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Experimental conditions affect the outcome of Plasmodium falciparum platelet-mediated clumping assays.

Arman M, Rowe JA - Malar. J. (2008)

Bottom Line: At low Ht (2%), the Pt of the culture has a large effect on clumping, with significantly higher clumping occurring at 12% Pt (mean 47% of IE in clumps) compared to 4% Pt (mean 26% IE in clumps) or 1% Pt (mean 7% IE in clumps) (ANOVA, p=0.0004).Similarly, at low Pt (1%), the Ht of the culture has a large effect on clumping, with significantly higher clumping occurring at 10% Ht (mean 62% IE in clumps) compared to 5% Ht (mean 25% IE in clumps) or 2% Ht (mean 10% IE in clumps) (ANOVA, p=0.0004).There was no significant difference in clumping when fresh platelets were used compared to platelets stored at 4 degrees C for 10 days.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, West Mains Rd, Edinburgh, EH9 3JT, UK.

ABSTRACT

Background: Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes (IE) is a parasite adhesion phenotype that has been associated with severe malaria in some, but not all, field isolate studies. A variety of experimental conditions have been used to study clumping in vitro, with substantial differences in parasitaemia (Pt), haematocrit (Ht), and time of reaction between studies. It is unknown whether these experimental variables affect the outcome of parasite clumping assays.

Methods: The effects of Pt (1, 4 and 12%), Ht (2, 5 and 10%) and time (15 min, 30 min, 1 h, 2 h) on the clumping of P. falciparum clone HB3 were examined. The effects of platelet freshness and parasite maturity were also studied.

Results: At low Ht (2%), the Pt of the culture has a large effect on clumping, with significantly higher clumping occurring at 12% Pt (mean 47% of IE in clumps) compared to 4% Pt (mean 26% IE in clumps) or 1% Pt (mean 7% IE in clumps) (ANOVA, p=0.0004). Similarly, at low Pt (1%), the Ht of the culture has a large effect on clumping, with significantly higher clumping occurring at 10% Ht (mean 62% IE in clumps) compared to 5% Ht (mean 25% IE in clumps) or 2% Ht (mean 10% IE in clumps) (ANOVA, p=0.0004). Combinations of high Ht and high Pt were impractical because of the difficulty assessing clumping in densely packed IE and the rapid formation of enormous clumps that could not be counted accurately. There was no significant difference in clumping when fresh platelets were used compared to platelets stored at 4 degrees C for 10 days. Clumping was a property of mature pigmented-trophozoites and schizonts but not ring stage parasites.

Conclusion: The Pt and Ht at which in vitro clumping assays are set up have a profound effect on the outcome. All previous field isolate studies on clumping and malaria severity suffer from potential problems in experimental design and methodology. Future studies of clumping should use standardized conditions and control for Pt, and should take into account the limitations and variability inherent in the assay.

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Comparison of HB3 platelet-mediated clumping frequencies detected by in vitro assays performed at a constant haematocrit (Ht) but varying parasitaemia (Pt). A. In vitro platelet-mediated clumping assays were set up at 2% Ht and varying Pt (1%, 4% and 12%) and the clumping frequency (percentage of IE in clumps out of at least 500 IE counted) was assessed by fluorescence microscopy of wet preparations at four time points (15 min, 30 min, 1 h and 2 h). The mean and standard deviation of three wet preparations from each time point are shown, and the range of clump sizes (number of IE forming the clumps) detected in each assay and each time point is indicated. Two replicates of each experiment, done on different days and with platelet-rich plasma from different donors, are shown. Each experiment included a negative control performed with PPP (platelet-poor plasma) and 12% Pt to determine the potential for non-platelet mediated clumping. B. As above but at 5% Ht. C. As above but at 10% Ht.
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Figure 2: Comparison of HB3 platelet-mediated clumping frequencies detected by in vitro assays performed at a constant haematocrit (Ht) but varying parasitaemia (Pt). A. In vitro platelet-mediated clumping assays were set up at 2% Ht and varying Pt (1%, 4% and 12%) and the clumping frequency (percentage of IE in clumps out of at least 500 IE counted) was assessed by fluorescence microscopy of wet preparations at four time points (15 min, 30 min, 1 h and 2 h). The mean and standard deviation of three wet preparations from each time point are shown, and the range of clump sizes (number of IE forming the clumps) detected in each assay and each time point is indicated. Two replicates of each experiment, done on different days and with platelet-rich plasma from different donors, are shown. Each experiment included a negative control performed with PPP (platelet-poor plasma) and 12% Pt to determine the potential for non-platelet mediated clumping. B. As above but at 5% Ht. C. As above but at 10% Ht.

Mentions: Most of the clumping field studies published to date [6-8] have included a comparison of clumping frequencies amongst field isolates set up at a standardized Ht (1, 2 or 5%, Table 1) but with a wide range of different Pt, reflecting the varying admission Pt of malaria patients. In order to determine if this is an optimal experimental situation, the effect of Pt on clumping was investigated using the P. falciparum laboratory strain HB3. A total of six experiments were performed on different days where the Ht of the assays was fixed (2, 5 or 10%) and different Pt levels were compared within the experiment (1, 4, and 12%, Figure 2). In every experiment, all the assays were prepared from a single HB3 culture stained with ethidium bromide, and the clumping assay was allowed to proceed for four different time periods (15 min, 30 min, 1 h, and 2 h). Three wet preparations were made from each tube at each time point and clumping frequency was assessed by fluorescence microscopy.


