Rapid identification and detection of parasitized human red cells by automated flow cytometry

Rapid and reliable identification of various human red cells parasites is important in many chemotherapeutic and immunologic studies. Because manual microscopic counting is tedious and imprecise, we have developed a simple diagnostic procedure for the automated flow cytometric detection of in vitro infected red cells, using a nucleic acid-binding fluorescent dye, acridine orange. Human malaria (Plasmodium falciparum)-infected red cells from continuous human erythrocyte culture were incubated at room temperature in acridine orange stain for 5 min after which the samples were analyzed by flow cytometry. Since mature red cells contain no DNA, infected red cells were identified with a distinct fluorescent signal. A total of 200,000 cells per sample were counted and analyzed in less than 2 min. Rings, trophozoites, and schizonts were assessed and identified in synchronized infected red cell cultures by flow cytometry. In addition, various stages of infected red cells were isolated with a cell sorter. This rapid method permits accurate and reliable assessment of data with the exclusion of anomalous data such as damaged cells, extraneous material, and contaminating particles.