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

Abstract

Rapid and reliable identification of various human red cell 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 fakiparum)-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 distinctThe advent of continuous erythrocyte culture of human Plasmodium falciparum malaria in 1976 made possible subsequent immunologic, pharmacologic, and biochemical investigation of host-parasite relations. Simultaneously, the problems and chores associated with parasite counting have become obvious. These tasks are tedious and subject to human error and fatigue and to peculiarities of slide preparations with maldistribution of infected red cells in very thin or very thick areas of a blood film. With the popularization, clinical applicability, and access of flow cytometry, it is now possible to study parasite counting by flow cytometry to improve precision, speed, and reliability.Other investigators (1, 4-6) have reported on flow cytometric studies of intracellular red cell parasites using other dyes and instruments. One group had attempted unsuccessfully the use of acridine orange (AO), a nucleic acid-binding dye, for plasmodia1 studies (7). We now report a rapid automated instrumentation method of identifying the percentage of P. falciparum-infected red cells in culture using acridine orange incorporated in complex nutrient media.We chose A 0 because this dye is well established in studies of flow cytometry of nucleated blood elements 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.Key terms: Automated flow cytometry. acridine orange, population identification, human malaria-infected red cells (2). In our system, the mature red cell lacks DNA and RNA and will be unstained by AO, whereas the parasitized red cell has DNA and RNA and will bind A 0 and fluorescence on exposure to 488-nm light. Moreover, the intensity of fluorescence is proportional to the amount of parasite DNA and RNA present in the red cell. Thus the result obtained are both quantitative and qualitative. MATERIALS AND METHODSCells for these studies were obtained from continuous long term culture of group 0 + human red cells infected wi...

Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO‐1

“…Recognizing these needs, many flow cytometric assays using different DNA-specific dyes have been evaluated. Several reports show YOYO-1 is better than Hoechst 33258 to easily differentiate between uninfected and infected RBC when parasitaemia is low (6,11). Our published data by using FCM indicated that the infected rat erythrocytes with YOYO-1 can be completely separated from uninfected erythrocytes because of its high relative fluorescence intensity of the dye.…”

“…Thus, if the parasite is inside the cell, its DNA can be stained with specific nucleic acid-binding dyes and detected by flow cytometry (FCM). Different dyes, such as Hoechst 33258 (5), acridine orange (6), thiazole orange (7), or hydroethidine (8), have been considered for the determination of parasitemia in cultures of P. falciparum by FCM. Jacobberger et al (9) used DiOC1, a membrane potential respon-sive dye and Hoechst 33342 to evaluate parasitemia levels in mice (7).…”

Plasmodium species: Flow cytometry and microfluorometry assessments of DNA content and synthesis

“…Flow cytometry of malaria parasites has appeared to be a sensitive and rapid method to detect, count, and sort cells, infected with Plasmodium spp., after staining of their DNA with fluorescent dyes (Howard and Battye 1979;Brown et al 1980;Saul et al 1982;Myler et al 1982;Whaun et al 1983;Franklin et al 1986). However, the use of this method to establish DNA contents of different stages and the rate of DNA synthesis in parasite populations has been hampered by asynchronicity of infections, presence of multiple infected host cells, and lack of knowledge concerning the process of DNA synthesis in malaria parasites.…”

Improvement of detection specificity of Plasmodium‐infected murine erythrocytes by flow cytometry using autofluorescence and YOYO‐1

“…However, they require flow cytometers equipped with ultraviolet lasers that are not available in most laboratories. Nonspecific DNA staining agents, such as thiazole orange (7), acridine orange (8)(9)(10), ethidium bromide (11), propidium iodide (12), or hydroethidine (13)(14)(15), have also been used to stain nucleic acids from infected erythrocytes. Recently, YOYO-1, a cyanine dimer, has been added to this list (16).…”