predicting S - s + sD +. The baby was GYP*Mur/sD and his RBCs were Mia +, s + w with anti-s (P3BER) and JENU+w . RBCs from two GYPB*sD -positive blood donors reacted with anti-sD (Dreyer). Proteolytic enzyme α-chymotrypsin-treated sD + cells did not react with anti-sD (Wat) produced by the GP.Mur/Mur mother but reacted with the original anti-sD (Dreyer). DISCUSSION: This is the first report of HDFN due to anti-sD in the Asian population. The genotype frequency for GYPB*sD in a selected Thai blood donor population is 2.2% (4/184). Anti-sD should be considered in mothers with Southeast Asian or East Asian background when antibody identification is unresolved in pregnancies affected by HDFN.

A new high-prevalence LW antigen detected by an antibody in an Indigenous Australian homozygous for LW*A c.309C>A variant.

BACKGROUND AND OBJECTIVES: The LW gene encodes the LW glycoprotein that carries the antigens of the LW blood group system. LW antigens are distinct from D antigen, however, they are phenotypically related and anti-LW antibodies are often mistaken as anti-D. An antibody was detected in an Australian patient of Aboriginal descent who consistently typed as LW(a+b-). This study aimed to describe the antibody recognizing a high-prevalence antigen on the LW glycoprotein. STUDY DESIGN AND METHODS: Samples from the patient and her four siblings were investigated. DNA was genotyped by single nucleotide polymorphism (SNP)-microarray and massively parallel sequencing (MPS) platforms. Red blood cells (RBCs) were phenotyped using standard haemagglutination techniques. Antibody investigations were performed using a panel of phenotyped RBCs from adults and cord blood cells. RESULTS: SNP-microarray and MPS genotyped all family members as LW*A/A, (c.299A), predicting LW(a+b-). In addition, a novel LW*A c.309C>A single nucleotide variant was detected in all family members. The patient and one of her siblings (M4) were LW c.309C>A homozygous. Antibody from the patient reacted positive to all reagent panel RBCs and cord blood cells but negative with RBCs from LW(a-b-), Rhnull and sibling M4. Antibody failed to react with RBCs treated with dithiothreitol. CONCLUSION: Antibody detected in the patient recognized a novel high-prevalence antigen, LWEM, in the LW blood group system. LWEM-negative patients who developed anti-LWEM can be safely transfused with D+ RBCs, however, D- is preferred. Accurate antibody identification can help better manage allocation of blood products especially when D- RBCs are in short supply.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

BACKGROUND AND OBJECTIVES: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021. MATERIALS AND METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented. RESULTS: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030). CONCLUSION: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

Non-invasive prenatal testing for management of haemolytic disease of the fetus and newborn induced by maternal alloimmunisation.

Anti-D immunoglobulin prophylaxis reduces the risk of RhD negative women becoming alloimmunised to the RhD antigen and is a major preventative strategy in reducing the burden of haemolytic disease of the fetus and newborn (HDFN). HDFN also arises from other maternal red cell antibodies, with the most clinically significant, after anti-D, being anti-K, anti-c and anti-E. Among the 39 human blood group systems advanced genomic technologies are still revealing novel or rare antigens involved in maternal alloimmunisation. Where clinically significant maternal antibodies are detected in pregnancy, non-invasive prenatal testing (NIPT) of cell-free fetal DNA provides a safe way to assess the fetal blood group antigen status. This provides information as to the risk for HDFN and thus guides management strategies. In many countries, NIPT fetal RHD genotyping as a diagnostic test using real-time PCR has already been integrated into routine clinical care for the management of women with allo-anti-D to assess the risk for HDFN. In addition, screening programs have been established to provide antenatal assessment of the fetal RHD genotype in non-alloimmunised RhD negative pregnant women to target anti-D prophylaxis to those predicted to be carrying an RhD positive baby. Both diagnostic and screening assays exhibit high accuracy (over 99 %). NIPT fetal genotyping for atypical (other than RhD) blood group antigens presents more challenges as most arise from a single nucleotide variant. Recent studies show potential for genomic and digital technologies to provide a personalised medicine approach with NIPT to assess fetal blood group status for women with other (non-D) red cell antibodies to manage the risk for HDFN.

Frequency of Mia (MNS7) and Classification of Mia-Positive Hybrid Glycophorins in an Australian Blood Donor Population.

