Re‐evaluating acridine orange for rapid flow cytometric enumeration of parasitemia in malaria‐infected rodents

Abstract

Methods facilitating research in malaria are of pivotal relevance. Flow cytometry offers the possibility of rapid enumeration of parasitemia. It relies on staining the parasite DNA to distinguish between infected and non-infected red blood cell (RBC) populations. Unfortunately, in rodents abundant reticulocyte RNA interferes with the application of the method. This results in time-consuming sample preparation protocols that offer no clear advantage over microscopic counting. We re-evaluated the use of the DNA/RNA discriminating vital fluorochrome acridine orange (AO) for rapid flow cytometric enumeration of parasitemia in rodents. Whole blood from rodents infected with Plasmodium berghei and Plasmodium yoelii was stained with AO and analyzed by flow cytometer. A newly developed two-channel (FL1/FL3) detection method was compared with conventional one-channel (FL1) detection and microscopic counting. The new AO two-channel detection method clearly discriminated between infected and non-infected RBC populations. It showed to be linear above parasitemias of 0.3%. Sample processing time amounted to approximately 5 min. It is shown that AO can be used for rapid, precise, and accurate enumeration of parasitemia in rodents. Due to its ease of handling the method might find widespread application in malaria research.

Considerations for point-of-care diagnostics: evaluation of acridine orange staining and postprocessing methods for a three-part leukocyte differential test

“…11,17 This has renewed the interest in the use of acridine orange (AO), a fluorescent vital dye that has been used in medical diagnostics since the 1950s for uses in diagnosing and detecting gynecological cancer, microorganisms in cerebrospinal fluid, and malaria. [29][30][31][32] AO only stains nucleated cells and, therefore, does not require additional reagents (ex. RBC lysis and dilution reagents), in contrast to other POC systems.…”

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds

“…Several methods of detection of malaria parasite by flow cytometry have been developed taking advantage of the absence of DNA in erythrocytes. Different dyes such as acridine orange (17,18), hydroethidine (19,20), SYTO 16 (21) and thiazole orange (22) were already employed for the determination of parasitemia in cultures of P. falciparum by FCM. Some previous studies using YOYO-1 showed that this dye possesses high affinity for DNA and discriminates infected RBC from the uninfected cells in a very effective way (14,23,24).…”

Feasibility of Flow Cytometry for Measurements of Plasmodium falciparum Parasite Burden in Studies in Areas of Malaria Endemicity by Use of Bidimensional Assessment of YOYO-1 and Autofluorescence

“…Recent improvements in parasite staining methods have identified means of excluding background from noninfected populations. The analysis of the emission in two different wavelengths of blood samples stained with a single dye allow for the greater characterization of infected and noninfected events by separating the infected erythrocytes from nucleic acid-containing noninfected erythrocytes (6,9,15,16,20,32). This method exploits the difference in autofluorescent patterns of erythrocyte subpopulations to distinguish reticulocytes from mature erythrocytes.…”

“…This pattern has been established in multiple mouse models of Plasmodia as well as P. falciparum, both from parasite cultures and in a humanized mouse model of malaria (2,15,16,20). Similar approaches using different dyes, such as acridine orange, have demonstrated promise but as yet have not shown superiority to YOYO-1 and suffer from issues such as hemolytic activity (6,9).…”