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Automated counting for Plasmodium falciparum cytoadherence experiments

BACKGROUND: The ability of mature forms of Plasmodium falciparum infected erythrocytes to bind to a range of host receptors including those displayed on endothelial cells has been associated with the pathology of this infection. Investigations into this adhesive phenomenon have used protein and cell...

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Autores principales: Paton, Douglas, Faragher, Brian, Mustaffa, Khairul MF, Szestak, Tadge, Barrett, Steve D, Craig, Alister G
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3094228/
https://www.ncbi.nlm.nih.gov/pubmed/21496305
http://dx.doi.org/10.1186/1475-2875-10-91
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author Paton, Douglas
Faragher, Brian
Mustaffa, Khairul MF
Szestak, Tadge
Barrett, Steve D
Craig, Alister G
author_facet Paton, Douglas
Faragher, Brian
Mustaffa, Khairul MF
Szestak, Tadge
Barrett, Steve D
Craig, Alister G
author_sort Paton, Douglas
collection PubMed
description BACKGROUND: The ability of mature forms of Plasmodium falciparum infected erythrocytes to bind to a range of host receptors including those displayed on endothelial cells has been associated with the pathology of this infection. Investigations into this adhesive phenomenon have used protein and cell-based adhesion assays to quantify the ability of infected red blood cells to bind. These adhesion assays tend to have relatively high inherent variability and so require multiple experiments in order to provide good quantitation. This means that investigators doing these experiments must count many fields of adherent parasites, a task that is time-consuming and laborious. To address this issue and to facilitate cytoadherence research, developed automated protocols were developed for counting parasite adhesion. METHODS: Parasite adhesion assays were mainly carried out under static conditions using purified receptors, which is the simplest form of these assays and is translatable to the field. Two different software platforms were used, one commercial (Image Pro-Plus (Media Cybernetics)) and one available in the public domain (ImageSXM) based on the freely available NIH Image software. The adhesion assays were performed and parasite binding quantified using standard manual techniques. Images were also captured using video microscopy and analysed using the two automated systems. The results generated by each system were compared using the Bland and Altman method for assessing the agreement between two methods. RESULTS: Both automated counting programs showed concordance compared to the 'gold standard' manual counting within the normal range of adhesion seen with these assays, although the ImageSXM technique had some systematic bias. There was some fall-off in accuracy at very high parasite densities, but this can be resolved through good design of the experiments. Cell based assays were also used as inputs to one of the automated systems (ImageSXM) and produced variable, but encouraging, results. CONCLUSIONS: The automated counting programs are an accurate and practical way of quantifying static parasite binding assays to purified proteins. They are less accurate when applied to cell based systems, but can still provide a reasonable level of accuracy to give a semi-quantitative readout.
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spelling pubmed-30942282011-05-14 Automated counting for Plasmodium falciparum cytoadherence experiments Paton, Douglas Faragher, Brian Mustaffa, Khairul MF Szestak, Tadge Barrett, Steve D Craig, Alister G Malar J Methodology BACKGROUND: The ability of mature forms of Plasmodium falciparum infected erythrocytes to bind to a range of host receptors including those displayed on endothelial cells has been associated with the pathology of this infection. Investigations into this adhesive phenomenon have used protein and cell-based adhesion assays to quantify the ability of infected red blood cells to bind. These adhesion assays tend to have relatively high inherent variability and so require multiple experiments in order to provide good quantitation. This means that investigators doing these experiments must count many fields of adherent parasites, a task that is time-consuming and laborious. To address this issue and to facilitate cytoadherence research, developed automated protocols were developed for counting parasite adhesion. METHODS: Parasite adhesion assays were mainly carried out under static conditions using purified receptors, which is the simplest form of these assays and is translatable to the field. Two different software platforms were used, one commercial (Image Pro-Plus (Media Cybernetics)) and one available in the public domain (ImageSXM) based on the freely available NIH Image software. The adhesion assays were performed and parasite binding quantified using standard manual techniques. Images were also captured using video microscopy and analysed using the two automated systems. The results generated by each system were compared using the Bland and Altman method for assessing the agreement between two methods. RESULTS: Both automated counting programs showed concordance compared to the 'gold standard' manual counting within the normal range of adhesion seen with these assays, although the ImageSXM technique had some systematic bias. There was some fall-off in accuracy at very high parasite densities, but this can be resolved through good design of the experiments. Cell based assays were also used as inputs to one of the automated systems (ImageSXM) and produced variable, but encouraging, results. CONCLUSIONS: The automated counting programs are an accurate and practical way of quantifying static parasite binding assays to purified proteins. They are less accurate when applied to cell based systems, but can still provide a reasonable level of accuracy to give a semi-quantitative readout. BioMed Central 2011-04-16 /pmc/articles/PMC3094228/ /pubmed/21496305 http://dx.doi.org/10.1186/1475-2875-10-91 Text en Copyright ©2011 Paton et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology
Paton, Douglas
Faragher, Brian
Mustaffa, Khairul MF
Szestak, Tadge
Barrett, Steve D
Craig, Alister G
Automated counting for Plasmodium falciparum cytoadherence experiments
title Automated counting for Plasmodium falciparum cytoadherence experiments
title_full Automated counting for Plasmodium falciparum cytoadherence experiments
title_fullStr Automated counting for Plasmodium falciparum cytoadherence experiments
title_full_unstemmed Automated counting for Plasmodium falciparum cytoadherence experiments
title_short Automated counting for Plasmodium falciparum cytoadherence experiments
title_sort automated counting for plasmodium falciparum cytoadherence experiments
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3094228/
https://www.ncbi.nlm.nih.gov/pubmed/21496305
http://dx.doi.org/10.1186/1475-2875-10-91
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