Ultrastructural and geometrical comparison of iRBCs and uRBCs from humans and M fascicularis monkeys with knowlesi malaria. (A) The sphericity (approximate aspect ratio) of iRBCs and uRBCs from 3 humans and 3 M fascicularis monkeys with knowlesi malaria, as determined by single-event flow cytometry (Amnis). In both species, sphericity was increased in iRBCs vs uRBCs. (In 3 humans, the median aspect ratio of iRBCs was 0.91, 0.87, and 0.93, respectively, vs 0.77, 0.84, and 0.84 for uRBCs, respectively; P < .0001. In 3 monkeys, the median aspect ratio of iRBCs was 0.87, 0.85, and 0.87, respectively, vs 0.83, 0.81, and 0.84 for uRBCs, respectively; P < .0001). Sphericity was increased in human iRBCs compared with monkey iRBCs (P < .0001). (B) Examples of images captured from the flow cytometer of P knowlesi iRBCs stained with Hoechst stain, ordered from earliest asexual forms (ring stage) to mature forms (schizonts). (C) Atomic force scans of P knowlesi iRBCs from humans (upper 4 images) and M fascicularis monkeys (lower 4 images) demonstrating increased sphericity of the human iRBCs. In each group of 4 images, the bottom 2 images (side = 2 μm) represent the magnified area in the image directly above (side = 8 μm) indicated by a red square, and show caveola pits.