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Automated counting for Plasmodium falciparum cytoadherence experiments.

Paton D, Faragher B, Mustaffa KM, Szestak T, Barrett SD, Craig AG - Malar. J. (2011)

Bottom Line: 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.Cell based assays were also used as inputs to one of the automated systems (ImageSXM) and produced variable, but encouraging, results.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.

ABSTRACT

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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Related in: MedlinePlus

Comparison of binding results. Bland and Altman plots comparing Image Pro Plus (ProPlus) and ImageSXM (SXM) automated counting techniques with manual counts for adhesion assays on plastic to ICAM-1 using the P. falciparum line ItG-ICAM.
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Figure 1: Comparison of binding results. Bland and Altman plots comparing Image Pro Plus (ProPlus) and ImageSXM (SXM) automated counting techniques with manual counts for adhesion assays on plastic to ICAM-1 using the P. falciparum line ItG-ICAM.

Mentions: The Bland and Altman plots (Figure 1) show clearly that the variation in the differences between the automatic and manual measurements tended to increase as the (average) count level increased (and hence as concentration increased) - and this was particularly marked for the SXM method.


Automated counting for Plasmodium falciparum cytoadherence experiments.

Paton D, Faragher B, Mustaffa KM, Szestak T, Barrett SD, Craig AG - Malar. J. (2011)

Comparison of binding results. Bland and Altman plots comparing Image Pro Plus (ProPlus) and ImageSXM (SXM) automated counting techniques with manual counts for adhesion assays on plastic to ICAM-1 using the P. falciparum line ItG-ICAM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094228&req=5

Figure 1: Comparison of binding results. Bland and Altman plots comparing Image Pro Plus (ProPlus) and ImageSXM (SXM) automated counting techniques with manual counts for adhesion assays on plastic to ICAM-1 using the P. falciparum line ItG-ICAM.
Mentions: The Bland and Altman plots (Figure 1) show clearly that the variation in the differences between the automatic and manual measurements tended to increase as the (average) count level increased (and hence as concentration increased) - and this was particularly marked for the SXM method.

Bottom Line: 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.Cell based assays were also used as inputs to one of the automated systems (ImageSXM) and produced variable, but encouraging, results.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus