Serologic reactivity and molecular analysis of samples with variant d phenotype

TRUCCO BOGGIONE, CAROLINA; LUJÁN BRAJOVICH, MELINA ELIANA; RACCA, AMELIA; COTORRUELO, CARLOS

Cancún

Congreso; XXXIIst International Congress of the ISBT; 2012

International Society of Blood Transfusion

Background: The D antigen is the most immunogenic of the Rh system. The possibility of D variants (Dvar) phenotypes must be considered if there is a discrepancy with different anti-D reagents, a reduced intensity of the hemagglutination reaction or if anti-D is detected in individuals typed D positive. The serologic assignment of the RhD status may be hindered in patients with a Dvar antigen. The implementation of molecular techniques has helped to identify alleles responsible for aberrant D phenotypes. Aim: The aim of this study was to characterize the molecular background of three D variant samples referred to our laboratory for D typing resolution. Materials and Methods: Serologic testing of patients? RBCs was performed to determine the complete Rh phenotype by standard tube hemagglutination technique including the indirect antiglobulin test (IAT). The D antigen status was evaluated with 4 commercially available monoclonal reagents: a blended IgM/IgG anti-D (clones TH-28 / MS-26), an IgM anti-D (clone MS-201), an IgM anti-D (clone RUM-1) and an IgM anti-D (clones LDM1 and ESD1M). Partial D phenotypes were evaluated using the Advanced Partial RhD Typing Kit (ALBAclone). Genomic DNA was isolated using a modified salting-out method. PCR SSP techniques were used to detect weak D types. Sequence analysis of the 10 RHD exons was also performed. Results: RBCs from the three samples were weakly reactive with all anti-D reagents by direct tube testing. The hemagglutination observed with the blended anti-D was enhanced by the IAT. The complete Rh phenotype of the three probands was Dvar, C-, c+, E+, e+. All samples reacted with the panel of 12 monoclonal anti-D reagents of the Advanced Partial RhD Typing Kit. The analysis of the genomic DNA sequence revealed a novel polymorphism characterized by the presence of the point mutation 359C>A in exon 3 of the RHD gene. Conclusions: We report a new RHD allele with a missense mutation that is responsible for the aminoacidic change Ala120Asp, predicted to be in the fourth transmembrane region of the RhD protein. Since the RBCs were reactive with all the anti-Ds used and taking into account the location of the point mutation, this allele could represent a weak D. The serologic and molecular results strongly suggest a genetic association between this new RHD variant and the RHcE allele. It is possible that difficulties of the polypeptide assembly to the erythrocyte membrane were responsible for the reduced expression of the D antigen. Our findings show that molecular studies are a necessary complement for characterizing the samples with variant D phenotypes. The RHD genotyping may contribute for clinical decision making in transfusion and obstetric medicine.