Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation.

Eculizumab inhibits the terminal, lytic pathway of complement by blocking the activation of the complement protein C5 and shows remarkable clinical benefits in certain complement-mediated diseases. However, several reports suggest that activation of C5 is not always completely suppressed in patients even under excess of eculizumab over C5, indicating that residual C5 activity may derogate the drug's therapeutic benefit under certain conditions. By using eculizumab and the tick-derived C5 inhibitor coversin, we determined conditions ex vivo in which C5 inhibition is incomplete. The degree of such residual lytic activity depended on the strength of the complement activator and the resulting surface density of the complement activation product C3b, which autoamplifies via the alternative pathway (AP) amplification loop. We show that at high C3b densities required for binding and activation of C5, both inhibitors reduce but do not abolish this interaction. The decrease of C5 binding to C3b clusters in the presence of C5 inhibitors correlated with the levels of residual hemolysis. However, by employing different C5 inhibitors simultaneously, residual hemolytic activity could be abolished. The importance of AP-produced C3b clusters for C5 activation in the presence of eculizumab was corroborated by the finding that residual hemolysis after forceful activation of the classical pathway could be reduced by blocking the AP. By providing insights into C5 activation and inhibition, our study delivers the rationale for the clinically observed phenomenon of residual terminal pathway activity under eculizumab treatment with important implications for anti-C5 therapy in general.

An update on the CD59 blood group system.

CONCLUSIONS: This update of the CD59 blood group system (Weinstock C, Anliker M, von Zabern I. CD59: a long-known complement inhibitor has advanced to a blood group system. Immunohematology 2015;31:145-51) increases the number of reported patients with CD59 deficiency from 10 to 14. All of these 14 patients suffered from severe illness. Recently, a new variant allele was found in heterozygosity. Flow cytometry data suggest that this variant was expressed on the red blood cells of the propositus. Although additional alleles have been found, the CD59 system (International Society of Blood Transfusion system 35) continues to have one antigen.

Complement activation by human red blood cell antibodies: hemolytic potential of antibodies and efficacy of complement inhibitors assessed by a sensitive flow cytometric assay.

BACKGROUND: Therapeutic intervention strategies in complement-mediated hemolytic diseases are still inappropriate, and lethal events cannot be reliably prevented. As an in vitro model of intravascular hemolysis, a sensitive flow cytometric assay was designed using red blood cells (RBCs) of patients with paroxysmal nocturnal hemoglobinuria (PNH) as target cells. Complement activation by human allo- and autoantibodies directed against RBC antigens and the effect of different complement inhibitors were studied. STUDY DESIGN AND METHODS: RBCs of patients with a PNH III RBC clone of more than 20% were coated with different human allo- or autoantibodies. Hemolysis was initiated with pooled normal human AB serum with or without the addition of complement inhibitors. Loss of PNH III RBCs was estimated by flow cytometry. RESULTS: RBC antibodies of 174 different patients representing 37 different specificities were tested for their potency to activate complement. In correlation with blood group specificities roughly three different patterns were observed: 1) strong and regular, 2) sporadic, and 3) weak or absent complement activation. Remarkably strong complement activators were among antibodies directed against high-prevalence blood group antigens. The C5 inhibitor eculizumab abrogated mild but not strong complement activation, even in presence of excess inhibitor. However, this residual complement activity could be further depressed by combining eculizumab with other inhibitors. CONCLUSION: The PNH hemolysis assay offers a sensitive tool for in vitro analyses of classical pathway-mediated complement activation. The recognition of additive effects of complement inhibitors may guide novel intervention strategies against unwanted complement damage.

CD59: A long-known complement inhibitor has advanced to a blood group system.

The blood group system number 35 is based on CD59, a 20-kDa membrane glycoprotein present on a large number of different cells, including erythrocytes. The major function of CD59 is to protect cells from complement attack. CD59 binds to complement components CS and C9 and prevents the polymerization of C9, which is required for the formation of the membrane attack complex (MAC). Other functions of CD59 in cellular immunity are less well defined. CD59 is inserted into the membrane by a glycosylphosphatidylinositol (GPI) anchor. A defect of this anchor causes lack of this protein from the cell membrane, which leads to an enhanced sensitivity towards complement attack. Patients with paroxysmal nocturnal hemoglobinuria (PNH) harbor a varying percentage of red blood cell clones with a defect in GPI-anchored proteins, including CD59. The most characteristic symptoms of this disease are episodes of hemolysis and thromboses. Although CD59 has been classified as a membrane protein for more than 25 years, an alloantibody directed against CD59 was found only recently. So far, the first and sole alloantibody described was detected in a CD59-deficient child. In 2014, CD59 received the status of a blood group system by the International Society for Blood Transfusion Red Cell Immunogenetics and Blood Group Terminology Working Party. Among a variety of almost 20 synonyms, the designation CD59 was chosen for the blood group system and CD59.l for the wild-type protein. The only three alleles published to date are null alleles. All CD59-deficient individuals recognized so far were severely ill, two of whom have died. Most of the reported cases present with a typical clinical picture within the first year of life that includes neuropathy, strokes, and mild Coombs-negative hemolysis. In one published case, the application of the complement inhibitor eculizumab caused a pronounced improvement of the clinical situation.

