Patients with Asian-type DEL can safely be transfused with RhD-positive blood.

Red blood cells (RBCs) of Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D-) phenotype in routine testing. RhD-positive (D+) RBC transfusion for patients with Asian-type DEL has been proposed but has not been generally adopted because of a lack of direct evidence regarding its safety and the underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in patients with Asian-type DEL; none of the recipients (0/42; 95% confidence interval, 0-8.40) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4045 serologic D- pregnant women throughout China; alloanti-D was found only in individuals with true D- (2.63%, 79/3009), but not in those with Asian-type DEL (0/1032). We further retrospectively examined 127 serologic D- pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs after transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in patients with Asian-type DEL, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as #NCT03727230.

40 years of researching the Del phenotype results in a change of transfusion practice.

Anti-D cannot agglutinate red cells of any Del phenotype in routine serology. Many individuals with East Asian ancestry who type D-negative in serology harbor a Del phenotype. Almost all such individuals carry one distinct DEL variant, dubbed Asian-type DEL, known as RHD*01EL.01, RHD*DEL1, RHD:c.1227G>A, formerly known as RHD(K409K). Clinical evidence strongly suggests that Asian-type DEL individuals can safely be transfused with RhD-positive blood and do not need anti-D prophylaxis in pregnancy.

Neutrophil antigen antibodies affect engraftment and secondary graft failure in hematopoietic progenitor cell transplantation.

BACKGROUND: Research is limited on the role of antibodies against human neutrophil antigen (HNA) in hematopoietic progenitor cell (HPC) transplantation outcomes. STUDY DESIGN AND METHODS: A retrospective review was conducted on medical records of patients at the NIH Clinical Center enrolled in six research protocols. This case-control study included 21 patients tested for HNA antibodies from January 2010 to March 2022 who underwent HPC transplantation. In addition, 42 patients following the same research protocols were randomly selected as a control group. RESULTS: The cumulative incidence of time to neutrophil engraftment was significantly impacted by the patients' anti-HNA status (p = .042), with the patients with anti-HNA experiencing delayed engraftment. Secondary graft failure occurred in 4 out of 42 patients (9.52%; 95% confidence interval [CI]: 3.7-22.1) of the control group, while 5 out of 9 patients (55.5%; 95% CI: 26.7-81.1) with anti-HNA experienced secondary graft failure (p = .005). Furthermore, patients with anti-HNA had a lower proportion (p = .008 for full and p = .002 for partial chimerism) and cumulative incidence (p = .016 for full and p = .010 for partial chimerism) of achieving donor chimerism compared to the control group. DISCUSSION: The study reveals a potential link between anti-HNA and HPC transplantation outcomes not previously reported. Patients with anti-HNA had a lower proportion and cumulative incidence of achieving donor chimerism. Additionally, anti-HNA status affected the time for neutrophil engraftment, with a slower rate of neutrophil engraftment and increased risk of secondary failure in patients with anti-HNA.

CD59 gene: 143 haplotypes of 22,718 nucleotides length by computational phasing in 113 individuals from different ethnicities.

BACKGROUND: CD59 deficiency due to rare germline variants in the CD59 gene causes disabilities, ischemic strokes, neuropathy, and hemolysis. CD59 deficiency due to common somatic variants in the PIG-A gene in hematopoietic stem cells causes paroxysmal nocturnal hemoglobinuria. The ISBT database lists one nonsense and three missense germline variants that are associated with the CD59-null phenotype. To analyze the genetic diversity of the CD59 gene, we determined long-range CD59 haplotypes among individuals from different ethnicities. METHODS: We determined a 22.7 kb genomic fragment of the CD59 gene in 113 individuals using next-generation sequencing (NGS), which covered the whole NM_203330.2 mRNA transcript of 7796 base pairs. Samples came from an FDA reference repository and our Ethiopia study cohorts. The raw genotype data were computationally phased into individual haplotype sequences. RESULTS: Nucleotide sequencing of the CD59 gene of 226 chromosomes identified 216 positions with single nucleotide variants. Only three haplotypes were observed in homozygous form, which allowed us to assign them unambiguously as experimentally verified CD59 haplotypes. They were also the most frequent haplotypes among both cohorts. An additional 140 haplotypes were imputed computationally. DISCUSSION: We provided a large set of haplotypes and proposed three verified long-range CD59 reference sequences, based on a population approach, using a generalizable rationale for our choice. Correct long-range haplotypes are useful as template sequences for allele calling in high-throughput NGS and precision medicine approaches, thus enhancing the reliability of clinical diagnostics. Long-range haplotypes can also be used to evaluate the influence of genetic variation on the risk of transfusion reactions or diseases.

