Enumeration of the Invasion Efficiency of Plasmodium falciparum In Vitro in Four Different Red Blood Cell Populations Using a Three-Color Flow Cytometry-Based Method.

A novel in vitro culture system using variable concentrations of biotin/streptavidin to label red blood cells (RBCs) that allows for the simultaneous comparison of growth rates in Plasmodium falciparum malaria parasite in four heterogeneous target RBC populations is described. Donor RBCs containing both P. falciparum-infected RBCs and non-infected RBCs at 0.5% parasitemia were first labeled with 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE) followed by co-culture with a mixture of equal numbers of four differentially biotin/streptavidin labeled RBC populations. After two to three schizogonic growth cycles, co-cultures were harvested and stained with streptavidin-phycoerythrin (SA-PE) followed by staining of parasite-infected RBCs with nucleic acid fluorochrome SYBR Green I. To demonstrate the application of this method, some target RBC populations that had sialic acid residues removed using neuraminidase treatment were mixed with RBC populations without enzymatic treatment and incubated with donor parasitized RBCs strain W2 (sialic acid-dependent) or 3D7 (sialic acid-independent). Significant less susceptibility to malaria parasite invasion was obtained with enzyme-treated RBC populations when compared with non-treated RBCs in blood samples from the same individual when using malaria parasite strain W2, whereas no difference in percent parasitemias was noted following infection with malaria parasite strain 3D7. This novel malaria culture method is cheap and provides increased sensitivity for direct comparison of parasite growth over time of any of the four RBC populations under identical conditions and eliminates the experimental bias due to contaminated donor RBCs. The application of biotin-labeled RBCs will therefore provide a better understanding of invasion phenotype-specific host-parasite interactions and the extent of complex malaria invasion mechanism. © 2019 International Society for Advancement of Cytometry.