Adaptive sampling (AS), a computational enrichment method developed for Oxford Nanopore Technologies sequencing platforms, offers a promising advance in molecular blood group diagnostics. By leveraging long-read sequencing, AS has the potential to accurately resolve complex structural variants in the RH and MNS blood group systems, while characterizing the entire blood group genome through a simple, fast and locus-adjustable protocol. As proof-of-principal, we evaluated the performance of AS using five samples with suspected complex variants in the RH and MNS systems, unresolved by standard immunohematological methods. Samples were sequenced on a PromethION P2 Solo with up to two samples per flowcell, generating 37.0-52.4 Gb of data with mean on-target coverages of 18.9-53.4x, allowing reliable variant detection. Hybrid alleles were characterized using a de novo assembly approach, whereas variants in non-recombinant regions were analyzed using both a custom in-house and the EPI2ME reference-based workflow. With reference to field-specific allele collections, 10-15% of detected alleles contained novel nonsynonymous single nucleotide variants (SNVs) or unreported exonic SNV combinations. All suspected hybrid alleles were successfully assembled and identified as GYP*401.02, RHD*03N.01, and RHD*01EL.44, representing the first fully characterized haplotypes for these variants publicly available. Overall, AS showed significant potential for advancing blood group genomics by enabling high-resolution, full-gene analysis. Its ability to support high-throughput donor genotyping and precise patient-donor matching may reduce the risk of alloimmunization and delayed hemolytic transfusion reactions, particularly in chronically transfused patients. These findings highlight AS as a powerful tool for both research and clinical applications in transfusion medicine.

Transfusion management of Kx- individuals with McLeod phenotype (MLP) is highly challenging, particularly as cryopreservation affects red blood cell (RBC) concentrate quality. We developed a concept to provide non-cryopreserved Kx- RBCs over the complete period of hematopoietic stem cell transplantation (HSCT) for treatment of X-linked chronic granulomatous disease (X-CGD) with MLP. An infant with a large deletion affecting 12 protein-coding genes, including DMD, PRRG1, LANCL3, XK, CYBB, and DYNLT3, leading to CGD, Duchenne muscular dystrophy, and MLP, was scheduled for HSCT with the need of Kx- blood supply. No Kx- and RhD compatible donors were identified by rare donor programs, and autologous blood collection was not possible. In an interdisciplinary multicenter effort pre- and post-HSCT blood management, including procurement of non-cryopreserved allogeneic Kx- RBCs from an individual with MLP, was orchestrated, balancing donations, storage, pediatric RBC preparation, and irradiation with the clinical schedule. Our concept ensured compatible blood supply from 100 days prior HSCT to the peritransplant phase. The patient received 5 non-cryopreserved Kx- pediatric RBCs and was discharged with complete chimerism at day +68. The screen was repeatedly negative for antibodies to high frequency RBC antigens. After 2.8 years, the patient remained independent of transfusions and was without signs of graft-versus-host disease. Close coordination between institutions and disciplines and process optimization allow readily available provision of non-cryopreserved Kx- RBCs to support HSCT to a patient with unique contiguous gene deletion syndrome of X chromosome.

