Reticulocyte tropism of P vivax is restricted to CD71⁺ reticulocytes. (A) Flow cytometry dot plot showing the CD71 and TO staining used to define the different erythrocyte subsets: TO⁺/CD71⁺ for the immature reticulocytes (green box), TO^low/CD71⁻ for the mature reticulocytes (red box), and TO⁻/CD71⁻ for the normocytes (black box). The insets on the right show the NMB-stained reticulocytes sampled from these gates by flow cytometry sorting. (B) Histogram showing the fold increase reinvasion, normalized to that measured for normocytes (TO⁻/CD71⁻) (mean 2.17 ± 1.23 standard deviation [SD] vs 9.47 ± 4.81 SD), for the 3 reticulocyte subsets harvested by cell sorting. Bar represents 8 μm. (C) Flow cytometry dot plot showing the reticulocyte sampling based on the CD71 expression (CD71⁻, CD71^low, CD71^medium, CD71^high). The top insets show each population stained by NMB. Note the red arrows in the right insets showing the newly reinvaded erythrocytes stained with Hoechst. (D) Histogram showing the fold increase reinvasion, normalized to that measured for the CD71⁻ population and for the 3 younger reticulocyte populations (mean CD71^low: 5.09 ± 1.75 SD; CD71^medium: 6.53 ± 2.41 SD; CD71^high: 7.94 ± 3.14 SD; n = 3). Note the red arrows in the right insets showing the newly reinvaded erythrocytes stained with Hoechst. Bar represents 8 μm. (E) Representative flow cytometry dot plot showing Hoechst and ethidium levels observed at the start (0 hours) and after 24 hours of the reinvasion assay with CD71⁻ erythrocytes, and CD71⁺ reticulocytes treated or not with anti-Duffy antibodies. For the 0-hour data, the black thick-lined squares show the percentage of schizonts; the black thin-lined squares show the percentage of rings before invasion. For the 24 hours of data, the black thin-lined squares at 24 hours show the percentage of unburst schizonts; the orange thick-lined squares show the percentage of rings after invasion. (F) The percentage of rings after reinvasion (n = 5). The value observed with CD71⁺ reticulocytes incubated with anti-Duffy antibodies was taken as the threshold of reinvasion (0.74%). hr, hour. *P < .05; **P < .01.