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

Background and objectives Weak D phenotypes with very low antigen densities and DEL phenotype may not be detected in RhD typing routine and could be typed as D‐negative, leading to D alloimmunization of D‐negative recipients. The present study aimed to investigate the presence of RHD‐positive genotypes in blood donors typed as D‐negative by an automated system using the solid‐phase methodology as a confirmatory test. Methods Two screenings were performed in different selected donor populations. For the first screening, we selected 1403 blood donor samples typed as D‐negative regardless of the CE status, and in the second screening, we selected 517 donor samples typed as D‐negative C+ and/or E+. RhD typing was performed by microplate in an automated equipment (Neo‐Immucor®), and the confirmatory test was performed by solid‐phase technique using Capture R® technology. A multiplex PCR specific to RHD and RHDψ was performed in a pool of 6 DNA samples. Sequencing of RHD exons was performed in all RHD‐positive samples, and a specific PCR was used to identify the D‐CE(4‐7)‐D hybrid gene. Results and conclusion No weak D type was found in either screening populations. Additionally, 353 (18·4%) D‐negative samples presented previously reported non‐functional RHD genes, 2 samples had a DEL allele, and 6 samples demonstrated new alleles, including one novel DEL allele. Our study identified six new RHD alleles and showed that the inclusion of a confirmatory test using serological methodology with high sensitivity can reduce the frequency of weak D samples typed as D‐negative.