RHD alleles and D antigen density among serologically D− C+ Brazilian blood donorsDear 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 anti-D MoAbs, by gel cards using anti-D MoAbs IgG (clone ESD1) (DiaMed, Latino América S.A., Brazil) and by haemagglutination Correspondence: Sidneia Costa,
Introduction: The RH blood group system is the most polymorphic and immunogenic among blood groups and some rare Rh phenotypes are found exclusively in the black population. The RH 18 phenotype (Hr-negative) that is one of these phenotypes is characterized by a high incidence antigen. The production of anti-RH 18 imposes to use equivalent rare antigen-negative red blood cells (RBCs), or deleted-Rh RBCs (Rhnull or D- -) for transfusion. Case Report: A 24-year-old female patient with two previous abortions and one ectopic pregnancy and miscarriage had to be transfused with a RBC unit. She was typed as blood group A, Dccee phenotype with weak D, positive RBC antibody screening, auto-control negative and TAD negative. The antibody was anti-e (title 1/64) determined by testing serum against a commercial RBC panel (Fresenius-Kabi) by IgG indirect antiglobulin test tube (PEG-IAT), and nontreated (LissCoombs) and papain-treated RBCs on a gel matrix (DiaMed, Latino America). After adsorption of serum on Rhe-negative RBC (DccEE), the retrieving antibody was an anti-e-like antibody reacting with all normal Rhe-positive RBCs. Molecular and serologic Rh typing was also performed on blood samples obtained from five relatives of the patient and her husband. Results: Patient and relatives cDNA sequence of gene RHD and RHCE transcripts were analyzed for RHCE exons 4 to 5 and exon 6; for RHD exons 4 to 5, 6 and 7. Sequencing of cDNA from the patient, her father and her sister showed in RHD the presence of the DAR allele point mutation 602 C>G in exon 4; 667 T>G and 744 C>T in exon 5; 957 G>A and 1025 T>C in exon 7; while on gene RHCE we found the ceAR allele carrying 712 A>G, 733 C>G e 787 A>G in exon 5. Hemoglobin tests revealed that the patient, her father and sister had a sickle cell trait (patient HbA1=55%; HbA2=2,8%; HbS=41%; HbF=1,2%; father HbA1=58,5%; HbA2=1,4%; HbS=37,7%; HbF=2,4%; sister HbA1=57,4%; HbA2=2,5%; HbS=38,6%; HbF=1,5%). Discussion: The family study showed that the haplotype DAR-ceAR was inherited from her father. Individuals carrying the ceAR allele may produce anti-hrs antibody that is defined as a Rhe-like which does not react with rare Rhe positive variant RBCs. The antibody may be clinically significant and represents a great risk for those patients who are not transfused with a similar rare phenotype variant. Serologically, DAR has shown weaker reactions with anti-D, it is characterized by complete loss of at least 9 of 37 RhD epitopes, and may produce anti-D. Both mutated alleles show a replacement of RHD exons by RHCE counterparts.
rev bras hematol hemoter. 2 0 1 6;3 8(1):79-81 w w w . r b h h . o r g
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