The demand for donor blood is high, and it is essential to match transfusions between donors and recipients based on red blood cell (RBC) antigens and plasma antibodies to prevent hemolytic adverse reactions. For over 40 years, the Enzyme-Conversion to group O-like (ECO) RBC concept has been pursued with the aim of producing ABO-universal blood. Although A and B glycans can successfully be converted to their precursor structure, H antigen, incompatible crossmatches with many plasmas persist. To assess the cause of remaining reactivities, deeper understanding of RBC glycophenotypes is necessary.

In this study, differences in H levels on RBCs between group O donors (n=99) were investigated. Flow cytometry revealed extensive variation in this cohort and donors were tentatively classified into categories; one with low H levels, another with high H expression and the remaining donors in a medium category. A strong correlation was noted between the two different monoclonal anti-H reagents used, and statistical significance was observed between serological titer score and flow cytometric results for the three categories. Similarly, differences were also observed for H-reactive glycoproteins, using SDS-PAGE/Western blot. Neither donor age nor secretor status (defined by Lewis phenotype) significantly influenced H levels. However, a trend with decreasing p-values was noticed when analyzing three age-defined groups instead of six, so age may need further study. Sequencing of FUT1 exon 4 that encodes H-synthesizing fucosyltransferase showed no significant differences, and neither did FUT1-mRNA levels. Therefore, the underlying cause of H variation on RBCs remains unresolved. Suggested future approaches include sequencing FUT1 promoter/enhancer regions, focusing on binding sites for erythroid transcription factors like KLF1 and GATA1, digital PCR, and CD34+ erythroid differentiation from donors with different H levels. Lastly, understanding the consequences of H variation on ECO treatment will be crucial for enabling conversion of A/B/AB phenotypes into universal O blood.