Zusammenfassung Aufgrund der starken Immunogenität des KEL1-Antigens ist dessen Erhebung oft Teil der routinemäßigen Spendertypisierung. Am Blutspendezentrum Zürich wird KEL1 serologisch als auch genetisch mittels Hochdurchsatzgenotypisierung bestimmt. Genotyp-Phänotypdiskrepanzen werden normalerweise durch eine aufwendige Sanger-Sequenzierung aller 19 Exons gelöst, welche jedoch keine Erstellung von Haplotypen zulässt. Hier präsentieren wir ein alternatives Vorgehen, das auf der neuesten Sequenzierungstechnologie von Oxford Nanopore Technologies basiert und die Generierung von Haplotypen ganzer Gene ermöglicht. Zur Ermittlung der KEL1-Expression kamen serologische Standardmethoden zur Anwendung. Vier Varianten innerhalb des KEL-Gens waren Teil der auf MALDI-TOF Massenspektrometrie basierenden Hochdurchsatz genotypisierung, darunter c.578C>T, welches die KEL1/2-Expression bestimmt. Die Bestätigung diskrepanter Ergebnisse erfolgte mittels PCR-SSP und serologischen Untersuchungen zur Antigenexpressionsstärke wie Adsorptions-Elutionsanalysen und Durchflusszytometrie. Zur Auflösung einer Diskrepanz bei einem Spender amplifizierten wir das ~21 kb lange KEL mit zwei sich um 4.4 kb überlappenden «long-range» PCRs von 12.7 kb und 14.3 kb Länge. Die Überlappung war dabei für die Haplotypisierung wesentlich. Die Nanopore-Sequenzierung der PCR-Amplifikate erfolgte auf einer Flongle flow cell, und die detektierten exonischen Varianten wurden durch Sanger-Sequenzierung bestätigt. Wir identifizierten einen heterozygoten KEL*01/02-Blutspender mit einem KEL:-1,2 (K-k+) Phänotyp. Diese Diskrepanz wies auf ein Null-Allel (KEL*01N) hin. Die Analyse der Probe ergab eine bisher bei der ISBT noch nicht beschriebene Missense-Variante in Exon 11 (c.1241C>A, p.Thr414Lys, rs1384232704), welche dem KEL*01-Allel zugeordnet werden konnte. Da kein KEL1-Antigen auf der Oberfläche der Erythrozyten nachweisbar war, wurde die Genvariante als Null-Allel definiert. Mit Hilfe der Nanopore-Sequenzierung konnten wir eine Diskrepanz zwischen Genotyp und Phänotyp innerhalb kurzer Zeit auflösen und ein neues KEL*01N-Allel beschreiben. Die Long-Read Technologie vereinfachte maßgeblich die Haplotypisierung des KEL-Gens und dies in einem kostengünstigen sowie zeitsparenden Verfahren, welches sich auch für die Abklärung von Genotyp-Phänotypdiskrepanzen in vielen anderen Blutgruppensystemen eignet.

Due to substantial improvements in read accuracy, third-generation long-read sequencing holds great potential in blood group diagnostics, particularly in cases where traditional genotyping or sequencing techniques, primarily targeting exons, fail to explain serological phenotypes. In this study, we employed Oxford Nanopore sequencing to resolve all genotype–phenotype discrepancies in the Kidd blood group system (JK, encoded by SLC14A1) observed over seven years of routine high-throughput donor genotyping using a mass spectrometry-based platform at the Blood Transfusion Service, Zurich. Discrepant results from standard serological typing and donor genotyping were confirmed using commercial PCR-SSP kits. To resolve discrepancies, we amplified the entire coding region of SLC14A1 (~24 kb, exons 3 to 10) in two overlapping long-range PCRs in all samples. Amplicons were barcoded and sequenced on a MinION flow cell. Sanger sequencing and bridge-PCRs were used to confirm findings. Among 11,972 donors with both serological and genotype data available for the Kidd system, we identified 10 cases with unexplained conflicting results. Five were linked to known weak and null alleles caused by variants not included in the routine donor genotyping. In two cases, we identified novel null alleles on the JK*01 (Gly40Asp; c.119G>A) and JK*02 (Gly242Glu; c.725G>A) haplotypes, respectively. Remarkably, the remaining three cases were associated with a yet unknown deletion of ~5 kb spanning exons 9–10 of the JK*01 allele, which other molecular methods had failed to detect. Overall, nanopore sequencing demonstrated reliable and accurate performance for detecting both single-nucleotide and structural variants. It possesses the potential to become a robust tool in the molecular diagnostic portfolio, particularly for addressing challenging structural variants such as hybrid genes, deletions and duplications.