Experimental conditions affect the outcome of Plasmodium falciparum platelet-mediated clumping assays.

Arman M, Rowe JA - Malar. J. (2008)

Comparison of HB3 platelet-mediated clumping frequencies detected by in vitro assays performed at a constant haematocrit (Ht) but varying parasitaemia (Pt). A. In vitro platelet-mediated clumping assays were set up at 2% Ht and varying Pt (1%, 4% and 12%) and the clumping frequency (percentage of IE in clumps out of at least 500 IE counted) was assessed by fluorescence microscopy of wet preparations at four time points (15 min, 30 min, 1 h and 2 h). The mean and standard deviation of three wet preparations from each time point are shown, and the range of clump sizes (number of IE forming the clumps) detected in each assay and each time point is indicated. Two replicates of each experiment, done on different days and with platelet-rich plasma from different donors, are shown. Each experiment included a negative control performed with PPP (platelet-poor plasma) and 12% Pt to determine the potential for non-platelet mediated clumping. B. As above but at 5% Ht. C. As above but at 10% Ht.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2599902&req=5

Figure 2: Comparison of HB3 platelet-mediated clumping frequencies detected by in vitro assays performed at a constant haematocrit (Ht) but varying parasitaemia (Pt). A. In vitro platelet-mediated clumping assays were set up at 2% Ht and varying Pt (1%, 4% and 12%) and the clumping frequency (percentage of IE in clumps out of at least 500 IE counted) was assessed by fluorescence microscopy of wet preparations at four time points (15 min, 30 min, 1 h and 2 h). The mean and standard deviation of three wet preparations from each time point are shown, and the range of clump sizes (number of IE forming the clumps) detected in each assay and each time point is indicated. Two replicates of each experiment, done on different days and with platelet-rich plasma from different donors, are shown. Each experiment included a negative control performed with PPP (platelet-poor plasma) and 12% Pt to determine the potential for non-platelet mediated clumping. B. As above but at 5% Ht. C. As above but at 10% Ht.
Mentions: Most of the clumping field studies published to date [6-8] have included a comparison of clumping frequencies amongst field isolates set up at a standardized Ht (1, 2 or 5%, Table 1) but with a wide range of different Pt, reflecting the varying admission Pt of malaria patients. In order to determine if this is an optimal experimental situation, the effect of Pt on clumping was investigated using the P. falciparum laboratory strain HB3. A total of six experiments were performed on different days where the Ht of the assays was fixed (2, 5 or 10%) and different Pt levels were compared within the experiment (1, 4, and 12%, Figure 2). In every experiment, all the assays were prepared from a single HB3 culture stained with ethidium bromide, and the clumping assay was allowed to proceed for four different time periods (15 min, 30 min, 1 h, and 2 h). Three wet preparations were made from each tube at each time point and clumping frequency was assessed by fluorescence microscopy.

Bottom Line: At low Ht (2%), the Pt of the culture has a large effect on clumping, with significantly higher clumping occurring at 12% Pt (mean 47% of IE in clumps) compared to 4% Pt (mean 26% IE in clumps) or 1% Pt (mean 7% IE in clumps) (ANOVA, p=0.0004).Similarly, at low Pt (1%), the Ht of the culture has a large effect on clumping, with significantly higher clumping occurring at 10% Ht (mean 62% IE in clumps) compared to 5% Ht (mean 25% IE in clumps) or 2% Ht (mean 10% IE in clumps) (ANOVA, p=0.0004).There was no significant difference in clumping when fresh platelets were used compared to platelets stored at 4 degrees C for 10 days.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, West Mains Rd, Edinburgh, EH9 3JT, UK.

ABSTRACT

Background: Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes (IE) is a parasite adhesion phenotype that has been associated with severe malaria in some, but not all, field isolate studies. A variety of experimental conditions have been used to study clumping in vitro, with substantial differences in parasitaemia (Pt), haematocrit (Ht), and time of reaction between studies. It is unknown whether these experimental variables affect the outcome of parasite clumping assays.

Methods: The effects of Pt (1, 4 and 12%), Ht (2, 5 and 10%) and time (15 min, 30 min, 1 h, 2 h) on the clumping of P. falciparum clone HB3 were examined. The effects of platelet freshness and parasite maturity were also studied.

Results: At low Ht (2%), the Pt of the culture has a large effect on clumping, with significantly higher clumping occurring at 12% Pt (mean 47% of IE in clumps) compared to 4% Pt (mean 26% IE in clumps) or 1% Pt (mean 7% IE in clumps) (ANOVA, p=0.0004). Similarly, at low Pt (1%), the Ht of the culture has a large effect on clumping, with significantly higher clumping occurring at 10% Ht (mean 62% IE in clumps) compared to 5% Ht (mean 25% IE in clumps) or 2% Ht (mean 10% IE in clumps) (ANOVA, p=0.0004). Combinations of high Ht and high Pt were impractical because of the difficulty assessing clumping in densely packed IE and the rapid formation of enormous clumps that could not be counted accurately. There was no significant difference in clumping when fresh platelets were used compared to platelets stored at 4 degrees C for 10 days. Clumping was a property of mature pigmented-trophozoites and schizonts but not ring stage parasites.

Conclusion: The Pt and Ht at which in vitro clumping assays are set up have a profound effect on the outcome. All previous field isolate studies on clumping and malaria severity suffer from potential problems in experimental design and methodology. Future studies of clumping should use standardized conditions and control for Pt, and should take into account the limitations and variability inherent in the assay.

Show MeSH
Related in: MedlinePlus