Rh flow cytometry: An updated methodology for D antigen density applied to weak D types 164 and 165.

BACKGROUND: An original methodology for determining the D antigen density on red cells was published in 2000 and has been applied in many publications since. This flow cytometry-based assay remained largely unrevised utilizing monoclonal anti-Ds that are not readily available anymore. We updated the methodology to quantify erythrocyte D antigen sites using microspheres and monoclonal anti-Ds that are commercially available today. METHODS: The absolute D antigen density of a frozen standard CcDEe cell, drawn in 2003, a fresh blood donation from the same individual, drawn in 2022, and an internal control CcDEe cell, was quantified by flow cytometry using fluorescence-labeled microspheres. The internal control CcDEe cell was used in conjunction with 9 commercial anti-Ds to determine D antigen densities of 7 normal D, 4 partial D, and 11 weak D type samples, including 2 novel alleles. RESULTS: The reproducibility of the updated assay was evaluated with red cells of published D antigen densities. The current results matched the known ones closely. The new weak D types 164 and 165 carried 4500 and 1505 D antigens/red cell, respectively. The absolute D antigen density decreased from 27,231 to 26,037 in an individual over 19 years. DISCUSSION: The updated assay gave highly reproducible results for the D antigen densities of Rh phenotypes. Readily available anti-Ds allowed for the determination of the D antigen densities of 7 weak D types. The assay is suitable to evaluate the effects of distinct amino acid substitutions on the RhD phenotype.

Asian-type DEL (RHD*DEL1) with an allo-anti-D: A paradoxical observation in a healthy multiparous woman.

BACKGROUND: The DEL phenotype is the D variant expressing the least amounts of D antigen per red cell. Asian-type DEL (RHD:c:1227G > A) is the most prevalent DEL in East Asia without any anti-D alloimmunization reported before. We investigated the first observation of an anti-D in any DEL phenotype, reported in the Japanese language at a 1987 conference, only 3 years after the discovery of DEL. METHODS: We contacted the proband 35 years after the initial report. Standard hemagglutination, adsorption/elution, and flow cytometry tests were performed, as was nucleotide sequencing for the RHD, RHCE, and HLA class I and class II genes. RESULTS: The healthy multiparous Japanese woman, a regular blood donor, still had the anti-D of titer 8 representing an alloantibody by standard serologic methods. Unexpectedly, she carried an Asian-type DEL without any additional RHD gene variation. All 12 HLA alleles identified were known in the Japanese population. Interestingly, one of her HLA-DRB1 and a variant of her HLA-DQB1 alleles had previously been associated with anti-D immunization. CONCLUSION: We described an allo-anti-D, maintained for more than three decades, in an Asian-type DEL. The combination of two implicated HLA alleles were rare and could have contributed to the anti-D immunization. Continued monitoring of anti-D immunization events in patients with DEL is warranted, and we discuss possible mechanisms for further study. As only this single observation has been recognized in the last 35 years, the current recommendation is affirmed: Individuals with Asian-type DEL should be treated as Rh D-positive for transfusion and Rh immune prophylaxis purposes.

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

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

Cataloguing experimentally confirmed 80.7 kb-long ACKR1 haplotypes from the 1000 Genomes Project database.

