Quantification and visualization of malaria-infected erythroblasts by imaging flow cytometry

Although malaria parasites are traditionally known to infect enucleated red blood cells, recent evidence-including our previous work-demonstrates their ability to invade nucleated erythroid precursors (erythroblasts). However, visualizing and quantifying parasitized erythroblasts (pEBs) in vivo remains challenging due to their rarity in peripheral blood and the limitations of existing techniques. In this study, we employed imaging flow cytometry (ImageStream) to identify and characterize pEBs in mice infected with Plasmodium yoelii 17XNL expressing green fluorescent protein and compared with conventional flow cytometry (FACS). At 14 days post-infection, splenic, bone marrow and peripheral blood cells were stained with antibodies against TER119 (erythroid marker) and MHC class I, which is expressed on nucleated host cells. To confirm the presence of nuclei, we also used Hoechst 33342 DNA staining. ImageStream analysis enabled simultaneous detection of GFP⁺TER119⁺MHC class I^hi Hoechst⁺ pEBs and morphological visualization. While no pEBs were detected in peripheral blood, a significant population was identified in the spleen and bone marrow. Importantly, quantification of pEB frequencies by ImageStream closely matched those obtained by FACS, validating the robustness of the imaging-based approach. Notably, ImageStream also revealed doublets and triplets involving pEBs and MHC class I⁺ cells, suggestive of erythroblastic islands or immunological interactions. Blebbing structures were occasionally observed, indicating apoptosis-like processes. These findings demonstrate that ImageStream offers a powerful platform for high-throughput, image-based detection of parasitized-erythroblasts in malaria. This approach may open new avenues for studying host-parasite interactions within hematopoietic tissues and their roles in parasite persistence and immune evasion.