A number of anti-CD47 drug candidates are currently being tested in clinical trials. As checkpoint inhibitors, they block the "don't eat me" signal that cancer cells send out in order to prevent macrophage-mediated killing, among other mechanisms of action. As CD47 is also expressed in high density on the surface of red blood cells, this type of therapy interferes with immunohematological diagnostics. Several methods to deal with this interference are currently being trialled. Here we report on the course of the interference in a patient with acute myeloid leukemia treated with magrolimab and present a method for masking CD47 to overcome the interference using a new reagent.

The Complement Receptor 1 (CR1) carries the Knops blood group antigens (KN; ISBT 022).1, 2 CR1 consists of 30 complement control protein (CCP) domains grouped into four long homologous repeats (LHR-A to -D). The antigens KN1 to KN10 are located in LHR-D while the antithetical antigens KN11 and KN12 are located in LHR-C.3 Antibodies to Knops antigens are not clinically significant but they are often found in patients causing problems in ruling out additional relevant antibodies. In a female patient (M.B.; index case) of Ethiopian origin with transfusion history and a severe COVID-19 pneumonia, we found an antibody to a high prevalence antigen. After ruling out antibodies to a number of high prevalence antigens, an antibody to a Knops antigen was suspected, because it could be inhibited by the Knops/DACY recombinant protein. Molecular analysis of CR1 was conducted for M.B. and a nonrelated African patient (Y.U.) who was nonreactive with M.B.'s plasma. Antibody identification was performed by the gel technique in the indirect antiglobulin test using different commercial panels (BioRad, Switzerland) with untreated and papain treated red cells negative for high prevalence antigens. Recombinant proteins Chido, Rodgers, JMH, Kn(a), and DACY (imunsyn GmbH, Hannover, Germany) were used for inhibition assays of M.B.'s plasma. Furthermore, recombinant CR1 proteins for LHR-C, LHR-C_1097Pro, LHR-C_1100Gly, and LHR-C_1097Pro-1100Gly were produced according to previously described procedures and used for inhibition assays.4 Molecular analysis of CR1 was included in targeted next generation sequencing of all exons of the blood group genes encoding the systems ISBT 001 to 043. Genomic DNA from M.B. and Y.U. was sequenced according to standard protocols for amplicon-based library generation with the iSeq 100 system (illumina Inc., Berlin, Germany). For data analysis, the Variant Interpreter (illumina Inc.) and the Integrative Genomics Viewer (IGV) tools were used.5 Genotyping of CR1 c.3290T>C (rs200111726) was performed according to standard PCR-SSP protocol with forward primers for the wild type allele (5′-GTGACCTACCGCTGCAATCT-3′) and the variant allele (5′-GTGACCTACCGCTGCAATCC-3′), a reverse primer (5′-TGGAGGCGTGCATTTGTTAGG-3′), and primers for an internal control amplified from the HBB gene.6 The antibody of M.B. was reactive with all test cells of the antibody identification panels and 29 test red cells negative for different high prevalence antigens. It was nonreactive with Y.U.'s red cells only and with all papain-treated red cells. An antibody to known KN antigens was ruled out. Recombinant Kn(a), Chido, Rodgers and JMH did not inhibit the antibody, whereas, it was inhibited by the DACY recombinant protein, indicating that the corresponding antigen is probably located in LHR-C. Molecular analysis revealed 5 homozygous missense variants in CR1 of both patients: 3 known CR1 variants (c.3623A>G, c.4801A>G, c.4843A>G) and 2 variants in exon 21 c.3290T>C (p.Leu1097Pro; rs200111726) and c.3298A>G (p.Arg1100Gly; rs202070239). PCR-SSP for c.3290T>C confirmed the sequencing results. Inhibition assays using the different recombinant CR1 proteins showed inhibition of the patient's antibody with LHR-C and LHR-C_1100Gly but no inhibition with LHR-C_1097Pro and LHR-C_1097Pro-1100Gly (Figure 1). The inhibition with LHR-C and LHR-C_1100Gly proved that the antibody is directed against p.1097Leu. Using an antibody to a high prevalence antigen found in a previously transfused Ethiopian patient we identified a new Knops blood group antigen located in LHR-C region of the Knops protein. In commemoration of the index patient who died from severe COVID-19 the provisional antigen name (KNMB) was derived from the initials. The antigen number KN13 was assigned by the ISBT working party on Red Cell Immunogenetics and Blood Group Terminology at the ISBT meeting in Gothenburg, Sweden, in June 2023. KNMB is defined by p.1097Leu and homozygosity for p.1097Pro in both patients caused the KNMB-negative phenotype. The underlying CR1 variant rs200111726T>C is rare (0.02%) in the European population, but more frequent (3.9%) in the African population.7 Accordingly, only 1 of 25,000,000 Europeans (0.000004%) but 1 of 625 Africans (0.16%) are expected to be KNMB negative. The authors have disclosed no conflicts of interest. Open Access funding enabled and organized by Projekt DEAL.

