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

Abstract

Background: Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large-scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO-1 that are observed in infected erythrocytes compared with noninfected erythrocytes. Methods: Samples of blood from Plasmodium yoeliiinfected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO-1 in 96-well plate format. After acquisition, erythrocytes gated in loga-

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

“…After having analyzed 242 samples from infected rats in the experiments during our methodological evaluation, we found that most of the parasitemias read by FCM were, on average, 14% higher than the counts of microscopy. The differences observed may be explained by the fact that microscopy is a subjective technique and many cells that do not show a healthy morphology may be discarded during counting, whereas FCM detects parasites irrespective of their health status (12). In fact, we found some schizontlike cells attached to the membrane of normal RBCs (Fig.…”

“…Therefore, the nucleated blood cells can easily be distinguished from infected cells on the basis of the difference in the fluorescence intensity (25). These features make YOYO-1 an extremely sensitive nucleic acid dye compared with many of the aforementioned dyes, a property that can be very useful in FCM (12,13).…”

“…In addition, this compensation exhibits a chromatograph of cell peaks (Fig. 2, A3) of increasing fluorescence intensities in which the single ring (first orange peak at 65.7 units), multiple ring or trophozoites (second orange peak at 91.2 units), and schizonts (third orange peak at 107.8 units) are separated from each other (12,22).…”

“…The most important parameters to be optimized from the original staining procedures (11)(12)(13)(14) were the cell fixation and digestion of reticulocyte RNA in the malaria infected rodent model. The optimal concentration of glutaraldehyde to obtain maximum resolution between uninfected and P. falciparum infected human erythrocytes was assessed.…”

“…YOYO-1 has an extremely high affinity for DNA, and it can be excited at 488 nm, which is the excitation wavelength available from most lasers employed in FCM. Its bright fluorescence signal and low background make it ideal for flow cytometric analysis of stained malaria nucleic acids (11,12). Compared to our previous experience, the parasitized erythrocytes infected with P. berghei labeled with YOYO-1 can be completely separated from uninfected erythrocytes because of the increased relative fluorescence intensity.…”