A new blood group antigen is defined by anti-CD59, detected in a CD59-deficient patient.

BACKGROUND: CD59 is a cell surface glycoprotein of approximately 20 kDa limiting the lytic activity of the terminal complement complex C5b-9. Although CD59 is known as a red blood cell (RBC) antigen defined by monoclonal antibodies, it so far has not been identified as a blood group antigen, since the description of a human alloantibody was missing. In this study we show the presence of an anti-CD59 in a patient affected by a homozygous CD59 deficiency. STUDY DESIGN AND METHODS: RBC CD59 and CD55 were determined by flow cytometry or by the column agglutination technique using monoclonal antisera. Commercially available His-tagged recombinant soluble CD59 protein was used to inhibit anti-CD59. RESULTS: Seven cases of an isolated CD59 deficiency due to three distinct null alleles of the CD59 gene have been published so far. Recently we described the CD59-null allele c.146delA in a young child of heterozygous parents. Her plasma contained an alloantibody directed against the high-prevalence RBC antigen CD59. The antibody specificity was identified using soluble recombinant human CD59 protein, which blocked the reactivity of the patient's antibody and of monoclonal anti-CD59 but not of monoclonal anti-CD55. In addition, RBC alloantibodies such as anti-K, anti-C, anti-c, or anti-Fy(a) remained unaffected. Therefore, inhibition by recombinant CD59 is a useful diagnostic tool to detect alloantibodies in the presence of anti-CD59. CONCLUSION: This is the first demonstration of a human anti-CD59 alloantibody, which defines CD59 as an RBC blood group antigen. CD59 represents a candidate for a new blood group system.

D category IV: a group of clinically relevant and phylogenetically diverse partial D.

BACKGROUND: The D typing strategies in several European countries protect carriers of D category VI (DVI) from anti-D immunization but not carriers of other partial D. Besides DVI, one of the clinically most important partial D is D category IV (DIV). A detailed description and direct comparison of the different DIV types was missing. STUDY DESIGN AND METHODS: RHD nucleotide sequences were determined from genomic DNA. D epitope patterns were established with commercial monoclonal anti-D panels. RESULTS: DIV comprises several variants of the D antigen with distinct serology, molecular structures, evolutionary origins, and ethnic prevalences. The DIV phenotype is determined by 350H shared by all, but not limited to, DIV variants which are further divided into DIVa and DIVb. The DIVa phenotype is expressed by DIV Type 1.0 harboring 350H and the dispersed amino acids 62F, 137V, and 152T. The DIVb phenotype is expressed by DIV Type 3 to Type 5 representing RHD-CE-D hybrids. Four of the six postulated DIV variants were encountered among 23 DIV samples analyzed. Of 12 DIV carriers with anti-D, 10 were female and seven likely immunized by pregnancy. Two DIV-related alleles are newly described: DWN, which differs from DIV Type 4 by 350D and epitope pattern. DNT carries 152T, known to cause a large D antigen density. CONCLUSION: DIV alleles arose from at least two independent evolutionary events. DIV Type 1.0 with DIVa phenotype belongs to the oldest extant human RHD alleles. DIV Type 2 to Type 5 with DIVb phenotype arose from more recent gene conversions. Anti-D immunization, especially dreaded in pregnancies, will be avoided not only in carriers of DVI but also in carriers of other D variants like DIV, if our proposed D typing strategy is adopted.

Molecular basis of two novel and related high-prevalence antigens in the Kell blood group system, KUCI and KANT, and their serologic and spatial association with K11 and KETI.

