PF4-associated parasite killing in clinical malaria samples. (A) Representative immunofluorescent images from Pf, Pv, Pk, and Pm patient blood smears illustrating PF4-associated parasite killing (PF4⁺TUNEL⁺ iRBCs). Scale bars, 5 µm. Arrows and arrowheads indicate platelets and parasites, respectively. Images were taken at 630× magnification on an Axio Scope A1 fluorescent microscope coupled to an Axiocam ICm-1 CCD camera, or an Axio Observer inverted fluorescence microscope coupled to an Axiocam 503 monochrome camera. ZEN 2 software was used for image acquisition and processing (all from Carl Zeiss, Germany). (B) Percentage of PF4⁺TUNEL⁺ parasites in clinical samples with Pf (Papua, n = 50; Sabah, n = 14), Pv (Papua, n = 32; Sabah, n = 13), Pm (n = 11), Pk (n = 15), and mixed species infection (n = 6). (C) Comparison of intraerythrocytic PF4 (PF4⁺) parasites as a percentage of dying (TUNEL⁺) parasites in Pf, Pv, Pm, Pk, and mixed species infection from Papua and Sabah (n as per B). (D) Inverse correlation of PF4⁺TUNEL⁺ iRBCs with parasitemia in Pf and Pv clinical samples (Spearman). (E) Proportions of PF4⁺TUNEL⁺ rings vs mature stages in Pv (rings, n = 15; mature, n = 16), Pm (rings, n = 6; mature, n = 9), and Pk (rings, n = 8; mature, n = 5). (F) Proportions of rings that were PF4⁺TUNEL⁺ and (G) PF4⁺ in Pf (Papua, n = 50; Sabah, n = 14), Pv (n = 15), Pm (n = 6), and Pk clinical samples (n = 8). Scatterplots indicate median ± interquartile range for each group. Parasitemia values are log transformed. Kruskal-Wallis or Mann-Whitney test used for statistical comparisons. Data presented in Table 4.