Background and Objectives Mixed-field agglutination in ABO phenotyping (A(3), B-3) has been linked to genetically different blood cell populations such as in chimerism, or to rare variants in either ABO exon 7 or regulatory regions. Clarification of such cases is challenging and would greatly benefit from sequencing technologies that allow resolving full-gene haplotypes at high resolution.Materials and MethodsWe used long-read sequencing by Oxford Nanopore Technologies to sequence the entire ABO gene, amplified in two overlapping long-range PCR fragments, in a blood donor presented with A(3)B phenotype. Confirmation analyses were carried out by Sanger sequencing and included samples from other family members.ResultsOur data revealed a novel heterozygous g.10924C>A variant on the ABO*A allele located in the transcription factor binding site for RUNX1 in intron 1 (+5.8 kb site). Inheritance was shown by the results of the donor's mother, who shared the novel variant and the anti-A specific mixed-field agglutination.ConclusionWe discovered a regulatory variant in the 8-bp RUNX1 motif of ABO, which extends current knowledge of three other variants affecting the same motif and also leading to A(3) or B(3 )phenotypes. Overall, long-range PCR combined with nanopore sequencing proved powerful and showed great potential as an emerging strategy for resolving cases with cryptic ABO phenotypes.

Since the KEL1 antigen of the Kell blood group system is very immunogenic, it is usually determined in routine donor typing. At our blood service in Zurich, KEL1 is determined by both serology and high-throughput genotyping. Genotype-phenotype discrepancies are normally resolved by laborious Sanger sequencing of all 19 exons without haplotype phasing capability. Here, we present an alternative protocol relying on sequencing with Oxford Nanopore Technologies (ONT), which enables the generation of full-gene haplotypes. Expression of KEL1 antigen was measured by standard serological techniques. Four variants within the KEL gene were part of our MALDI-TOF mass spectrometry based high-throughput blood group genotyping routine, including c.578C>T determining KEL1/2 expression. Genotype-phenotype discrepancies were reassessed by commercially available PCR-SSP kits, adsorption-elution as well as flow cytometry experiments. To resolve a discrepancy in one donor, the entire KEL gene (similar to 21 kb) was amplified in two long-range PCRs (fragments of 12.7 and 14.3 kb, respectively), exhibiting a large overlap (similar to 4.4 kb) essential for haplotype phasing. Amplicons were sequenced on a Flongle flow cell (ONT) and detected exonic variants were confirmed with Sanger sequencing. We identified a heterozygous KEL*01/02 blood donor with a KEL:-1,2 (K-k+) phenotype. This discrepancy pointed to a null allele (KEL*01N ) as no KEL1 antigen could be detected on the erythrocyte surface. A few minutes of Nanopore sequencing already yielded enough data for reliable variant calling. A heterozygous variant in the overlap sequence allowed complete gene haplotype phasing. In exon 11, we identified a missense variant c.1241C>A (p.Thr414Lys, rs1384232704), which was phased to the KEL*01 allele. The variant was yet undescribed, despite the over 100 alleles collected by the ISBT, and was confirmed by Sanger sequencing. Using Nanopore sequencing, we resolved a genotype-phenotype discrepancy within short turnaround time and discovered a novel KEL*01N allele. The long reads allowed phasing of detected variants to the respective KEL*01/02 background and even constructing full-length KEL haplotypes. The use of a protocol and flow cell optimized for single-sample analysis kept time and expenses competitive. Overall, our approach proved very promising for resolving genotype-phenotype discrepancies in many blood group systems.

Single nucleotide polymorphisms (SNPs) in the cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) gene, an inhibitor of T-cell priming, are associated with auto and alloimmunity. Studies implied a role for these SNPs as surrogate markers for immunotherapy-outcome in patients with melanoma. However, no predictive SNPs are defined to date. We analyzed different CTLA-4 SNPs in a large multicenter cohort of patients with ipilimumab-treated melanoma and investigated possible correlations with treatment-related outcomes. Archival blood and/or tumor tissue samples were collected from 361 patients with advanced-stage ipilimumab-treated (±nivolumab) in 6 Swiss and Dutch hospitals. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry based DNA genotyping was performed for 10 different CTLA-4 SNPs: 49A>G, CT60G>A, Jo27T>C, Jo30G>A, Jo31G>T, −658C>T, −1722T>C, −1661A>G, 318C>T, and C>T rs1863800. Associations between different allele genotypes and occurrence of grade ≥3 adverse events (AEs) and survival were tested using univariable logistic regressions or Cox proportional hazard models. 262/361 (73%) patients could be analyzed; 65% of those were males, the median age was 58 years, 39% showed a partial or complete response, and 65% had ≥1 AEs. A TT-genotype of −1722T>C SNP was significantly associated with a lower incidence of grade ≥3 AEs ( P = 0.049), whereas the GG-genotype of CT60G>A correlated with a higher incidence of grade ≥3 AEs ( P = 0.026). The TT-genotype of Jo27T>C SNP ( P = 0.056) and GG-genotype of Jo31G>T ( P = 0.046) were associated with overall survival. CTLA-4 SNPs might predict treatment-related outcomes in patients with melanoma receiving ipilimumab. Confirmatory studies are needed to fully exploit those findings as predictive biomarkers for ipilimumab AEs.

