56. Jahrestagung der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie

Vorsitz

PD Dr.

Prof. Dr.

Molecular characterization of the complement receptor 1 gene in patients with alloantibodies against antigens of the knops blood group system

Background The antigens of the Knops blood group system (KN; ISBT 022) are located on the complement receptor 1 (CR1), a large glycoprotein composed of 4 Long-Homologous-Repeats (LHR-A, -B, -C, -D). Most KN antigens are located LHR-D, except for KN11/KN12 and KNMB located in LHR-C. Antibodies to KN antigens are not clinically relevant but they are common in patients. Here, we sequenced the CR1 gene in patients with suspected alloantibodies to KN antigens.Methods Serologic work-up identified 23 cases with suspected antibodies against a high prevalence antigen in the Knops blood group system. The ethnic background of the patients was European (15 cases) or African (8 cases). All cases were subjected to Massively Parallel Sequencing (MPS) of all 39 exons of the CR1 gene included in the targeted panel of all blood group genes. Allele frequencies of the identifed gene variants in different populations were taken from the gnomAD database.Results The CR1 sequence in 2 of the European cases showed homozygosity for the KN*01.–05 allele (c.4223C&gt;T; p.T1408M) indicating absence of the Yk(a) antigen. Homozygosity for the KN*01.–08 allele (c.4828T&gt;A; p.S1610T) indicating absence of the Sl3 antigen was identified in 2 European cases. In 8 cases we found heterozygosity for at least one known KN allele or rare CR1 variant. The other 3 European cases showed no CR1 variation. Two of the African cases were homozygous for CR1 c.3290T&gt;C (p.L1097P) and further serologic investigation showed that the variant defines a new high prevalence antigen named KNMB. Each of the other 6 African cases revealed heterozygosity for at least 2 known KN alleles or rare CR1 variants.Conclusion MPS simplified the molecular analysis especially of blood group systems with large genes such as CR1 for the KN system. The systematic case work enabled the identification of the new KN antigen KNMB. The significantly different frequency in African and European populations of CR1 variants encoding KN antigens may result in an increased risk of alloimmunization evoked by blood transfusion.Offenlegung Interessenkonflikt: No conflicts of interestInvited talks abstract/summary

Immunohematology

Freier Vortrag

KNMB, a novel Knops blood group antigen located on LHR-C

Background The complement receptor 1 (CR1) carries the antigens of the Knops blood group system (KN; ISBT 022). They are located on the long homologous repeats (LHR) C and D of the CR1. Antibodies to Knops antigens are not clinically significant but they are common in patients. In a previously transfused female patient of Ethiopian origin with a severe COVID-19 pneumonia we found an antibody to a new high prevalence antigen which we suspected to belong the Knops blood group system. Methods Antibody identification was performed in IAT in gel technique (BioRad, CH) using different commercial panels as well as untreated and papain treated red cells negative for high prevalence antigens. Different recombinant blood group proteins (imusyn, Germany) and specially produced recombinant proteins for LHR-C, LHR-C_1097Pro, LHR-C_1100Gly and LHR-C_1097Pro-1100Gly were used for inhibition assays. Massive parallel sequencing (MPS) of a blood group gene panel including all exons of the genes encoding the blood group systems ISBT001 to 043 was performed on the patient and a single non-related African patient non-reactive with patient´s plasma. Sequence-specific PCR (PCR-SSP) methods were established to confirm the mutations in CR1.Results The antibody was reactive with all test cells of the antibody identification panels and 29 test red cells negative for different high prevalence antigens. It was non-reactive with papain-treated red cells. Recombinant DACY protein inhibited, recombinant Kn(a) protein did not inhibit the antibody, indicating that the antigen is located on LHR-C. Molecular analysis revealed 2 unknown variants in CR1 exon 21: c.3290T&gt;C (p.Leu1097Pro; rs200111726) and c.3298A&gt;G (p.Arg1100Gly; rs202070239). Further assays showed inhibition of the antibody with LHR-C and LHR-C_1100Gly recombinant proteins, but not with LHR-C_1097Pro and LHR-C_1097Pro-1100Gly. This proved that the antibody is directed against p.1097Leu.Conclusion Using an antibody to a high prevalence antigen found in a previously transfused patient of Ethiopian origin we identified a new Knops blood group antigen located on LHR-C region of the Knops protein. The provisional antigen name (KNMB) was derived from the initials of the antibody producer. KNMB is defined by p.1097Leu and homozygosity for p.1097Pro caused the KNMB-negative phenotype. The CR1 variant rs200111726T&gt;C is rare in the European population but frequent in the African population.Offenlegung Interessenkonflikt: no conflict of interest

