A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum

Abstract

Variability in the ability of the malaria parasite Plasmodium falciparum to invade human erythrocytes is postulated to be an important determinant of disease severity. Both the parasite multiplication rate and erythrocyte selectivity are important parameters that underlie such variable invasion. We have established a flow cytometry-based method for simultaneously calculating both the parasitemia and the number of multiply-infected erythrocytes. Staining with the DNA-specific dye SYBR Green I allows quantitation of parasite invasion at the ring stage of parasite development. We discuss in vitro and in vivo applications and limitations of this method in relation to the study of parasite invasion.

Figure 1. Accuracy of flow cytometry determined assessment of first round invasion parasitemia

(A) Flow cytometry provides accurate determinations of P. falciparum parasitemia. There is a high correlation between microscopy- and flow cytometry-determined parasitemia. This representative experiment depicts an R-squared value of 0.9925 between the two methods. Individual data points are the average of triplicate samples and error bars represent the standard deviation. (B) Flow cytometry resolves singly and multiply infected erythrocytes. Following staining with SYBR Green I, 100,000 events were counted and three peaks emerge, corresponding to singly, doubly, and triply infected erythrocytes. (C) Parasitemia within singly, doubly, and triply infected erythrocytes was measured by flow cytometry and microscopy. The resulting parasitemia calculated by both methods was highly correlated and highly reproducible between experiments.

Figure 2. Accuracy of flow cytometry determined assessment of in vitro parasitemia and first round invasion parasitemia ex vivo from Senegalese patient isolates

(A) Flow cytometry provides accurate determinations of ex vivo invasion assay P. falciparum parasitemia. Three representative Senegalese samples are shown to demonstrate the similarity between microscopy- and flow cytometry-determined parasitemia. Individual data points represent the average of duplicate samples and error bars represent the range. (B) Following staining with SYBR Green I, 100,000 events were counted for both in vivo (initial) and ex vivo (re-invasion) parasitemia. For in vitro parasitemia, four peaks are observed, corresponding to singly, doubly, and triply, and quadruply infected erythrocytes. (C) Parasitemia within singly, doubly, triply, and quadruply infected erythrocytes was measured by flow cytometry and microscopy. The resulting parasitemia calculated by both methods was highly correlated for a given patient isolate.