American Society of Hematology

Fig. 3.

$Fig. 3. Osmotic fragility curves of IRBCs and noninfected (cohorts) cells from the same. / P falciparum culture. Hemolysis was induced by delivery of 10 μL cell suspension (5% or 10% hematocrit) to each of 24 wells containing 250 μL lysing media with different tonicities (“Materials and methods”). The lysing media were prepared by mixing 2 solutions: one containing 150 mM NaCl and 2 mM HEPES-Na, pH 7.5, equiosmolal with the culture medium used, regarded as relative tonicity (RT) = 1, and the other containing 2 mM HEPES-Na, pH 7.5, assumed equivalent to RT approximately 0. The experiment was performed on a nonsynchronized culture containing 18% parasitized cells. Separation of IRBCs and cohorts was done by gelatin flotation (“Materials and methods”). Parasitemia in IRBC and cohort fractions was 96% and 3.2%, respectively. Differential counts (500 cells) in IRBCs rendered 5% rings, 20% young trophozoites, 35% mature trophozoites, 36% schizonts plus segmentors, and 4% uninfected cells, whereas in the cohort fraction, 81% of the 3.2% IRBCs were rings. The y-axis reports percentage of Hb released by cell lysis. For cohorts, this is equivalent to percentage of cells lysed. For IRBCs, on the other hand, the Hb contribution from the cells with mature parasites is less than that from cells with younger parasites. Therefore, the right shift of the IRBC hemolysis curve relative to cohorts tends to underrepresent the true number of cells lysed. Inset: differential counts of unlysed IRBCs recovered from selected hemolysis wells (“Materials and methods”). After removal of residual lysis fluid, the cell pellets were resuspended in supplemented RPMI for smearing and staining. In all such recovered samples there was a variable abundance of lysed ghosts with stained parasite fragments. Differential counts (200 cells) were performed only on clearly recognizable intact cells. Differential counts were performed in 3 of the 6 experiments of this series in which crossover patterns were observed, with similar results to those shown in this inset.$

Osmotic fragility curves of IRBCs and noninfected (cohorts) cells from the same

P falciparum culture. Hemolysis was induced by delivery of 10 μL cell suspension (5% or 10% hematocrit) to each of 24 wells containing 250 μL lysing media with different tonicities (“Materials and methods”). The lysing media were prepared by mixing 2 solutions: one containing 150 mM NaCl and 2 mM HEPES-Na, pH 7.5, equiosmolal with the culture medium used, regarded as relative tonicity (RT) = 1, and the other containing 2 mM HEPES-Na, pH 7.5, assumed equivalent to RT approximately 0. The experiment was performed on a nonsynchronized culture containing 18% parasitized cells. Separation of IRBCs and cohorts was done by gelatin flotation (“Materials and methods”). Parasitemia in IRBC and cohort fractions was 96% and 3.2%, respectively. Differential counts (500 cells) in IRBCs rendered 5% rings, 20% young trophozoites, 35% mature trophozoites, 36% schizonts plus segmentors, and 4% uninfected cells, whereas in the cohort fraction, 81% of the 3.2% IRBCs were rings. The y-axis reports percentage of Hb released by cell lysis. For cohorts, this is equivalent to percentage of cells lysed. For IRBCs, on the other hand, the Hb contribution from the cells with mature parasites is less than that from cells with younger parasites. Therefore, the right shift of the IRBC hemolysis curve relative to cohorts tends to underrepresent the true number of cells lysed. Inset: differential counts of unlysed IRBCs recovered from selected hemolysis wells (“Materials and methods”). After removal of residual lysis fluid, the cell pellets were resuspended in supplemented RPMI for smearing and staining. In all such recovered samples there was a variable abundance of lysed ghosts with stained parasite fragments. Differential counts (200 cells) were performed only on clearly recognizable intact cells. Differential counts were performed in 3 of the 6 experiments of this series in which crossover patterns were observed, with similar results to those shown in this inset.

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