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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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Platelet-mediated clumps of P. falciparum infected erythrocytes (IE) detected by in vitro clumping assays. A. Platelet-mediated clumps of infected erythrocytes (IEs) from P. falciparum clone HB3 viewed by ethidium bromide staining and fluoresence microscopy (400×) of a wet preparation. Uninfected erythrocytes (Es) present in the clumping assay are unstained. B. Platelet-mediated clumps of HB3 IEs observed by Giemsa-stained thin smears and light microscopy (1000×).
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Figure 1: Platelet-mediated clumps of P. falciparum infected erythrocytes (IE) detected by in vitro clumping assays. A. Platelet-mediated clumps of infected erythrocytes (IEs) from P. falciparum clone HB3 viewed by ethidium bromide staining and fluoresence microscopy (400×) of a wet preparation. Uninfected erythrocytes (Es) present in the clumping assay are unstained. B. Platelet-mediated clumps of HB3 IEs observed by Giemsa-stained thin smears and light microscopy (1000×).

Mentions: One of the most recent P. falciparum cytoadherence phenotypes to be described is the ability of IE to bind to platelets in suspension assays in vitro to form platelet-mediated clumps of infected cells [6] (Figure 1). This phenotype has been demonstrated in a wide range of P. falciparum laboratory strains and field isolates [6-10]. An association of the platelet-mediated clumping phenotype with severe or cerebral malaria has been reported after analysing field isolates from Kenya [6], Thailand [7] and Malawi [10]. However, a recent study in Mali showed a strong positive correlation between P. falciparum clumping in vitro and admission parasitaemia (percentage of erythrocytes infected with P. falciparum parasites), however, no significant association with severe malaria was seen [8]. In each of the above field isolate studies the clumping assay has been carried out in a different way. For example, the exact experimental conditions of haematocrit (Ht) (the percentage volume of the reaction occupied by erythrocytes), parasitaemia (Pt) and time of the clumping assay has varied between studies (Table 1).


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

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

Platelet-mediated clumps of P. falciparum infected erythrocytes (IE) detected by in vitro clumping assays. A. Platelet-mediated clumps of infected erythrocytes (IEs) from P. falciparum clone HB3 viewed by ethidium bromide staining and fluoresence microscopy (400×) of a wet preparation. Uninfected erythrocytes (Es) present in the clumping assay are unstained. B. Platelet-mediated clumps of HB3 IEs observed by Giemsa-stained thin smears and light microscopy (1000×).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Platelet-mediated clumps of P. falciparum infected erythrocytes (IE) detected by in vitro clumping assays. A. Platelet-mediated clumps of infected erythrocytes (IEs) from P. falciparum clone HB3 viewed by ethidium bromide staining and fluoresence microscopy (400×) of a wet preparation. Uninfected erythrocytes (Es) present in the clumping assay are unstained. B. Platelet-mediated clumps of HB3 IEs observed by Giemsa-stained thin smears and light microscopy (1000×).
Mentions: One of the most recent P. falciparum cytoadherence phenotypes to be described is the ability of IE to bind to platelets in suspension assays in vitro to form platelet-mediated clumps of infected cells [6] (Figure 1). This phenotype has been demonstrated in a wide range of P. falciparum laboratory strains and field isolates [6-10]. An association of the platelet-mediated clumping phenotype with severe or cerebral malaria has been reported after analysing field isolates from Kenya [6], Thailand [7] and Malawi [10]. However, a recent study in Mali showed a strong positive correlation between P. falciparum clumping in vitro and admission parasitaemia (percentage of erythrocytes infected with P. falciparum parasites), however, no significant association with severe malaria was seen [8]. In each of the above field isolate studies the clumping assay has been carried out in a different way. For example, the exact experimental conditions of haematocrit (Ht) (the percentage volume of the reaction occupied by erythrocytes), parasitaemia (Pt) and time of the clumping assay has varied between studies (Table 1).

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