High-throughput multi-parameter flow-cytometric analysis from micro-quantities of Plasmodium-infected blood

Abstract Despite significant technological and conceptual advances over the last century, evaluation of the efficacy of anti-malarial vaccines or drugs continues to rely principally on direct microscopic visualisation of parasites on thick and/or thin Giemsa-stained blood smears. This requires technical expertise of the microscopist, is highly subjective and error-prone, and does not account for aberrations such as anaemia. Many published methods have shown that flow cytometric analysis of blood is a highly versatile method that can readily detect nucleic acid-stained parasitised red blood cells within cultured cell populations and in ex-vivo samples. However several impediments, including the difficulty in distinguishing reticulocytes from infected red blood cells and the fickle nature of red blood cells, have precluded the development and universal adoption of flow-cytometric based assays for ex-vivo sample analysis. We have developed a novel high-throughput assay for the flow cytometric assessment of blood that overcomes these impediments by utilising the unique properties of the nucleic acid stain DAPI to differentially stain RNA and DNA, combined with novel fixation and analysis protocols. The assay allows the rapid and reliable analysis of multiple parameters from micro-volumes of blood, including: parasitaemia, platelet count, reticulocyte count, normocyte count, white blood cell count and delineation of subsets and phenotypic markers including, but not limited to, CD4 + and CD8 + T cells, and the expression of phenotypic markers such as PD-L1 or intracellular cytokines. The assay requires less than one drop of blood and is therefore suitable for short interval time-course experiments and allows the progression of infection and immune responses to be closely monitored in the laboratory or cytometer-equipped field locations. Herein, we describe the technique and demonstrate its application in vaccinology and with a range of rodent and human parasite species including Plasmodium yoelii , Plasmodium chabaudi , Plasmodium berghei and Plasmodium falciparum.