title: 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.
  
  
  CONCLUSIONS
  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.
authors.forename: Regis W
authors.initials: RW
authors.lastname: Tiendrebeogo
authors.forename: Bright
authors.initials: B
authors.lastname: Adu
authors.forename: Susheel K
authors.initials: SK
authors.lastname: Singh
authors.forename: Daniel
authors.initials: D
authors.lastname: Dodoo
authors.forename: Morten H
authors.initials: MH
authors.lastname: Dziegiel
authors.forename: Benjamin
authors.initials: B
authors.lastname: Mordmüller
authors.forename: Issa
authors.initials: I
authors.lastname: Nébié
authors.forename: Sodiomon B
authors.initials: SB
authors.lastname: Sirima
authors.forename: Michael
authors.initials: M
authors.lastname: Christiansen
authors.affiliation: Department of Clinical Biochemistry, Immunology and Genetics, Statens Serum Institut, Copenhagen, Denmark. mth@ssi.dk.
authors.forename: Michael
authors.initials: M
authors.lastname: Theisen
mesh_terms: D000328:Adult; D002648:Child; D002675:Child, Preschool; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007223:Infant; D016778:Malaria, Falciparum / Q000175:diagnosis / Q000469:parasitology*; D059208:Parasite Load / Q000379:methods*; D018512:Parasitemia / Q000175:diagnosis / Q000469:parasitology*; D010963:Plasmodium falciparum / Q000302:isolation & purification*
chemical_list: D005456:Fluorescent Dyes