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.

Influence of donor age, sex and ethnicity on high-titre anti-A and -B: Review of 6 million donations from two national blood providers.

BACKGROUND AND OBJECTIVES: Some blood operators routinely screen blood donations for high-titre (HT) anti-A/B to reduce the risk of a haemolytic transfusion reaction due to out-of-group plasma-rich components. We assessed donor factors associated with an increased likelihood of screening positive and compared routine data between England and Australia. MATERIALS AND METHODS: Data were assessed from HT screening during 2018-2020 in Australia and 2018-2021 in England, totalling nearly 6 million blood donations. Screening was performed using a Beckman Coulter PK7300 analyser with a micro-titre plate saline direct agglutination test in both countries, although different reagent red cells were chosen. HT-positive was defined as testing positive at a titre of 128 or above. RESULTS: The likelihood of a donor testing HT-positive was greater for females than males, declined with age and was dependent on the ABO group. However, the proportion of donors testing HT-positive was consistently higher in Australia than in England: overall, 14% of group O donations and 5% of group A donations in England tested HT-positive, compared with 51% and 22%, respectively in Australia. English data also showed that donors from Black, Asian or mixed ethnic backgrounds were more likely to test HT-positive than White donors. CONCLUSION: These data demonstrate that donor sex, age, ABO group and ethnicity affect the likelihood of testing HT-positive. Differences in testing methods likely had a significant impact on the proportion of donors testing as HT-positive or -negative rather than any differences in donor populations.

Frequency of red blood cell phenotypes from genotyped Australian blood donors.

BACKGROUND: Australian Red Cross Lifeblood (Lifeblood) performs human erythrocyte antigen (HEA) genotyping for a subset of repeat whole-blood donors through preferential selection which aims to maximise variation of results and possibility of identifying donors lacking high frequency red cell antigens. MATERIALS AND METHODS: The HEA Molecular Bead chip™ assay is used by Lifeblood for donor genotyping. A review of all donor HEA genotype data from March 2019 to May 2022 (3 years) was conducted. RESULTS: HEA genotyping was performed for 20,185donors. Due to selective genotyping of donors, a higher frequency of R1R1 predicted phenotype was identified. However, frequencies of other red cell phenotypes were relatively similar to previous reported in the Australian population. A small number of donors with rare red cell phenotypes was identified. CONCLUSION: Genotyping of blood donors provides an available pool of extended matched red blood cell products for matching to recipients. Additionally genotyping can improve the identification of donors with rare phenotypes. Whilst limitations exist, genotyping may reduce the need for labour intensive serotyping, improve blood inventory management, and may be useful in donor recruitment and retention.

A case of haemolytic disease of the fetus and newborn attributed to a novel antigen in the RHAG blood group system.

BACKGROUND AND OBJECTIVES: A newborn presented with jaundice in Thailand. The cord red cells tested positive by direct antiglobulin test (DAT) for an unknown maternal red cell antibody. Initial blood group sequencing suggested that the infant carried a novel variant RHAG c.140T>C, responsible for a low-prevalence antigen in the RHAG blood group system (ISBT 030). We report here on testing of samples from the infant's parents and older sibling to define a new antigen in the RHAG system. MATERIALS AND METHODS: Massive parallel sequencing (MPS) using a custom-designed panel was performed on all four family members. Extended serological testing was also performed to determine whether family members with the same variant as the infant showed reactivity with the antibody in the maternal plasma. RESULTS: We identified a novel single nucleotide variant (SNV) (RHAG c.140T>C, p.[Phe47Ser]) in samples from three of the four family members tested (the infant, the older sibling and the father). The variant was not detected in the mother's sample. Maternal plasma showed positive agglutination with all family members tested; however, when tested with routine panel cells, no reactivity was observed. CONCLUSION: This case study showed that the presence of the novel variant (RHAG c.140T>C), encoding a p.(Phe47Ser) change in the RhAG glycoprotein, was the apparent cause of incompatibility between maternal plasma and that of red cells from the proband, father and older sibling of the proband. We propose this variant to be a new low-prevalence antigen in the RHAG blood group system.