BACKGROUND: MNS blood group system genes GYPA and GYPB share a high degree of sequence homology and gene structure. Homologous exchanges between GYPA and GYPB form hybrid genes encoding hybrid glycophorins GP(A-B-A) and GP(B-A-B). Over 20 hybrid glycophorins have been characterised. Each has a distinct phenotype defined by the profile of antigens expressed including Mia. Seven hybrid glycophorins carry Mia and have been reported in Caucasian and Asian population groups. In Australia, the population is diverse; however, the prevalence of hybrid glycophorins in the population has never been determined. The aims of this study were to determine the frequency of Mia and to classify Mia-positive hybrid glycophorins in an Australian blood donor population. METHOD: Blood samples from 5,098 Australian blood donors were randomly selected and screened for Mia using anti-Mia monoclonal antibody (CBC-172) by standard haemagglutination technique. Mia-positive red blood cells (RBCs) were further characterised using a panel of phenotyping reagents. Genotyping by high-resolution melting analysis and DNA sequencing were used to confirm serology. RESULT: RBCs from 11/5,098 samples were Mia-positive, representing a frequency of 0.22%. Serological and molecular typing identified four types of Mia-positive hybrid glycophorins: GP.Hut (n = 2), GP.Vw (n = 3), GP.Mur (n = 5), and 1 GP.Bun (n = 1). GP.Mur was the most common. CONCLUSION: This is the first comprehensive study on the frequency of Mia and types of hybrid glycophorins present in an Australian blood donor population. The demographics of Australia are diverse and ever-changing. Knowing the blood group profile in a population is essential to manage transfusion needs.

Developments beyond blood group serology in the genomics era.

Blood group serology and single nucleotide polymorphism-based genotyping platforms are accurate but do not provide a comprehensive cover for all 36 blood group systems and do not cover the antigen diversity observed among population groups. This review examines the extent to which genomics is shaping blood group serology. Resources for genomics include the Human Reference Genome Sequence assembly; curated blood group tables listing variants; public databases providing information on genetic variants from world-wide studies; and massively parallel sequencing technologies. Blood group genomic studies span the spectrum, from bioinformatic data mining of huge data sets containing whole genome and whole exome information to laboratory investigations utilising targeted sequencing approaches. Blood group predictions based on genome sequencing and genomic studies are proving accurate, and have shown utility in both research and reference settings. Overall, studies confirm the potential for blood group genomics to reshape donor and patient transfusion management strategies to provide more compatible blood transfusions.

Comprehensive blood group antigen profile predictions for Western Desert Indigenous Australians from whole exome sequence data.

BACKGROUND: The distribution of RBC antigens, which define blood group types, differs among populations. In contrast to many world populations, blood group profiles for Indigenous Australians have not been well studied. As it is now possible to predict comprehensive blood group antigen profiles from genomic data sets, we aimed to apply this for Indigenous Australians and to provide a comparison to other major world populations. STUDY DESIGN AND METHODS: Whole exome sequence data for 72 Western Desert Indigenous Australians was provided by the Telethon Kids Institute. Variants (against hg19) were annotated using computer software (ANNOVAR, Qiagen Bioinformatics) and filtered to include only variants in genes for 36 blood group systems, and the transcription factors KLF1 and GATA1. The RHCE*C allele and RHD zygosity were identified by copy number variant analysis of sequence alignments. The impact of missense variants was investigated in silico using a meta-predictor of disease-causing variants (Meta-SNP). RESULTS: For 21 blood group systems the predicted blood group antigen frequencies were comparable to those for other major world populations. For 13 systems, interesting points of contrast were identified. Furthermore, we identified 12 novel variants, one novel D allele, and four rare variants with potential clinical significance. CONCLUSION: This is the first systematic assessment of genomic data to elucidate blood group antigen profiles for Indigenous Australians who are linguistically and culturally diverse. Our study paves the way to understanding the geographic distribution of blood group variants in different Indigenous groups and the associated RBC phenotypes. This in turn is expected to guide transfusion practice for Indigenous individuals.

Severe hemolytic disease of the fetus and newborn due to allo-anti-D in a patient with a partial DEL phenotype arising from the variant allele described as RHD*148+1T (RHD*01EL.31).

BACKGROUND: RhD DEL variants may show complete or partial expression of RhD epitopes. There have been only rare reports of anti-D causing hemolytic disease of the fetus and newborn (HDFN) in this context. We report a case of severe HDFN associated with a recently described DEL variant. CASE REPORT: A multiparous woman presented with an allo-anti-D and showed incongruent phenotyping and genotyping results on initial study. Further investigations identified the RHD mutation, defined as RHD*148+1T and named RHD*01EL.31, which had been previously associated with a DEL phenotype. Extended RhD phenotyping by adsorption-elution showed that there was reactivity with four of nine monoclonal anti-D antibodies, suggesting a partial DEL phenotype. The first child showed no clinical evidence of HDFN, although the cord direct antiglobulin test was positive. The second child developed fetal anemia treated with intrauterine transfusion, and neonatal hyperbilirubinemia requiring exchange transfusion. CONCLUSION: The RHD allele, RHD*148+1T, results in a partial Del phenotype, and the anti-D formed in pregnant women with this phenotype is capable of causing severe HDFN.

