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

Abstract

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,

Characterization of RHD alleles present in serologically RHD‐negative donors determined by a sensitive microplate technique

“…The serologic D antigen typing currently used in our donor routine assured good sensitivity to detect weak expressions of D, since no weak D type was molecularly detected among D‐negative donors. Weak D type 38 and 11, which are common in Brazilians, have low antigen density and can be typed as D‐negative in donor screening, as shown by other studies conducted in the Brazilian population . Mota et al detected weak D alleles in 0·57% of D‐negative donors, without restricting the RhCE phenotype and Costa et al showed that 2·88% of D‐C+ donors carried weak D alleles, with RHD*01W.38 being responsible for 73% of cases.…”

RHD-Positive Alleles among D- C/E+ Individuals from India

“…Although the main RHD alleles resulting in a D- phenotype, i.e. RHDΨ and (C)ce s , are most frequently cis -associated with RHCE*ce , other RHD gene variants in D- individuals, including several hybrid genes, have been shown to segregate with RHCE alleles expressing C and/or E (C/E+) antigens in Caucasian, Asian, African, and mixed populations [4,7,8,9,10,11,12,13,14,15,16,17,18,19,20]. …”

RHD*Weak D Type 38: A Family Study

“…In conclusion, this study found a relatively high prevalence of RHD*weak D type 38 in Brazilians, remembering that Brazil was colonized by the Portuguese. 6,7 This is the first report showing the antigen densities of this weak D type with different MoAbs. The low D density found (60-80 sites/cell) could explain why weak D type 38 subjects are routinely mistyped as D negative by standard serologic methods including IAT.…”

“…This RHD allele shows reduced expression of the D antigen on the RBC surface and may be erroneously typed as D negative by standard serologic methods. [4][5][6] The frequencies of weak D type 38 vary in different populations. The weak D type 38 was found in * Corresponding author at: Rua Diogo de Faria, 824, 04023-061 São Paulo, SP, Brazil.…”

“…1.5% of Caucasians with the Ccee phenotype, 4 while a relatively higher frequency of 2.6% was recently detected among Brazilians with the Ccee phenotype. 6 Although the RHD*weak D type 38 is considered a very rare allele, it is relatively common in the Portuguese population. 7 In this study we analyzed three generations of a family with RHD*weak D type 38 using serologic and molecular methods.…”