A novel flow cytometry approach to quantify malaria-derived particles

Abstract

Quantifying malarial parasite density is crucial for diagnosis and treatment in endemic areas. While Malaria-derived particles (MDPs) have been linked to malaria pathology, a direct quantification method for routine laboratory use remains unestablished. To address this, our study optimized a flow cytometry approach to enumerate MDPs per microliter of blood. Specimens were incubated with propidium iodide and red blood cell (RBC) lysis solution. The number of MDPs was quantified using a CytoFlex flow cytometer, size-standard beads, and counting beads. Electron microscopy was used to study the ultrastructures of the malarial parasites in the lysed RBC specimens. A significant increase in MDP levels was detected in blood samples from P. falciparum and P. vivax infections, but fewer than 1 particle/μL of MDPs were detected in the controls. The number of MDPs correlated with the percentage of infected red blood cells (iRBCs) obtained by manual counting (R² = 0.94). The dilution assay demonstrated a strong correlation between the measured and expected values of the MDPs. An electron microscopic study demonstrated that different stages of malarial parasites exist in lysed RBCs in the form of membrane-bound spherical cells. A positive association was established between parasite density and MDPs across both P. falciparum (R² = 0.94) and P. vivax (R² = 0.91) infections. We demonstrated the potential use of flow cytometry for determining the MDP concentration. The developed approach is reliable and straightforward for the diagnosis and treatment of patients with malarial parasite infection in routine laboratory settings.