Flow cytometry is a potentially efficient approach for the quantification of
parasitemias in experimental
malaria infections and
drug susceptibility assays using rodent
malaria models such as Plasmodium berghei. In this study, we used two red
DNA-binding
fluorochromes,
rhodamine 800 (R800) and LD700, to measure
parasitemia levels in whole blood samples from mice infected with P. berghei. Blood samples were treated with
RNAse A to eliminate
RNA-derived signals.
Propidium iodide, which stains both
DNA and
RNA, was used as a positive control. The
parasitemia levels determined by R800 and LD700 were comparable to those calculated by microscopic analysis of blood smears and flow cytometry using
Hoechst 33258.
RNAse treatment did not affect these measurements. We also used R800 or LD700 to quantify
parasitemias in mice infected with a GFP-expressing P. berghei line to correlate the
parasitemia levels determined by
DNA staining versus parasite numbers using GFP fluorescence as a surrogate measurement. A positive correlation was found between levels determined by flow cytometry using these
dyes and those measured by GFP expression. Similar results were obtained when
parasitemias determined by flow cytometry were compared to those determined by conventional microscopy. The limit of detection of infected red blood cells using R800 or LD700 staining was 0.1% and 0.15%, respectively. This study demonstrates that red
laser-based flow cytometry using R800 or LD700 can be used for effective quantification of
parasitemia levels in Plasmodium infected red blood cells. Furthermore, this method has the advantage that it does not require
RNAse pretreatment and allows for a greater amount of cells to be analyzed for parasite burden than otherwise measured by conventional microscopy. © 2011 International Society for Advancement of Cytometry.