BACKGROUND:Administration of anti-CD38 antibodies is a state-of-the-art therapy for patients diagnosed with multiple myeloma (MM). However, this treatment frequently leads to pan-agglutination of red blood cells (RBCs) in patients' serological testing making accurate blood typing and timely transfusion of compatible blood a challenging effort. The antigen masking indirect antiglobulin test (AMIAT) is an approach to address this diagnostic challenge.STUDY DESIGN AND METHODS:A new reagent, called DaraEx plus, uses anti-CD38 Fab fragments to mitigate the anti-CD38 antibody interference in serological assays by masking CD38 on the cell surface. Its performance is extensively examined with commercial sera as well as with patient samples, and compared to the current standard method using dithiothreitol (DTT), which denatures the CD38 antigens on test panel erythrocytes.RESULTS:In the Bio-Rad ID System, DaraEx plus effectively mitigated the interference caused by anti-CD38 antibodies in 86% of patient samples tested while DTT was successful in only 68%. Moreover, there was no negative influence on DTT-sensitive blood group systems such as KEL upon DaraEx plus treatment. The agglutination reactions of all tested anti-CD38 antibodies (Daratumumab, Felzartamab, and Isatuximab) were inhibited by DaraEx plus. The treatment was successful only if DaraEx plus was added to the test cells before the sample. Some of the other gel card systems tested showed background reactions with DaraEx plus-treated cells.CONCLUSION:DaraEx plus treatment is straightforward and quick to perform. In the Bio-Rad ID System, it is superior to DTT treatment in the prevention of anti-CD38 antibody interference.

Anti-CD38 antibodies are increasingly used in the clinic for the treatment of multiple myeloma and other indications. CD38 is a surface molecule expressed on a number of cells and tissues, including erythrocytes. After administration of anti-CD38 antibodies such as Daratumumab, interference regularly occurs in the indirect antihuman globulin (IAT) assays, hampering screening of irregular antibodies while also levering out the safety of the cross match. Several methods to deal with this interference are currently recommended by the German Society for Transfusion Medicine and Immunohematology (DGTI). We present here a comparison between the DTT method and the masking of CD38 by a new reagent in a patient with non-secretory multiple myeloma.

BACKGROUND:All antigens described in the KN blood group system are located in the long homologous repeat D (LHR-D) of complement receptor 1 (CR1). While there have been reports that some sera react only with the long homologous repeat C (LHR-C), the antigens in LHR-C are unknown.STUDY DESIGN AND METHODS:Recombinant LHR-C and LHR-D were used to identify antibodies directed against LHR-C of CR1, into which a point mutation was introduced to characterize the underlying blood group antigens. In addition, database studies to define haplotypes of CR1 were performed.RESULTS:Several antisera were identified that were specific against CR1 p.1208His and against CR1 p.1208Arg, located in LHR-C. Fifteen KN haplotypes were found in the Ensembl genome browser. It was shown that due to a linkage disequilibrium anti-CR1 p.1208His may be mistaken for anti-KCAM.CONCLUSION:A novel antithetical KN blood group antigen pair was found at position p.1208 of CR1, for which the names DACY and YCAD are proposed. Antibodies against these two novel antigens seem to contribute to more than a quarter of all KN sera in Europe.

