High-Throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays

Abstract

Background: Unbiased flow cytometry-based methods have become the technique of choice in many laboratories for high-Throughput, accurate assessments of malaria parasites in bioassays. A method to quantify live parasites based on mitotracker red CMXRos was recently described but consistent distinction of early ring stages of Plasmodium falciparum from uninfected red blood cells (uRBC) remains a challenge. Methods: Here, a high-Throughput, three-parameter (tri-colour) flow cytometry technique based on mitotracker red dye, the nucleic acid dye coriphosphine O (CPO) and the leucocyte marker CD45 for enumerating live parasites in bioassays was developed. The technique was applied to estimate the specific growth inhibition index (SGI) in the antibody-dependent cellular inhibition (ADCI) assay and compared to parasite quantification by microscopy and mitotracker red staining. The Bland-Altman analysis was used to compare biases between SGI estimated by the tri-colour staining technique, mitotracker red and by microscopy. Results: CPO allowed a better separation between early rings and uRBCs compared to mitotracker red resulting in a more accurate estimate of total parasitaemia. The tri-colour technique is rapid, cost effective and robust with comparable sensitivity to microscopy and capable of discriminating between live and dead and/or compromised parasites. Staining for CD45 improved parasitaemia estimates in ADCI assay since high numbers of leucocytes interfered with the accurate identification of parasitized RBC. The least bias (?1.60) in SGI was observed between the tri-colour and microscopy. Conclusion: An improved methodology for high-Throughput assessment of P. falciparum parasitaemia under culture conditions that could be useful in different bioassays, including ADCI and growth inhibition assays has been developed. © 2014 Tiendrebeogo et al.; licensee BioMed Central Ltd.