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

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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 yoelii–infected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO‐1 in 96‐well plate format. After acquisition, erythrocytes gated in logarithmic side/scatter plots were analyzed in bidimensional FL‐2/YOYO‐1 plots in comparison with unidimensional YOYO‐1 analysis.Results: The infected erythrocytes showed a characteristic pattern of staining different from that of noninfected erythrocytes. In routine evaluation, the limit of sensitivity was 0.01% and the measurements of parasitemia were linear at parasitemias above 0.1%. Interestingly, using this approach, infected reticulocytes could be differentiated from infected normocytes.Conclusions: The method described is robust, increases the specificity and sensitivity of detection in routine testing, and is especially well suited for detection of low parasitemias in murine models of malaria.