BACKGROUND: The numerous antigens in the Kell blood group system result from missense nucleotide changes in KEL. Antibodies to antigens in this system can be clinically important. We describe six probands whose plasma contained antibodies to high-prevalence Kell antigens and discuss their relationship. STUDY DESIGN AND METHODS: Polymerase chain reaction amplification, direct sequencing, restriction fragment length polymorphism assays, hemagglutination, flow cytometry, and protein modeling were performed by standard methods. RESULTS: Proband 1 (KUCI) and her serologically compatible sister were heterozygous for a nucleotide change in Exon 11 (KEL*1271C/T; Ala424Val). Proband 2 (KANT) was heterozygous for KEL*1283G/T (Arg428Leu) and KEL*1216C/T (Arg406Stop) in Exon 11. Red blood cells (RBCs) from Proband 1 and her sister were not agglutinated by plasma from Proband 2; however, RBCs from Proband 2 were agglutinated by plasma from Proband 1. Probands 3, 4, 5, and 6 had the KEL*1391C>T change associated with the previously reported KETI- phenotype. Proband 5 was also homozygous for KEL*905T>C encoding the K11-K17+ phenotype. Hemagglutination studies revealed an association between KUCI, KANT, KETI, and K11. Protein modeling indicated that whereas Ala424 and Arg428 are clustered, Val302 and Thr464 are not. CONCLUSION: Ala424 in the Kell glycoprotein is associated with the high-prevalence Kell antigen, KUCI (ISBT 006032), which is detected by the antibody of Proband 1. Arg428 is associated with the high-prevalence Kell antigen, KANT (ISBT 006033). The association between KUCI, KANT, KETI, and K11 and the results of protein modeling are discussed.

Six years' experience performing RHD genotyping to confirm D- red blood cell units in Germany for preventing anti-D immunizations.

BACKGROUND: Red blood cell (RBC) units of D+ donors are falsely labeled D- if regular serologic typing fails to detect low D antigen expression or chimerism. The limitations of serology can be overcome by molecular typing. STUDY DESIGN AND METHODS: In January 2002, we introduced a polymerase chain reaction (PCR)-based assay for RHD as a routine test for first-time donors who typed D- by serologic methods including the indirect antiglobulin test. Samples were tested in pools of 20 for the RHD-specific polymorphism in Intron 4. RHD alleles were identified by PCR and nucleotide sequencing. RESULTS: Within 6 years, 46,133 serologically D- first-time donors were screened for the RHD gene. The prevalence of RHD gene carriers detected by this method was 0.21 percent. Twenty-three RHD alleles were found of which 15 were new. Approximately one-half of the RHD gene carriers harbored alleles expressing a DEL phenotype resulting in a prevalence of 0.1 percent. CONCLUSION: The integration of RHD genotyping into the routine screening program was practical. We report 6 years' experience of this donor testing policy, which is not performed in most transfusion services worldwide. RBC units of donors with DEL phenotype have been reported to anti-D immunize D- recipients. We transferred those donors to the D+ donor pool with the rationale of preventing anti-D immunizations, especially dreaded in pregnancies. For each population, it will be necessary to adapt the RHD genotyping strategy to the spectrum of prevalent alleles.

RhCE protein variants in Southwestern Germany detected by serologic routine testing.

BACKGROUND: Variant RHCE alleles with diminished expression of C, c, E, and e antigens have been described and indicate the genetic diversity of this gene locus in several populations. In this study the molecular background of variant RhCE antigens identified by standard serologic routine testing in German blood donors and patients was determined. STUDY DESIGN AND METHODS: Samples from blood donors and patients were routinely analyzed for RhCE phenotype using the PK7200 analyzer with two sets of monoclonal anti-C, -c, -E, and -e reagents. Samples with confirmed variant RhCE antigens were analyzed by nucleotide sequencing of the 10 RHCE exons. A multiplex polymerase chain reaction with sequence-specific priming (PCR-SSP) method was established for rapid typing of the rare RHCE alleles. RESULTS: We identified 43 samples with serologic RhCE variants. Molecular analysis revealed variant RHCE alleles in 34 samples. Altogether 22 RHCE alleles were detected; 10 have not been published before. Twenty alleles harbored distinct single-nucleotide substitutions, 18 of which encoded amino acid changes and 2 of which occurred in noncoding regions. Two samples represented RHCE-D-CE hybrid alleles involving different segments of the RHCE Exon 5. A multiplex PCR-SSP screening for 17 RHCE alleles was negative in 1344 samples of the DNA bank GerBS. The cumulative phenotype frequency was estimated between 1 in 488 (0.20%) and 1 in 8449 (0.012%). CONCLUSION: Single-amino-acid substitutions were the molecular basis for variant RhCE antigen expression in most samples. Nucleotide substitutions in RHCE exons were excluded as possible mechanism of diminished RhCE antigen expression in one-fifth of the serologically identified samples.