Investigation of the variable In(Lu) phenotype caused by KLF1 variants.

INTRODUCTION: KLF1 is an essential transcriptional activator that drives erythropoiesis. KLF1 variants can result in the Inhibitor of Lutheran, or In(Lu), phenotype where red blood cells (RBCs) have reduced BCAM (LU) and CD44 (IN). Other RBC surface molecules also have changed expression; however, there is controversy in the literature regarding which are truly impacted. We aimed to investigate KLF1 variants in the Australian population. STUDY DESIGN AND METHODS: In(Lu) samples were sourced through screening and through the RBC reference laboratory. Blood donor samples (8036) were screened to identify weakened/absent Lub antigen. Samples were genotyped by massively parallel sequencing, while surface carbohydrates and blood group molecules were assessed by flow cytometry. Hemoglobin (Hb) types were analyzed by high-performance liquid chromatography. RESULTS: Four of 8036 donors were identified to be In(Lu), and two previously identified In(Lu) samples were provided from the RBC reference laboratory. Five different KLF1 variants were identified; two were novel: c.954G>C/p.Trp318Cys and c.421C>T/p.Arg141*. BCAM and CD44 were reduced in all samples, consistent with previous reports. As a group, In(Lu) RBCs had reduced CD35 (KN), ICAM4 (LW), and CD147 (OK), and demonstrated increased binding of lectins ECA and SNAI. One In(Lu) sample had elevated HbF and another elevated HbA2. CONCLUSION: Different KLF1 variants may potentially produce variable phenotypes. A framework for investigating KLF1 variants and their phenotypic impact has been provided. In the future, given available international databases, further testing algorithms (as advocated here) will allow for correlation of phenotype with genotype and therefore accurately document this variability between KLF1 variants.

Genotyping analysis of MNS blood group GP(B-A-B) hybrid glycophorins in the Chinese Southern Han population using a high-resolution melting assay.

BACKGROUND: MNS hybrid GP(B-A-B) glycophorins are more commonly found in Southeast Asians and alloantibodies to antigens they carry are clinically significant. Detection of hybrid glycophorins by serologic techniques is limited due to lack of commercial reagents. In this study, a genotyping method for GP(B-A-B) hybrid glycophorins based on high-resolution melting (HRM) analysis was applied for genotyping analysis in the Chinese Southern Han population. STUDY DESIGN AND METHODS: DNA samples from 3104 Chinese Southern Han blood donors were collected. GYP(B-A-B) genotypes were analyzed by HRM assay. Parts of samples (n = 106) were also tested by multiplex ligation-dependent probe amplification (MLPA) assay. Direct sequencing was conducted in samples with variant melting curve profiles. RESULTS: A total of five GYP(B-A-B) genotypes (201/3104, 6.5%) were identified, which were GYP*Mur heterozygote (n = 194), GYP*Mur homozygote (n = 3), GYP*Bun heterozygote (n = 2), GYP*HF heterozygote (n = 1), and a novel GYP(B-A-B) hybrid allele (n = 1). Genotyping results for GYP*Mur and wild-type GYPB samples obtained by HRM were consistent with MLPA, while GYP*Bun and GYP*HF heterozygote identified by HRM could only be identified to have one copy of 5' inactive splice site of GYPB Pseudoexon 3 by MLPA. In addition, 10 single-nucleotide polymorphisms (SNPs) including four known and six novel SNPs were identified in 31 samples. One sample was identified carrying both GYP*Mur and GYP*Sch alleles. CONCLUSION: The HRM assay could distinguish the GYP(B-A-B) hybrid alleles successfully. Polymorphisms identified within the GYPB gene should be taken into consideration when developing GYP(B-A-B) genotyping kits for the Chinese population.

A DEL phenotype attributed to RHD Exon 9 sequence deletion: slipped-strand mispairing and blood group polymorphisms.

BACKGROUND: The RhD blood group antigen is extremely polymorphic and the DEL phenotype represents one such class of polymorphisms. The DEL phenotype prevalent in East Asian populations arises from a synonymous substitution defined as RHD*1227A. However, initially, based on genomic and cDNA studies, the genetic basis for a DEL phenotype in Taiwan was attributed to a deletion of RHD Exon 9 that was never verified at the genomic level by any other independent group. Here we investigate the genetic basis for a Caucasian donor with a DEL partial D phenotype and compare the genomic findings to those initial molecular studies. STUDY DESIGN AND METHODS: The 3'-region of the RHD gene was amplified by long-range polymerase chain reaction (PCR) for massively parallel sequencing. Primers were designed to encompass a deletion, flanking Exon 9, by standard PCR for Sanger sequencing. Targeted sequencing of exons and flanking introns was also performed. RESULTS: Genomic DNA exhibited a 1012-bp deletion spanning from Intron 8, across Exon 9 into Intron 9. The deletion breakpoints occurred between two 25-bp repeat motifs flanking Exon 9 such that one repeat sequence remained. CONCLUSION: Deletion mutations bordered by repeat sequences are a hallmark of slipped-strand mispairing (SSM) event. We propose this genetic mechanism generated the germline deletion in the Caucasian donor. Extensive studies show that the RHD*1227A is the most prevalent DEL allele in East Asian populations and may have confounded the initial molecular studies. Review of the literature revealed that the SSM model explains some of the extreme polymorphisms observed in the clinically significant RhD blood group antigen.