Key Points • The first comprehensive collection of full-length haplotype sequences for all 6 main ABO allele groups will support ABO genetic analyses.• ABO genetic diversity patterns revealed putatively ABO∗A1-diagnostic variants, which could finally enable direct genetic typing of A1.

Due to substantial improvement in read accuracy, third-generation long-read sequencing holds great potential in blood group diagnostics, particularly in cases where traditional genotyping or sequencing techniques, primarily targeting exons, are unable to explain serologic phenotypes. In this study, we employed Oxford Nanopore sequencing to resolve all genotype-phenotype discrepancies in the Kidd blood group system (JK, SLC14A1 ) observed over seven years of routine high-throughput donor genotyping using a mass spectrometry based platform at Blood Transfusion Service Zurich. Discrepant results of standard serological typing and donor genotyping were confirmed by commercial PCR-SSP kits. To resolve discrepancies, we amplified the entire coding region of SLC14A1 (∼24 kb, exons 3 to 10) in two overlapping long-range PCRs in all samples. Amplicons were barcoded and sequenced on a MinION flow cell. Sanger sequencing and bridge-PCRs were used to confirm findings. Among 11,972 donors who had both serology and genotypic data available for the Kidd system, we identified 10 cases with unexplained conflicting results. Five were linked to known weak and null alleles caused by variants not included in the routine donor genotyping. In two cases, we identified novel null alleles on the JK*01 (Gly40Asp; c.119G>A) and JK*02 (Gly242Glu; c.725G>A) haplotype, respectively. Remarkably, the remaining three cases were linked to a yet unknown deletion of ∼5 kb spanning over exon 9-10 of the JK*01 allele, which other molecular methods had failed to detect. Overall, nanopore sequencing demonstrated reliable and accurate performance for detecting both single nucleotide and structural variants. It possesses the potential to become a robust tool in the molecular diagnostic portfolio, particularly for addressing challenging structural variation such as hybrid genes, deletions and duplications.

Acute porphyrias are a group of monogenetic inborn errors of heme biosynthesis, characterized by acute and potentially life-threatening neurovisceral attacks upon exposure to certain triggering factors. Biochemical analyses can determine the type of acute porphyria, and subsequent genetic analysis allows for the identification of pathogenic variants in the specific gene, which provides information for family counselling. In 2017, a male Swiss patient was diagnosed with an acute porphyria while suffering from an acute attack. The pattern of porphyrin metabolite excretion in urine, faeces, and plasma was typical for an acute intermittent porphyria (AIP), which is caused by inherited autosomal dominant mutations in the gene for hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. However, the measurement of HMBS enzymatic activity in the erythrocytes was within the normal range and Sanger sequencing of the HMBS gene failed to detect any pathogenic variants. To explore the molecular basis of the apparent AIP in this patient, we performed third-generation long-read single-molecule sequencing (nanopore sequencing) on a PCR product spanning the entire HMBS gene, including the intronic sequences. We identified a known pathogenic variant, c.77G>A, p.(Arg26His), in exon 3 at an allelic frequency of ~22% in the patient’s blood. The absence of the pathogenic variant in the DNA of the parents and the results of additional confirmatory studies supported the presence of a de novo mosaic mutation. To our knowledge, such a mutation has not been previously described in any acute porphyria. Therefore, de novo mosaic mutations should be considered as potential causes of acute porphyrias when no pathogenic genetic variant can be identified through routine molecular diagnostics.