The anticancer drug daratumumab, an anti-CD38 antibody, has recently been approved by the European Commission for treatment of multiple myeloma. CD38 is a surface molecule expressed on a number of tissues and cells, including erythrocytes. Daratumumab in patient sera leads to a strong interference in the indirect antiglobulin test (IAT), where most, if not all, of the reactions turn positive. This occurs in red blood cell crossmatches as well as antibody screens and identifications and may happen up to 6 months after the last treatment with daratumumab. The current standard technique using dithiothreitol (DTT) for this problem has considerable drawbacks and technical challenges (i.e. damage of the red cell surface with partial blood group loss and hemolysis, time consuming procedure). We introduce here a new, simple and time-saving test for removing the interference of daratumumab that circumvents the DTT associated red blood cell damage.

Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

BACKGROUND:Until now, it was not possible to identify antibodies to red blood cells (RBCs) except with pretyped RBCs. Here, a novel method with particles coated with recombinant Lu(b) protein for detection of anti-Lu(b) is described.STUDY DESIGN AND METHODS:Prokaryotic recombinant Lu(b) proteins were generated and coupled onto superparamagnetic particles coated with streptavidin. The coated particles were tested in the presence of different serum and plasma samples (13 anti-Lu(b), 6 anti-Lu(a), 20 other antibodies, and 35 serum samples from blood donors) with the particle gel immunoassay (ID-PaGIA).RESULTS:Lu(b)-coated particles reacted with all 13 samples containing anti-Lu(b), but not with any samples lacking anti-Lu(b). In addition, the anti-Lu(b) titers were higher with Lu(b)-coated particles than with Lu(a-b+) RBCs in almost all cases.CONCLUSION:Recombinant blood group proteins may be able to dispense with the need for RBCs for identification of certain RBC alloantibodies.

BACKGROUND: Little is known about the mechanism by which amino acid polymorphisms outside the catalytically active cleft of ABO glycosyltransferases cause weak ABO phenotypes.STUDY DESIGN AND METHODS: Extensive ABC phenotyping and genotyping were performed to classify the blood of a healthy blood group 0 donor with weak iso-agglutinins. ABO antigen and glycosyltransferase expression profiles were then studied in eukaryotic transfection experiments, and the topology of ABO glycosyltransferase was analyzed.RESULTS: The donor's red blood cells were retyped as A(weak), and his serum contained weakly reactive anti-A and anti-B. Sequence analysis revealed two novel ABO alleles. A donor splice-site mutation detected at the exon 6/intron 6 junction of an ABO* A101 allele was predicted to result in skipping exon 6 in the mRNA. The other haplotype displayed a single 688G>C substitution predicting a Gly230Arg exchange in the catalytic domain in an otherwise normal ABO* B101 allele. The transfection studies revealed very weak expression of B antigen by the novel ABO* B allele. According to the topologic analysis, steric hindrance due to the Gly230Arg exchange may cause conformational changes in the variant B transferase. Compared to the wild-type B transferase, the transfected cells exhibited lower-level protein expression and intracellular dislocation.CONCLUSION: This study provides first evidence that aberrant trafficking of variant ABC transferases may be involved in the formation of weak ABC phenotypes.

BACKGROUND: Binding of CCAAT‐binding factor NF‐Y (CBF/NF‐Y) to a 43‐bp repeat unit in the minisatellite region in the 5′ region of the ABO gene (CBF/NF‐Y enhancer region) plays an important role in regulating the transcription of ABO genes. The common ABO alleles were found to have CBF/NF‐Y enhancer regions with specific numbers of 43‐bp minisatellite repeats.