BACKGROUND: Clinically effective and safe genotyping relies on correct reference sequences, often represented by haplotypes. The 1000 Genomes Project recorded individual genotypes across 26 different populations and, using computerized genotype phasing, reported haplotype data. In contrast, we identified long reference sequences by analyzing the homozygous genomic regions in this online database, a concept that has rarely been reported since next generation sequencing data became available. STUDY DESIGN AND METHODS: Phased genotype data for a 80.6 kb region of chromosome 1 was downloaded for all 2,504 unrelated individuals of the 1000 Genome Project Phase 3 cohort. The data was centered on the ACKR1 gene and bordered by the CADM3 and FCER1A genes. Individuals with heterozygosity at a single site or with complete homozygosity allowed unambiguous assignment of an ACKR1 haplotype. A computer algorithm was developed for extracting these haplotypes from the 1000 Genome Project in an automated fashion. A manual analysis validated the data extracted by the algorithm. RESULTS: We confirmed 902 ACKR1 haplotypes of varying lengths, the longest at 80,584 nucleotides and shortest at 1,901 nucleotides. The combined length of haplotype sequences comprised 19,895,388 nucleotides with a median of 16,014 nucleotides. Based on our approach, all haplotypes can be considered experimentally confirmed and not affected by the known errors of computerized genotype phasing. CONCLUSIONS: Tracts of homozygosity can provide definitive reference sequences for any gene. They are particularly useful when observed in unrelated individuals of large scale sequence databases. As a proof of principle, we explored the 1000 Genomes Project database for ACKR1 gene data and mined long haplotypes. These haplotypes are useful for high throughput analysis with next generation sequencing. Our approach is scalable, using automated bioinformatics tools, and can be applied to any gene.

DEL in China: the D antigen among serologic RhD-negative individuals.

BACKGROUND: Providing RhD-negative red cell transfusions is a challenge in East Asia, represented by China, Korea, and Japan, where the frequency of RhD-negative is the lowest in the world. FINDINGS: Among 56 ethnic groups in China, the RhD-negative frequency in Han, the prevalent ethnicity, is 0.5% or less, similar to most other ethnic groups. The Uyghur ethnic group has the highest reported RhD-negative frequency of up to 4.7%, as compared to 13.9% in the US. However, an estimated 7.15 million RhD-negative people live in China. The RhD-negative phenotype typically results from a loss of the entire RHD gene, causing the lack of the RhD protein and D antigen. The DEL phenotype carries a low amount of the D antigen and types as RhD-negative in routine serology. The DEL prevalence in RhD-negative individuals averages 23.3% in the Han, 17% in the Hui and 2.4% in the Uyghur ethnicities. The Asian type DEL, also known as RHD*DEL1 and RHD:c.1227G > A allele, is by far the most prevalent among the 13 DEL alleles observed in China. CONCLUSION: The purpose of this review is to summarize the data on DEL and to provide a basis for practical strategy decisions in managing patients and donors with DEL alleles in East Asia using molecular assays.

Pharmacogenomics with red cells: a model to study protein variants of drug transporter genes.

The PharmacoScan pharmacogenomics platform screens for variation in genes that affect drug absorption, distribution, metabolism, elimination, immune adverse reactions and targets. Among the 1,191 genes tested on the platform, 12 genes are expressed in the red cell membrane: ABCC1, ABCC4, ABCC5, ABCG2, CFTR, SLC16A1, SLC19A1, SLC29A1, ATP7A, CYP4F3, EPHX1 and FLOT1. These genes represent 5 ATP-binding cassette proteins, 3 solute carrier proteins, 1 ATP transport protein and 3 genes associated with drug metabolism and adverse drug reactions. Only ABCG2 and SLC29A1 encode blood group systems, JR and AUG, respectively. We propose red cells as an ex vivo model system to study the effect of heritable variants in genes encoding the transport proteins on the pharmacokinetics of drugs. Altered pharmacodynamics in red cells could also cause adverse reactions, such as haemolysis, hitherto unexplained by other mechanisms.

HNA-3a and HNA-3b antigens among 9 ethnic populations and the Han population in Southwest China.