The U antigen (MNS5) is one of 49 antigens belonging to the MNS blood group system (ISBT002) carried on glycophorins A (GPA) and B (GPB). U is present on the red blood cells in almost all Europeans and Asians but absent in approximately 1.0% of Black Africans. U negativity coincides with negativity for S (MNS3) and s (MNS4) on GPB, thus be called S-s-U-, and is thought to arise from homozygous deletion of GYPB. Little is known about the molecular background of these deletions. Bioinformatic analysis of the 1000 Genomes Project data revealed several candidate regions with apparent deletions in GYPB. Highly specific Gap-PCRs, only resulting in positive amplification from DNAs with deletions present, allowed for the exact genetic localization of 3 different breakpoints; 110.24- and 103.26-kb deletions were proven to be the most frequent in Black Americans and Africans. Among 157 CEPH DNAs, deletions in 6 out of 8 African ethnicities were present. Allele frequencies of the deletions within African ethnicities varied greatly and reached a cumulative 23.3% among the Mbuti Pygmy people from the Congo. Similar observations were made for U+var alleles, known to cause strongly reduced GPB expression. The 110- and 103-kb deletional GYPB haplotypes were found to represent the most prevalent hereditary factors causative of the MNS blood group phenotype S-s-U-. Respective GYPB deletions are now accessible by molecular detection of homo- and hemizygous transmission.

Extended blood group genotyping is an invaluable tool used for prevention of alloimmunization. Genotyping is particularly suitable when antigens are weak, specific antisera are unavailable, or accurate phenotyping is problematic because of a disease state or recent transfusions. In addition, genotyping facilitates establishment of mass-scale patient-matched donor databases. However, standardization of genotyping technologies has been hindered by the lack of reference panels. A well characterized renewable reference panel for standardization of blood group genotyping was developed. The panel consists of genomic DNA lyophilized and stored in glass vials. Genomic DNA was extracted in bulk from immortalized lymphoblastoid cell lines, generated by Epstein-Barr virus transformation of peripheral blood Lymphocytes harvested from volunteer blood donors. The panel was validated by an international collaborative study involving 28 laboratories that tested each DNA panel member for 41 polymorphisms associated with 17 blood group systems. Overall, analysis of genotyping results showed >98% agreement with the expected outcomes, demonstrating suitability of the material for use as reference. Highest levels of discordance were observed for the genes CR1, CD55, BSG, and RHD. Although limited, observed inconsistencies and procedural limitations reinforce the importance of reference reagents to standardize and harmonize results. Results of stability and accelerated degradation studies support the suitability of this panel for use as reference reagent for blood group genotyping assay development and standardization.

Recently, we reported a pregnant SCD patient with a specific anti-public-antibody (anti-Fy5) amongst other alloantibodies. During her present pregnancy we were able to demonstrate that two positive crossmatches of two former compatible donors were caused by a new alloantibody against a low-prevalence antigen, namely anti-Rh23, derived from several Rh23+ RBC transfusions during the previous pregnancy.