Haemoglobin S testing using HEA BeadChip™ technology: Lifeblood comparison with clinical diagnosis.

BACKGROUND AND OBJECTIVES: Red cell antigen genotyping is commonly performed on patients requiring chronic transfusion support, such as sickle cell disease and thalassaemia. The Immucor HEA BeadChip™ test, in addition to assessing red cell antigen expression, can also detect the haemoglobin S (HbS) mutation. Our aim was to compare HbS results using HEA BeadChip™ performed at the Australian Red Cross Lifeblood with conventional haemoglobin studies. MATERIALS AND METHODS: Patients with thalassaemia and sickle cell trait (SCT) or disease (SCD) referred for red cell genotyping between 2017 and 2019 were assessed. The HbS result obtained from HEA BeadChip™ was compared with that obtained from high-performance liquid chromatography (HPLC) performed by the referring pathology provider. RESULTS: One-hundred and nineteen cases had comparable HPLC and HEA BeadChip™ results. On HEA BeadChip™ testing, 40 cases showed a negative HbS result, 31 cases showed HbS+ and 47 cases showed HbS++. There was one case with 'low signal' result. Of the negative HbS cases, there was none with SCT. The HbS+ group comprised a mixture of SCT and SCD due to compound heterozygosity for HbS and ß-thalassaemia mutations. The HbS++ group comprised predominantly SCD due to homozygosity for HbS. CONCLUSION: HEA BeadChip™ is an accurate screening test for the detection of HbS. There were no false positives or false negatives. The identification of donors with the HbS mutation through the targeted genotyping programme would enable early intervention, improved donor management and reduced wastage.

Routine donor red cell antibody screening: Considering the alternate strategy.

BACKGROUND AND OBJECTIVES: Australian Red Cross Lifeblood (Lifeblood) performs red blood cell (RBC) antibody screening on every whole blood donation. An alternate strategy has been proposed whereby an antibody screen is performed on the first donation and only repeated following pregnancy, transfusion or a significant break between donations (>2 years). We assess the blood safety risks associated with removing antibody screening for every whole blood donation. MATERIALS AND METHODS: A retrospective desktop analysis included all whole blood donations collected by Lifeblood between 01 May 2018 and 30 April 2019 to quantify the antibodies that would have been undetected with the alternate strategy. The strategy was further assessed using the Alliance of Blood Operators Risk-Based Decision-Making framework. RESULTS: One hundred and seventy-one routine donors had antibodies for the first time, but reported no sensitizing event since their last donation. Forty-seven of these had antibodies of a clinically significant specificity and titre that have the potential to cause a haemolytic transfusion reaction (HTR). The calculated risk of undetected antibodies being transfused to an incompatible recipient is 1 in 82,200. CONCLUSION: The estimated risk of HTRs with the alternate strategy results in an increased risk. While the alternate strategy is identified as the most cost-effective option within the Australian setting, this additional residual risk was not deemed to be acceptable. Blood services would need to determine whether the increase in residual risk stemming from implementation of such a strategy is tolerable.

The role of non-invasive prenatal testing (NIPT) for fetal blood group typing in Australia.

Maternal alloimmunisation against red blood cell antigens can cause haemolytic disease of the fetus and newborn (HDFN). Although most frequently caused by anti-D, since the implementation of rhesus D (RhD) immunoglobulin prophylaxis, other alloantibodies have become more prevalent in HDFN. Recent advances in non-invasive prenatal testing (NIPT) have allowed early prediction of HDFN risk in alloimmunised pregnancies and allow clinicians to focus health resources on those pregnancies that require intervention. This article aims to provide updates on the current status of NIPT in Australia as both a diagnostic and screening tool in pregnancy.

Understanding the demand for phenotyped red blood cell units and requests to perform molecular red blood cell typing for Australian patients.