BACKGROUND: Human neutrophil antigen 3 (HNA-3) is encoded by the SLC44A2 gene. Antibodies against HNAs can cause severe, often fatal, transfusion reactions, known as transfusion-related acute lung injury, and neonatal neutropenia. We explored the 2 common HNA-3 variants in 9 ethnic populations residing in Sichuan and Yunnan provinces of China as compared to the Han population. METHODS: We genotyped for SLC44A2 (rs2288904) by polymerase chain reaction sequence-based typing among blood donors, for a total of 2206 individuals in Yunnan and 376 in Sichuan. RESULTS: The SLC44A2*02 allele (HNA-3b antigen) frequency varied between 0.24 and 0.33 for all 9 ethnic populations in Yunnan, including Zhuang, Derung, Hani, Lisu, Bai, Miao, Dai, Naxi, and Yi. Specifically, the Yi ethnicity did not present an unusually great SLC44A2*02 frequency at any of the 4 locations examined in Yunnan. Except of the Yi ethnicity in Sichuan (0.40), the Han ethnicity, as the majority population group, had the greatest SLC44A2*02 frequency with 0.39 in Yunnan and 0.35 in Sichuan. CONCLUSION: The ethnic populations in Southwest China are not at an increased risk for anti-HNA3a compared to the Han population, with the possible exception of Yi in Sichuan. Our data, however, corroborated the known high prevalence of SLC44A2*02 in Han populations. Hence, the Han populations in Yunnan, Sichuan and elsewhere in China are at a comparatively great risk for developing HNA-3a antibodies.

Two large deletions extending beyond either end of the RHD gene and their red cell phenotypes.

Only two partial deletions longer than 655 nucleotides had been reported for the RHD gene, constrained within the gene and causing DEL phenotypes. Using a combination of quantitative PCR and long-range PCR, we examined three distinct deletions affecting parts of the RHD gene in three blood donors. Their RHD nucleotide sequences and exact boundaries of the breakpoint regions were determined. DEL phenotypes were caused by a novel 18.4 kb deletion and a previously published 5.4 kb deletion of the RHD gene; a D-negative phenotype was caused by a novel 7.6 kb deletion. Examination of the deletion-flanking regions suggested microhomology-mediated end-joining, replication slippage, and non-homologous end-joining, respectively, as the most likely mechanisms for the three distinct deletions. We described two new deletions affecting parts of the RHD gene, much longer than any previously reported partial deletion: one was the first deletion observed at the 5' end of the RHD gene extending into the intergenic region, and the other the second deletion observed at its 3' end. Large deletions present at either end are a mechanism for a much reduced RhD protein expression or its complete loss. Exact molecular characterization of such deletions is instrumental for accurate RHD genotyping.

Author Correction: Two large deletions extending beyond either end of the RHD gene and their red cell phenotypes.

The authors of the above paper noticed an error in publication. In Results section, under sub-section RHD genetic variations, the deletion nomenclature for Sample 1 was incorrectly given as [NC_000001.11(NG_007494.1):c.(1-15149_1-15153)_(148+3154_148+3158)del].

Transfusion strategy for weak D Type 4.0 based on RHD alleles and RH haplotypes in Tunisia.

BACKGROUND: With more than 460 RHD alleles, this gene is the most complex and polymorphic among all blood group systems. The Tunisian population has the largest known prevalence of weak D Type 4.0 alleles, occurring in one of 105 RH haplotypes. We aimed to establish a rationale for the transfusion strategy of weak D Type 4.0 in Tunisia. STUDY DESIGN AND METHODS: Donors were randomly screened for the serologic weak D phenotype. The RHD coding sequence and parts of the introns were sequenced. To establish the RH haplotype, the RHCE gene was tested for characteristic single-nucleotide positions. RESULTS: We determined all RHD alleles and the RH haplotypes coding for the serologic weak D phenotype among 13,431 Tunisian donations. A serologic weak D phenotype was found in 67 individuals (0.50%). Among them, 60 carried a weak D Type 4 allele: 53 weak D Type 4.0, six weak D Type 4.2.2 (DAR), and one weak D Type 4.1. An additional four donors had one variant allele each: DVII, weak D Type 1, weak D Type 3, and weak D type 100, while three donors showed a normal RHD sequence. The weak D Type 4.0 was most often linked to RHCE*ceVS.04.01, weak D Type 4.2.2 to RHCE*ceAR, and weak D Type 4.1 to RHCE*ceVS.02, while the other RHD alleles were linked to one of the common RHCE alleles. CONCLUSIONS: Among the weak D phenotypes in Tunisia, no novel RHD allele was found and almost 90% were caused by alleles of the weak D Type 4 cluster, of which 88% represented the weak D Type 4.0 allele. Based on established RH haplotypes for variant RHD and RHCE alleles and the lack of adverse clinical reports, we recommend D+ transfusions for patients with weak D Type 4.0 in Tunisia.

