Mitigation of therapeutic anti-CD38 antibody interference with fab fragments: How well does it perform?

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.

Two novel antithetical KN blood group antigens may contribute to more than a quarter of all KN antisera in Europe.

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.

Recombinant blood group proteins facilitate the detection of alloantibodies to high-prevalence antigens and reveal underlying antibodies: results of an international study.

BACKGROUND: Alloantibodies to high-prevalence red blood cell (RBC) antigens are not easily identified by routine serologic techniques. This multicenter study was conducted to test the effectiveness of recombinant blood group proteins (rBGPs) at regional and international RBC reference laboratories. STUDY DESIGN AND METHODS: Single or mixed soluble rBGPs (Lu, Yt, Kn, JMH, Sc, Rg, Ch, Do, and Cr) were assessed for their ability to inhibit the reactivity of antibodies to specific antigens. Initially, the effect of rBGPs was validated by testing panels of well-characterized patient serum samples containing antibodies to high-prevalence antigens in the hemagglutination inhibition assay. Subsequently, the rBGPs were prospectively used for routine antibody identification and the results were compared to those obtained with RBC-based diagnostics. RESULTS: Panels of predefined antibodies to high-prevalence antigens were completely and specifically neutralized by the corresponding rBGP specificities. For prospective identification, antibodies to high-prevalence antigens (n = 62) were specifically inhibited by the corresponding rBGP specificities except for some Complement Receptor 1-related antibodies, which may be directed to epitopes not expressed on the truncated recombinant Kn. In 14 cases, additional clinically relevant alloantibodies were identified. In cross-matching, the rBGPs were successfully used to inhibit the reactivity of clinically irrelevant antibodies to high-prevalence antigens to determine compatibility between donor and recipient. CONCLUSION: rBGPs enable the identification of antibodies to high-prevalence antigens without the need for rare RBC reagents, which are often unavailable. Underlying antibodies can be reliably detected and cross-matching results validated, resulting in a more efficient blood supply for immunized patients.

Easy identification of antibodies to high-prevalence Scianna antigens and detection of admixed alloantibodies using soluble recombinant Scianna protein.

BACKGROUND: Identification of antibodies against high-prevalence Scianna (Sc; ERMAP) antigens, like Sc1 and Sc5, is difficult and may incur delays in blood procurement and costs. The detection of additional clinically significant alloantibodies is hampered in the presence of anti-Scianna. Soluble recombinant Scianna protein is demonstrated to facilitate antibody diagnostics in both cases. STUDY DESIGN AND METHODS: Soluble recombinant Scianna protein (Sc:1,-2,3,-4,5,6,7) was produced comprising the antigenic extracellular domain fused to a V5-His tag. The protein was isolated from eukaryotic cell culture supernatants of stably transfected HEK293 cells. Seven serum samples with anti-Sc1, anti-Sc2, and anti-Sc5 and 30 serum samples with antibodies to other blood group antigens were evaluated in hemagglutination inhibition assays. Antisera with mixed antibody specificities and autoantibodies were also tested. RESULTS: Soluble Scianna protein inhibited specifically antibodies to the high-prevalence Scianna antigens Sc1 and Sc5. No antibodies were neutralized that were directed to the low-prevalence Sc2 antigen or to a large representative set of antigens from other blood group systems. Clinically relevant antibodies could be identified despite being masked by anti-Sc1 and anti-Sc5. A mixture of Scianna and JMH proteins allowed detecting a common antibody despite the presence of antibodies to high-prevalence antigens of the Scianna or JMH blood group systems. CONCLUSION: Antibody detection systems comprising soluble recombinant Scianna protein provide an easy single-step method for detection and identification of antibodies to high-prevalence Scianna antigens. Reagents with Scianna and other recombinant blood group proteins and mixtures of such proteins would be useful routine reagents in immunohematology.

Rapid detection of JMH antibodies with recombinant Sema7A (CD108) protein and the particle gel immunoassay.

BACKGROUND: At present, identification of antibodies against the high-prevalence JMH antigen is difficult and limited to reference laboratories having panels of rare red blood cell (RBC) specimens in stock. Here, a novel method is described for detection of anti-JMH with particles coated with recombinant semaphorin 7A (Sema7A, CD108), the protein that carries the JMH blood group antigens. STUDY DESIGN AND METHODS: Recombinant Sema7A protein was generated and coupled onto superparamagnetic particles coated with streptavidin. The coated particles were tested in the presence of different serum and plasma samples (11 anti-JMH, 20 other antibodies, and 50 samples from nonimmunized blood donors) with the particle gel immunoassay and flow cytometry. RESULTS: Sema7A-coated particles reacted with all 11 samples containing anti-JMH, but not with samples lacking anti-JMH. In addition, the anti-JMH agglutination scores were higher with Sema7A-coated particles than with JMH-positive RBCs in all cases. CONCLUSION: Recombinant blood group proteins have the potential to replace RBCs as antigen carriers for identification of certain RBC alloantibodies.

Rapid detection of anti-Lu(b) with recombinant Lu(b) protein and the particle gel immunoassay.

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.