BACKGROUNDBlood group phenotype variation has been attributed to potential resistance to pathogen invasion. Variation was mapped in blood donors from Lampang (northern region) and Saraburi (central region), Thailand, where malaria is endemic. The previously unknown blood group allele profiles were characterized and the data were correlated with phenotypes. The high incidence of the Vel‐negative phenotype previously reported in Thais was investigated.STUDY DESIGN AND METHODSDNA from 396 blood donors was analyzed by matrix‐assisted laser desorption/ionization–time‐of‐flight mass spectrometry. Outliers were investigated by serology and DNA sequencing. Allele discrimination assays for SMIM1 rs1175550A/G and ACKR1 rs118062001C/T were performed and correlated with antigen expression.RESULTSAll samples were phenotyped for Rh, MNS, and K. Genotyping/phenotyping for RhD, K, and S/s showed 100% concordance. Investigation of three RHCE outliers revealed an e‐variant antigen encoded by RHCE*02.22. Screening for rs147357308 (RHCE c.667T) revealed a frequency of 3.3%. MN typing discrepancies in 41 samples revealed glycophorin variants, of which 40 of 41 were due to Mia. Nine samples (2.3%) were heterozygous for FY*01W.01 (c.265C > T), and six samples (1.5%) were heterozygous for JK*02N.01. All samples were wildtype SMIM1 homozygotes with 97% homozygosity for rs1175550A.CONCLUSIONSMatrix‐assisted laser desorption/ionization–time‐of‐flight mass spectrometry is an efficient method for rapid routine genotyping and investigation of outliers identified novel variation among our samples. The expected high prevalence of the Mi(a+) phenotype was observed from both regions. Of potential clinical relevance in a region where transfusion‐dependent thalassemia is common, we identified two RHCE*02 alleles known to encode an e‐variant antigen.

BACKGROUND:High-frequency blood group antigens (HFA) are present in >90% of the human population, according to some reports even in >99% of individuals. Therefore, patients lacking HFA may become challenging for transfusion support because compatible blood is hardly found, and if the patient carries alloantibodies, the cross-match will be positive with virtual every red cell unit tested.METHODS:In this study, we applied high-throughput blood group SNP genotyping on >37,000 Swiss blood donors, intending to identify homozygous carriers of low-frequency blood group antigens (LFA).RESULTS:326 such individuals were identified and made available to transfusion specialists for future support of patients in need of rare blood products.CONCLUSION:Thorough comparison of minor allele frequencies using population genetics revealed heterogeneity of allele distributions among Swiss blood donors which may be explained by the topographical and cultural peculiarities of Switzerland. Moreover, geographically localized donor subpopulations are described which contain above-average numbers of individuals carrying rare blood group genotypes.

BACKGROUNDAlloimmunization against human platelet antigens (HPAs) during pregnancy is rare but can lead to severe bleeding disorders, such as fetal and neonatal alloimmune thrombocytopenia.STUDY DESIGN AND METHODSIn a cohort of 241 uncomplicated pregnancies, we investigated the immunogenicity of HPA mismatches and correlated HLA sensitization with HPA antibody formation. HPA antibodies were measured with a Luminex‐based multiplex assay.RESULTSHPA mismatches were observed in 109 of 241 pregnancies (45%), but child‐specific HPA antibodies were only found in two of 109 cases (2%), indicating a low immunogenicity. Only nine of 241 women (4%) had detectable HPA antibodies. HLA sensitization was identified as a strong and independent predictor for HPA antibody formation (hazard ratio, 10.2; 95% confidence interval, 1.8‐193; p = 0.006), whereas the number of pregnancies was not.CONCLUSIONOur observational data indicated a low immunogenicity of HPA and suggest that a broader immune response—inferred by HLA sensitization—is probably associated with HPA antibody induction.

Results of genotyping with true high-throughput capability for MNSs antigens are underrepresented, probably because of technical issues, due to the high level of nucleotide sequence homology of the paralogous genes GYPA , GYPB and GYPE . Eight MNSs-specific single nucleotide polymorphisms (SNP) were detected using matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) in 5800 serologically M/N and S/s pre-typed Swiss blood donors and 50 individuals of known or presumptive black African ethnicity. Comparison of serotype with genotype delivered concordance rates of 99·70% and 99·90% and accuracy of genotyping alone of 99·88% and 99·95%, for M/N and S/s, respectively. The area under the curve of peak signals was measured in intron 1 of the two highly homologous genes GYPB and GYPE and allowed for gene copy number variation estimates in all individuals investigated. Elevated GYPB : GYPE ratios accumulated in several carriers of two newly observed GYP*401 variants, termed type G and H, both encoding for the low incidence antigen St(a). In black Africans, reduced GYPB gene contents were proven in pre-typed S-s-U- phenotypes and could be reproduced in unknown specimens. Quantitative gene copy number estimates represented a highly attractive supplement to conventional genotyping, solely based on MNSs SNPs.