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Human erythrocytes are indispensable for  Plasmodium falciparum  development. Unlike other eukaryotic cells, there is no existing erythroid cell line capable of supporting long-term  P. falciparum  in vitro experiments. Consequently, invasion phenotyping experiments rely on erythrocytes of different individuals. However, the contribution of the erythrocytes variation in influencing invasion rates remains unknown, which represents a challenge for conducting large-scale comparative studies. Here, we used erythrocytes of different blood groups harboring different hemoglobin genotypes to assess the relative contribution of blood donor variability in  P. falciparum  invasion phenotyping assays. For each donor, we investigated the relationship between parasite invasion phenotypes and erythrocyte phenotypic characteristics, including the expression levels of surface receptors (e.g. the human glycophorins A and C, the complement receptor 1 and decay accelerating factor), blood groups (e.g. ABO/Rh system), and hemoglobin genotypes (e.g. AA, AS and AC). Across all donors, there were significant differences in invasion efficiency following treatment with either neuraminidase, trypsin or chymotrypsin relative to the control erythrocytes. Primarily, we showed that the levels of key erythrocyte surface receptors and their sensitivity to enzyme treatment significantly differed across donors. However, invasion efficiency did not correlate with susceptibility to enzyme treatment or with the levels of the selected erythrocyte surface receptors. Furthermore, we found no relationship between  P. falciparum  invasion phenotype and blood group or hemoglobin genotype. Altogether, our findings demonstrate the need to consider erythrocyte donor uniformity and anticipate challenges associated with blood donor variability in early stages of large-scale study design.

Blood donor variability is a modulatory factor for P. falciparum invasion phenotyping assays