D variants at the RhD vestibule in the weak D type 4 and Eurasian D clusters.

BACKGROUND: One branch of the RHD phylogenetic tree is represented by the weak D type 4 cluster of alleles with F223V as the primordial amino acid substitution. F223V as well as a large number of further substitutions causing D variants are located at the extracellular RhD protein vestibule, which represents the entrance to the transmembraneous channel of the RhD protein. STUDY DESIGN AND METHODS: RHD and RHCE nucleotide sequences were determined from genomic DNA and cDNA. D epitope patterns were established with commercial monoclonal anti-D panels. RESULTS: The RHD alleles DOL-1 and DOL-2 had the two amino acid substitutions M170T (509T>C) and F223V (667T>G) in common. DOL-2 harbored the additional substitution L378V (1132C>G). Both alleles were observed in Africans and are probably evolutionary related. DMI carried M170I (510G>A), which differed from the DOL-typical substitution. DFW and DFL harbored the substitutions H166P (497A>C) and Y165C (494A>G). The antigen densities of DOL-1, DFL, and DFW were only moderately reduced. CONCLUSION: DOL-1 and DOL-2 belong to the weak D type 4 cluster of RHD alleles. Together with DMI, DFL, and DFW they represent D variants with amino acid substitutions located at extracellular loops 3 or 4 lining the RhD protein vestibule. These substitutions were of minor influence on antigen density while adjacent substitutions in the transmembraneous section caused weak D antigen expression. All these D variants were partial D and alloanti-D immunizations have been observed in DOL-1, DMI, and DFL carriers. The substitution at position 170 causes partial D although located deep in the vestibule.

The Bloodgen Project of the European Union, 2003-2009.

The Bloodgen project was funded by the European Commission between 2003 and 2006, and involved academic blood centres, universities, and Progenika Biopharma S.A., a commercial supplier of genotyping platforms that incorporate glass arrays. The project has led to the development of a commercially available product, BLOODchip, that can be used to comprehensively genotype an individual for all clinically significant blood groups. The intention of making this system available is that blood services and perhaps even hospital blood banks would be able to obtain extended information concerning the blood group of routine blood donors and vulnerable patient groups. This may be of significant use in the current management of multi-transfused patients who become alloimmunised due to incomplete matching of blood groups. In the future it can be envisaged that better matching of donor-patient blood could be achieved by comprehensive genotyping of every blood donor, especially regular ones. This situation could even be extended to genotyping every individual at birth, which may prove to have significant long-term health economic benefits as it may be coupled with detection of inborn errors of metabolism.

DCS-1, DCS-2, and DFV share amino acid substitutions at the extracellular RhD protein vestibule.

BACKGROUND: RhD and RhCE are structurally related to ammonium transporter proteins, yet their physiologic function remains unclear. Recent three-dimensional homology modeling with Escherichia coli AmtB as a template defined a putative transmembraneous channel. Three RhD variants with amino acid substitutions located at the extracellular channel aperture are described. STUDY DESIGN AND METHODS: Blood samples were selected because of serologic abnormalities. RHD, RHCE, and LW nucleotide sequences were determined from genomic DNA. D epitope patterns were established with monoclonal anti-D panels. Three-dimensional Rh structures were calculated by alignment to AmtB. RESULTS: The RHD allele DCS-1 was found to carry the two amino acid substitutions F223V (667T > G) and A226P (676G > C) caused by missense mutations in RHD exon 5. This study compared DCS-1 with the D variants DFV (F223V) and DCS-2 (A226P), harboring solely one or the other of the two substitutions. All three D variants were associated with a cDE haplotype. The antigen densities were approximately 3,000 D antigens per red blood cell for DCS-1, 800 for DCS-2, and 9,300 for DFV. DCS-1 and DCS-2 were partial D, because they lacked distinct epitopes. DFV presented as an almost normal D phenotype; the sample contained allo-anti-LW(a). The D(w) antigen was absent from DCS-1, DFV, and DAU-4, but expressed by DAU-5. CONCLUSION: DCS-1, DCS-2, and DFV carry amino acid substitutions at the extracellular vestibule, visualized by 3-dimensional modeling. Proline at position 226 greatly influenced the D antigen density and may reduce the RhD membrane integration. Although the F223V substitution is regarded as the initial event in the evolution of the weak D Type 4 cluster, the current DFV allele probably evolved independently, as evident from different RHCE haplotypes.