Inge von Zabern

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.

The BloodGen project: toward mass-scale comprehensive genotyping of blood donors in the European Union and beyond.

Neil D. Avent, Antonio Martinez, Willy A. Flegel, Martin L. Olsson, Marion L. Scott, Núria Nogués, Martin Písǎcka, Geoff Daniels, Ellen van der Schoot, Eduardo Muñiz-Diaz, Tracey E. Madgett, Jill R. Storry, Sigrid H. Beiboer, Petra A. Maaskant-van Wijk, Inge von Zabern, Elisa Jiménez, Diego Tejedor, Mónica López, Emma Camacho, Goedele Cheroutre, Anita Hacker, Pavel Jinoch, Irena Svobodova, and Masja de Haas

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.

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 drugs 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.

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.

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.

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.

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

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.

IVS5-38del4 deletion in the RHD gene does not cause a DEL phenotype: relevance for RHD alleles including DFR-3.

We read with interest the report by Lee and colleagues regarding the molecular basis of KEL29 (KALT) and KEL30 (KTIM), two novel high-prevalence antigens in the Kell blood group system. KEL29 antigen was identified in a Mexican woman with a homozygous 1988G>A polymorphism in exon 17, which is predicted to encode lysine instead of arginine at amino acid position 623. Interestingly, her serum sample contained an alloantibody identified as anti-KEL29 (anti-KALT) that did not agglutinate trypsin-treated RBCs. KEL30 antigen was identified in an American woman with history of transfusion and pregnancies with a homozygous 1033G>A polymorphism in exon 8, which is predicted to encode asparagine instead of aspartic acid at amino acid position 305. Her serum sample contained anti-KEL30 (anti-KTIM), which agglutinated against papain, trypsin, or a-chymotrypsin–treated RBCs. Because there are no data about the frequency of the KEL*29 and KEL*30 alleles in different populations, we performed a study to determine the frequency of the wildtype and variant KEL*29 and KEL*30 alleles in Brazilian persons. Employing the PCR-TfiI-RFLP and PCR-TaqIRFLP techniques described by Lee and coworkers, we studied 300 random blood donors representing a Brazilian population with a high rate of racial admixture and 68 Brazilian patients of African descent with hemoglobinopathies (45 HbSS, 4 HbS-thal, 15 HbSC, and 4 HbCC). All 300 donors (100%) and 68 patients (100%) were homozygous for wild-type consensus nucleotides at the KEL*29 and KEL*30 positions. No variant 1988G>A KEL*29 or 1033G>A KEL*30 alleles were identified among the 736 alleles that were analyzed. The Kell system is distinguished by its complexity and at present includes 27 known antigens, which may be classified into five antithetical sets of highand low-incidence antigens: KEL1 (K) and KEL2 (k); KEL3 (Kp), KEL4 (Kp), and KEL21 (Kp); KEL6 (Js) and KEL7 (Js); KEL11 and KEL17; and KEL14 and KEL24. In addition, there are 16 independently expressed antigens, 4 low-incidence (KEL10 [Ul], KEL23, KEL25 [VLAN], KEL28 [VONG]), and 12 high-incidence (KEL5 [Ku], KEL12, KEL13, KEL16, KEL18, KEL19, KEL20 [Km], KEL22, KEL26 [TOU], KEL27 [RAZ], KEL29 [KALT], KEL30 [KTIM]). The low-incidence antigens reflect single-nucleotide polymorphisms in the KEL exons and some of them show ethnic or racial specificity. Kell antigens are important in transfusion medicine owing to their strong immunogenicity. Alloantibodies to Kell antigens can cause either hemolytic transfusion reactions or hemolytic disease of the newborn. Most of these alloantibodies are directed against KEL1 and other low-incidence Kell antigens. Alloantibodies to high-incidence Kell antigens have been reported, however. Our data suggest that the KEL29 and KEL30 antigens have little clinical impact, because the alloimmunization by these antigens is probably a very rare event, restricted to a few cases involving subjects with KEL: -29 or KEL: -30 phenotype. Edmir Boturão-Neto, MD José O. Bordin, MD, PhD Discipline of Hematology and Hemotherapy Federal University of Sao Paulo Escola Paulista de Medicina São Paulo, SP, Brazil e-mail: jobordin@hemato.epm.br

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

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.