BACKGROUND: Australian Red Cross Lifeblood has seen a 50 % increase in demand for phenotyped red blood cell (RBC) units between 2016-2018 and a 30 % increase in demand in 2018 to perform molecular RBC typing on patient samples. Lifeblood conducted a survey to understand transfusion laboratory practices for requesting patient phenotyping and/or molecular RBC typing and for selecting phenotyped RBC units in various patient groups. STUDY DESIGN AND METHODS: An electronic Qualtrics survey form was sent to 296 transfusion laboratories with questions designed to understand the practice of selecting phenotyped RBC units and reasons for requesting extended serology or molecular RBC typing. RESULTS: 49 (16.6 %) transfusion laboratories provided data. Reasons to request extended phenotyping and/or molecular RBC typing for patients included; chronic transfusion (n = 31 laboratories), sickle cell disease (n = 25), Thalassemia (n = 23), requirement for anti-CD38 or other MAB therapy (n = 23) or Myelodysplasia (n = 22). Forty-seven transfusion laboratories provided responses with reasons for requesting molecular RBC typing which included: predicting phenotype in patients with multiple antibodies (n = 31), prior to administering anti-CD38 or other MAB therapies (n = 29), for pregnancy related transfusions (n = 28) or for confirming the phenotype of recently transfused patients (n = 18). CONCLUSION: Transfusion laboratory practices indicated that phenotyped RBC units were selected for patients requiring chronic transfusion support and/or undergoing MAB therapy. Requests for molecular RBC typing occurred for more complex patient requirements where serological investigations were not suitable or possible due to reagent restrictions.

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.

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.

The prevalence of selected clinically significant red blood cell antigens among Australian blood donors.

Red blood cell (RBC) transfusion can cause some patients to form antibodies to RBC antigens when RBC phenotypes do not match that of the blood donor. Transfusion practitioners can order phenotyped RBC units for patients with known RBC antibodies or those who are at risk of forming them. However, with increasing demand for phenotyped RBC units, contemporary data on antigen prevalence is required to manage the changing supply. A total of 490,491 blood donors, including 103,798 (21.2%) first-time blood donors, from 2019 were analysed for the prevalence of selected clinically relevant blood group antigens. Prevalence of the phenotype R1R1 (D+ C+ E- c- e+) increased from the previous estimate of 17.3% to 24.0% in first-time blood donors. The prevalence of R1r (D+ C+ E- c+ e+) decreased from 35.3% to 30.8%. R1R1 was more common in blood donors born in Asia or the Middle East. The prevalence of Fy(a-b-) in donors where Fy antigens were tested was 0.2%. Of these, 71.8% stated their region of birth as Africa. The prevalence of Jk(a-b-) is 0.01% in donors where the Jk antigens were tested with region of birth stated as either Oceania or Asia. The increasing prevalence of the c-negative phenotype in R1R1 individuals is associated with the changing demographics of the Australian community. For R1R1 individuals with childbearing potential, the transfusion of RhD negative blood, which is usually c-positive, may increase the possibility of haemolytic disease of the fetus and newborn during pregnancy. Continued diversification of the Australian blood donor panel will support having the appropriate phenotyped RBC units available.

Probabilistic mathematical modelling to predict the red cell phenotyped donor panel size.

In the last decade, Australia has experienced an overall decline in red cell demand, but there has been an increased need for phenotyped matched red cells. Lifeblood and mathematicians from Queensland universities have developed a probabilistic model to determine the percentage of the donor panel that would need extended antigen typing to meet this increasing demand, and an estimated timeline to achieve the optimum required phenotyped (genotyped) panel. Mathematical modelling, based on Multinomial distributions, was used to provide guidance on the percentage of typed donor panel needed, based on recent historical blood request data and the current donor panel size. Only antigen combinations determined to be uncommon, but not rare, were considered. Simulations were run to attain at least 95% success percentage. Modelling predicted a target of 38% of the donor panel, or 205,000 donors, would need to be genotyped to meet the current demand. If 5% of weekly returning donors were genotyped, this target would be reached within 12 years. For phenotyping, 35% or 188,000 donors would need to be phenotyped to meet Lifeblood's demand. With the current level of testing, this would take eight years but could be performed within three years if testing was increased to 9% of weekly returning donors. An additional 26,140 returning donors need to be phenotyped annually to maintain this panel. This mathematical model will inform business decisions and assist Lifeblood in determining the level of investment required to meet the desired timeline to achieve the optimum donor panel size.