Targeted exome sequencing defines novel and rare variants in complex blood group serology cases for a red blood cell reference laboratory setting.

BACKGROUND: We previously demonstrated that targeted exome sequencing accurately defined blood group genotypes for reference panel samples characterized by serology and single-nucleotide polymorphism (SNP) genotyping. Here we investigate the application of this approach to resolve problematic serology and SNP-typing cases. STUDY DESIGN AND METHODS: The TruSight One sequencing panel and MiSeq platform was used for sequencing. CLC Genomics Workbench software was used for data analysis of the blood group genes implicated in the serology and SNP-typing problem. Sequence variants were compared to public databases listing blood group alleles. The effect of predicted amino acid changes on protein function for novel alleles was assessed using SIFT and PolyPhen-2. RESULTS: Among 29 unresolved samples, sequencing defined SNPs in blood group genes consistent with serologic observation: 22 samples exhibited SNPs associated with varied but known blood group alleles and one sample exhibited a chimeric RH genotype. Three samples showed novel variants in the CROM, LAN, and RH systems, respectively, predicting respective amino acid changes with possible deleterious impact. Two samples harbored rare variants in the RH and FY systems, respectively, not previously associated with a blood group allele or phenotype. A final sample comprised a rare variant within the KLF1 transcription factor gene that may modulate DNA-binding activity. CONCLUSION: Targeted exome sequencing resolved complex serology problems and defined both novel blood group alleles (CD55:c.203G>A, ABCB6:c.1118_1124delCGGATCG, ABCB6:c.1656-1G>A, and RHD:c.452G>A) and rare variants on blood group alleles associated with altered phenotypes. This study illustrates the utility of exome sequencing, in conjunction with serology, as an alternative approach to resolve complex cases.

Anti-D in a mother, hemizygous for the variant RHD*DNB gene, associated with hemolytic disease of the fetus and newborn.

BACKGROUND: Individuals with the partial D phenotype when exposed to D+ red blood cells (RBCs) carrying the epitopes they lack may develop anti-D specific for the missing epitopes. DNB is the most common partial D in Caucasians and the clinical significance for anti-D in these individuals is unknown. STUDY DESIGN AND METHODS: This article describes the serologic genotyping results and clinical manifestations in two group D+ babies of a mother presenting as group O, D+ with alloanti-D. RESULTS: The mother was hemizygous for RHD*DNB gene and sequencing confirmed a single-nucleotide change at c.1063G>A. One baby (group A, D+) displayed bilirubinemia at birth with a normal hemoglobin level. Anti-A and anti-D were eluted from the RBCs. For the next ongoing pregnancy, the anti-D titer increased from 32 to 256. On delivery the baby typed group O and anti-D was eluted from the RBCs. This baby at birth exhibited anemia, reticulocytosis, and hyperbilirubinemia requiring intensive phototherapy treatment from Day 0 to Day 9 after birth and was discharged on Day 13. Intravenous immunoglobulin was also administered. Both babies were heterozygous for RHD and RHD*DNB. CONCLUSION: The anti-D produced by this woman with partial D DNB resulted in a case of hemolytic disease of the fetus and newborn (HDFN) requiring intensive treatment in the perinatal period. Anti-D formed by women with the partial D DNB phenotype has the potential to cause HDFN where the fetus is D+. Women carrying RHD*DNB should be offered appropriate prophylactic anti-D and be transfused with D- RBCs if not already alloimmunized.

Evaluation of targeted exome sequencing for 28 protein-based blood group systems, including the homologous gene systems, for blood group genotyping.

BACKGROUND: Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS. STUDY DESIGN AND METHODS: Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems. RESULTS: The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement. CONCLUSION: The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting.

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

OBJECTIVE: To undertake a cost-effectiveness analysis of noninvasive fetal RHD genotyping to target pregnant women for antenatal anti-D prophylaxis therapy. METHOD: 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. RESULTS: 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 625 IU (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, 13 938 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$159 701 (US$111 791) per year (0.05%) were predicted for total health care costs. CONCLUSIONS: Given the vulnerable supply of donor plasma and other health concerns, RHD genotyping is an economically sound option for Australia.