Freier Vortrag (Reinhold-Eckstein-Investigator-Award)

Creation of an adequate structural model of the vel epitope with artificial intelligence

Background Vel is a clinically relevant blood group antigen known to be carried by the small integral membrane protein 1 (SMIM1). The presentation of the Vel epitope requires inter-molecular disulfide bonds and a functional GXXXG-motif, which reportedly supports the dimerization of membrane proteins. The exact structure of the Vel epitope, however, remains elusive so far.Methods A putative Vel epitope was modelled using artificial intelligence supported structure prediction of dimers of SMIM1 and SMIM1 variants.Results Two conditions were considered necessary for a model of the Vel epitope to be acceptable: firstly, the formation of an inter-molecular disulfide bond, and secondly, the correct orientation of the GXXXG-motif. Modelling a Vel dimer without constraints fulfilled neither the first nor the second condition. After an iterative process of adding other structural motifs and linkers, a model was obtained that fulfilled both conditions (Fig 1).Conclusion The dimerization of SMIM1 puts a certain apparent stress on the structure, which probably requires the disulfide bond to remain stable. This could explain why the disulfide bond is crucial for the presentation of the Vel antigen and anti-Vel antibodies seem to be mostly conformation dependent. The modelled Vel epitope still needs experimental validation.Offenlegung Interessenkonflikt: Angestellter bei imusyn GmbH &amp; Co. KGInvited talks abstract/summary

Recombinant blood group antigens in patient testing

Background Antibodies to high-prevalence red cell antigens can be challenging in serologic patient workup. Hemagglutination inhibition assays using recombinant blood group antigens (rBGAs) can identify the specificity of the antibody and uncover additional, possibly significant, alloantibodies. Data of use in routine immunohematologic laboratories are still scarce. We report the use of rBGAs in patient testing regarding feasibility, effectiveness and limitations.Methods Patients (n=12) with suspected or known antibodies to high-prevalence antigens were tested with single soluble rBGAs (Ch, Rg, Kna, JMH, Yta, Dob, CROM/DAF, Kell-Kpb-Jsa, Cellano-Kpb-Jsa, Lub/Aub, Sc1; inno-train Diagnostic GmbH, Kronberg, Germany) according to the manufacturer's instruction. 2 µl rBGA solution was mixed with 25 µl patient plasma, incubated at room temperature for 30 min and tested in a gel agglutination system (BioRad ID-System) in the indirect antiglobulin test with untreated or papain-treated test cells. Inhibition of reactivity occurred if the corresponding antibody was present and additional antibodies could further be identified in routine test systems. Choice of rBGAs was dependent on preceding serologic results.Results In 9 (75%) of 12 patients antibodies to high-prevalence antigens were identified with rBGAs of the specificities Ch (n=3), Yta (n=3), Kna (n=2), and Lub (n=1). Single or multiple additional underlying antibodies of 6 different specificities (Wra, Fya, C, Jkb, Fyb, and S) were identified in 4 (44%) of these 9 patients. In 3 cases (25%) inhibition was not successful and explained by a warm-reactive autoantibody plus anti-E (n=1), nonspecific reactivity (n=1) and reactions to a high-prevalence antigen not related to the rBGAs used (n=1). Specificities of rBGAs and antibodies are detailed in Table 1.Conclusion Hemagglutination inhibition assays using rBGAs can easily be carried out in a routine testing laboratory without the need of rare test cells or antisera, or sometimes complicated and time-consuming procedures. In 75% of our cases the antibody specificity to a high-prevalence antigen were determined and all additionally identified single or multiple alloantibodies were clinically significant. Recombinant blood group antigens are a valuable tool to increase transfusion safety.Offenlegung Interessenkonflikt: AS is a member of the scientific advisory board of Imusyn (manufacturer of the rBGAs). All other authors: None

A universal blood genotyping array tailored for transfusion services and validated in a pre-clinical study of a large, ethnically diverse cohort