Genotype frequency of human neutrophil antigen-3 polymorphisms in the Yi, Han, and Tibetan populations of China.

BACKGROUND: Human neutrophil antigen-3 (HNA-3) alloantibodies can cause fatal transfusion-related acute lung injury (TRALI). Most frequencies of SLC44A2 alleles encoding the HNA-3a/b antigens have been established in Han individuals by polymerase chain reaction with sequence-specific priming (PCR-SSP). We sequenced SLC44A2 gene fragments and determined allele frequencies in three ethnicities of China. STUDY DESIGN AND METHODS: Genomic DNA was extracted from 448 samples of 100 blood donors of Yi ethnicity in Xichang, Liangshan; 248 Han in Nanjing, Jiangsu; and 100 Tibetan in Lhasa, Tibet. A PCR-SSP was applied to determine the phase of two single-nucleotide polymorphisms (SNPs); SLC44A2 haplotypes were constructed. RESULTS: In the 567 nucleotides of the SLC44A2 gene covered by our sequencing approach in Han individuals, we detected the known 331-44G>A (rs12972963) and 461G>A (rs2288904) polymorphisms. In the 243 nucleotides sequenced in Yi and Tibetan populations, we detected the known 461G>A and 503-15T>C (rs1560711) polymorphisms. A PCR-SSP for the common HNA-3a/b SNP was 100% concordant. The frequencies of the HNA-3a allele were 0.58, 0.66, and 0.69 in Yi, Han (Nanjing), and Tibetan, respectively (0.42, 0.34, and 0.31 for HNA-3b). CONCLUSIONS: The Yi population of China had the highest frequency of blood donors at risk of harboring anti-HNA-3a compared to any population studied so far. We confirmed that the underlying SLC44A2*2 allele is more common in China than in any European or African populations.

The DAU cluster: a comparative analysis of 18 RHD alleles, some forming partial D antigens.

BACKGROUND: The Rh system is the most complex and polymorphic blood group system in humans with more than 460 alleles known for the RHD gene. The DAU cluster of RHD alleles is characterized by the single-nucleotide change producing the p.Thr379Met amino acid substitution. It is called the DAU-0 allele and has been postulated to be the primordial allele, from which all other alleles of the DAU cluster have eventually evolved. STUDY DESIGN AND METHODS: For two novel DAU alleles, the nucleotide sequences of all 10 exons as well as adjacent intronic regions, including the 5' and 3' untranslated regions (UTR), were determined for the RHD and RHCE genes. A phylogenetic tree for all DAU alleles was established using the neighbor-joining method with Pan troglodytes as root. Standard hemagglutination and flow cytometry tests were performed. RESULTS: We characterized two DAU alleles, DAU-11 and DAU-5.1, closely related to DAU-3 and DAU-5, respectively. A phylogenetic analysis of the 18 known DAU alleles indicated point mutations and interallelic recombination contributing to diversification of the DAU cluster. CONCLUSIONS: The DAU alleles encode a group of RhD protein variants, some forming partial D antigens known to permit anti-D in carriers; all are expected to cause anti-D alloimmunization in recipients of red blood cell transfusions. The DAU alleles evolved through genomic point mutations and recombination. These results suggest that the cluster of DAU alleles represent a clade, which is concordant with our previous postulate that they derived from the primordial DAU-0 allele.