RHD alleles and D antigen density among serologically D− C+ Brazilian blood donors

Dear Sir, The D-negative phenotype may be caused by the lack of functional RhD protein or by the presence of aberrant forms of RhD not expressing the D antigen. Numerous single nucleotide polymorphism (SNPs) changes in the RHD gene are currently known. The weak D has been shown to be caused by SNPs in most cases, leading to amino acid changes in the transmembraneous, or intracellular, parts of the D protein, consequently showing reduced D antigen density (Wagner et al., 1999; Wagner et al., 2000). Very weakly expressed weak D, or D only detectable by adsorption-elution techniques, named DELs, were frequently unidentified by routine serologic procedures, and large groups of completely unexpressed RHD alleles only became evident by DNA-typing methods (Gassner et al., 2005). Several studies have confirmed unexpressed RHD alleles in association with D − Ce or cE phenotypes (Gassner et al., 2005; Christiansen et al., 2010). The frequency of phenotypes dCcee and dccEe in the Brazilian population is approximately 0·6 and 0·2%, respectively (Cruz et al., 2012). At our Blood Centre, we consider C/E+ and D− blood donors as RhD-negative and therefore their blood is transfused into RhD-negative patients. Thus, in the present study we decided to investigate the molecular background in Brazilian blood donors phenotyped as D−C/E+. In addition, qualitative and quantitative analyses of the D antigen expressed by RBCs of D variants were performed. We investigated 520 blood donors from Southeast (São Paulo, SP) Brazil labelled D−C/E+ confirmed by the commercial anti-D Immunoglobulin G (IgG) (MS-26) + IgM (TH-28) and polyclonal antibodies against C, c, E and e (DiaMed, Latino América S.A., Brazil). The samples were screened by polymerase chain reaction (PCR) with sequence-specific primers for the presence of RHD in two genomic regions, intron 4 (primers RHI41 and RHI42) and exon 10 (primers EX10F, RHD3′-UTR and RHCE 3′-UTR). Samples identified as RHD gene-positive were sequenced in full length for all 10 RHD exons (Legler et al., 2001; Qun et al., 2005). The sequences were analysed using a sequencing Kit (Big Dye Terminator v1.1, applied Biosystems, Weiterstadt, Germany) and a genetic analyser (ABI 3100, Applied Biosystems, Foster City, CA, USA). The samples that have demonstrated the RHD variants by the molecular methods were further analysed with different antiD MoAbs, by gel cards using anti-D MoAbs IgG (clone ESD1) (DiaMed, Latino América S.A., Brazil) and by haemagglutination