High-throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays
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- 作者：Regis W Tiendrebeogo (1) (2)
Bright Adu (1) (2)
Susheel K Singh (1) (2)
Daniel Dodoo (3)
Morten H Dziegiel (4)
Benjamin Mordm眉ller (5) (6)
Issa N茅bi茅 (7)
Sodiomon B Sirima (7)
Michael Christiansen (1)
Michael Theisen (1) (2)
1. Department of Clinical Biochemistry ; Immunology and Genetics ; Statens Serum Institut ; Copenhagen ; Denmark
2. Centre for Medical Parasitology at Department of International Health ; Immunology ; and Microbiology and Department of Infectious Diseases ; Rigshospitalet ; University of Copenhagen ; Copenhagen ; Denmark
3. Noguchi Memorial Institute for Medical Research ; University of Ghana ; Legon ; Ghana
4. Blood Bank KI 2034 ; Copenhagen University Hospital ; Copenhagen ; Denmark
5. Institute of Tropical Medicine ; University of T眉bingen ; Wilhelmstra脽e 27 ; T眉bingen ; D-72074 ; Germany
6. Centre de Recherche M茅dicale de Lambar茅n茅 (CERMEL) ; Lambar茅n茅 ; BP 118 ; Gabon
7. Centre National de Recherche et de Formation sur le Paludisme ; Ouagadougou ; Burkina Faso - 关键词：Malaria ; Plasmodium falciparum ; Mitotracker red ; Coriphosphine ; O ; ADCI ; Bioassay ; Flow cytometry ; CD45 ; Tri ; colour
- 刊名：Malaria Journal
- 出版年：2014
- 出版时间：December 2014
- 年：2014
- 卷：13
- 期：1
- 全文大小：2,108 KB
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- 出版者：BioMed Central
- ISSN：1475-2875
文摘
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.