Background Extended blood group matching substantially reduces sensitization to non-self antigens as side effect of transfusion. For broad application, affordable high-throughput typing of relevant antigens is required. Following an earlier proof-of-concept study, our consortium presents here the development of a tailored universal blood donor genotyping array, including the results of an international Pre-Clinical accreditation Study (PCS) comprising an ethnically highly diverse panel of samples.Methods The custom-designed Axiom array contains 50,000 probes tagging 20,000 variants relevant for blood services. It has a 384-sample format and runs on GeneTitan instrument, which can generate data for 3,000 samples/week. The array allows simultaneous typing of clinically relevant human erythroid (HEA), platelet (HPA), and leukocyte (HLA) antigens. For the PCS, DNA samples and clinical antigen typing data from 13,908 donors provided by seven blood services were analysed. Samples representing diverse ethnic groups (74% European, 11% African, 15% others) were genotyped at three blood services in the Netherlands, USA, and UK. Array-inferred antigen types were analysed for concordance with provided clinical antigen types for the first 6,953 samples.Results Reproducibility between sample results from the testing laboratories was outstanding for the 20k variants. The overall concordance for HEA, HPA, and HLA antigens with previous testing was very high. Of ~100,000 comparisons between blood service determined HEA types and array determined types, the concordance was 99.8%. Over 80% of the discordances can be resolved by simple algorithmic modifications and over half of the remaining ones were caused by incorrect serology. The results of the PCS proofed a robust validation of also non-European genotypes configurations. The 778 samples of African ancestry showed a HEA concordance rate &gt;99.7%. Also HLA class I and II concordance level, which could be assessed for 1319 DNA samples, was excellent.Conclusion An affordable and comprehensive DNA-based test for automated high-throughput typing of donors and patients is reported here. The universal blood typing array is tailored for the needs of transfusion services and validated in an international, diverse cohort. Among others, the array represents a promising new tool to facilitate a broader application practise of extended blood matching to reduce sensitization rates and to identify rare donors.Offenlegung Interessenkonflikt: The Blood transfusion Genomics Consortium was financially supported by Thermo Fisher Scientific (Santa Clara, USA) for the development of the Axiom array.

Adaptive sampling to resolve complex structural variants in targeted blood group genes by nanopore sequencing

Background Adaptive sampling is a software-driven enrichment method unique to Oxford Nanopore Technologies (ONT) and allows simultaneous sequencing of the entire blood group genome. Without any restrictions on read length, it can excel in resolving complex structural variants (SV) like hybrid alleles, which are a hallmark of some blood group systems like the RH. Here, we used adaptive sampling to tackle RHCE genotype-phenotype discrepancies unresolved by other methods and suspected to harbor a SV.Methods We reassessed three RHCc genotype-phenotype discrepancies, which we observed in routine high-throughput donor typing, by PCR-SSP, MLPA, and extensive serological analyses. Suspecting a SV, we employed high density MALDI-TOF MS analysis along the RHCE gene to get hints on copy number variation. As unresolvable, we took advantage of latest developments of ONT's adaptive sampling for sequencing the entire RHCE gene as single reads for one of the samples. The sequencing library was built with high-molecular weight gDNA. Beside RHCE we targeted the entire blood group genome (48 genes; ~8 Mb incl. 50 kb flanking regions) in adaptive sampling. Structural variant breakpoints in all three samples were confirmed by bridge-PCRs and Sanger sequencing.Results All three donors were serologically typed as C+c- but genotyped as RHCE*Cc. The high-density MALDI-TOF MS assay pointed towards an identical potential SV located near the end of the RHCE gene. The type or location of the SV, however, could not be determined. ONT sequencing of one sample exhibited high mean read length (N50 of &gt;30 kb) with a median coverage of ~15x across all 48 genes. The availability of particularly long reads (20 reads &gt;50 kb; max. 250 kb) spanning the RH locus allowed identifying a large deletion. Specifically, we identified a novel ~8.6 kb deletion spanning from intron 8 to intron 9 of RHCE. Bridge-PCRs and Sanger sequencing confirmed the exact breakpoint locations, with an identical deletion in the other two samples.Conclusion Adaptive sampling is a promising enrichment method for simultaneously targeting all blood group genes without restrictions on read length, which opens new avenues for identifying SVs. It has allowed us resolving long-tackled RHCE genotype-phenotype discrepancies by discovering a large novel deletion. Although SVs are a hallmark of RH, to our knowledge only two other large deletions have yet been reported for RHCE, suggesting the need for suitable approaches like the one presented here.Offenlegung Interessenkonflikt: keine Interessenskonflikte

Immunohematology 1 - Red Cells

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