Resolution of Haplotype Ambiguities in Transfusion Settings.

Red blood cell (RBC) transfusion, limited by patient alloimmunization, demands accurate blood group typing. The Rh system requires specific attention due to the limitations of serological phenotyping methods. Although these have been compensated for by molecular biology solutions, some RhCE ambiguities remain unresolved. The mRNA length is compatible with full-length analysis and haplotype discrimination, but the mRNA analyses reported so far are based on reticulocyte isolation and molecular biology protocols that are fastidious to implement in a routine context. We aim to present the most efficient reticulocyte isolation method, combined with an RT-PCR sequencing protocol that embraces the phasing of all haplotype configurations and identification of any allele. Two protocols were tested for reticulocyte isolation based either on their size/density properties or on their specific antigenicity. We show that the reticulocyte sorting method by antigen specificity from EDTA blood samples collected up to 48 h before processing is the most efficient and that the combination of an -specific RT-PCR followed by allele-specific sequencing enables analysis of cDNA haplotypes. All samples analyzed show full concordance between phenotype and haplotype sequencing. Two samples from the immunohematology laboratory with ambiguous results were successfully analyzed and resolved, one of them displaying a novel allele (*03 c.340C>T).