High-throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays
Research output: Contribution to journal › Journal article › Research › peer-review
Standard
High-throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays. / Tiendrebeogo, Regis W; Adu, Bright; Singh, Susheel K; Dodoo, Daniel; Dziegiel, Morten H; Mordmüller, Benjamin; Nébié, Issa; Sirima, Sodiomon B; Christiansen, Michael; Theisen, Michael.
In: Malaria Journal, Vol. 13, No. 1, 2014, p. 412.Research output: Contribution to journal › Journal article › Research › peer-review
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - High-throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays
AU - Tiendrebeogo, Regis W
AU - Adu, Bright
AU - Singh, Susheel K
AU - Dodoo, Daniel
AU - Dziegiel, Morten H
AU - Mordmüller, Benjamin
AU - Nébié, Issa
AU - Sirima, Sodiomon B
AU - Christiansen, Michael
AU - Theisen, Michael
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
U2 - 10.1186/1475-2875-13-412
DO - 10.1186/1475-2875-13-412
M3 - Journal article
C2 - 25331683
VL - 13
SP - 412
JO - Malaria Journal
JF - Malaria Journal
SN - 1475-2875
IS - 1
ER -
ID: 125745092