Experimental conditions affect the outcome of Plasmodium falciparum platelet-mediated clumping assays
Tóm tắt
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. 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. 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°C for 10 days. Clumping was a property of mature pigmented-trophozoites and schizonts but not ring stage parasites. 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.
Tài liệu tham khảo
Greenwood B, Mutabingwa T: Malaria in 2002. Nature. 2002, 415: 670-672.
Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI: The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature. 2005, 434: 214-217.
Warrell DA: Pathophysiology of severe falciparum malaria. Papua New Guinea Medical Journal. 1992, 35: 229-232.
Miller LH, Baruch DI, Marsh K, Doumbo OK: The pathogenic basis of malaria. Nature. 2002, 415: 673-679.
Rowe JA, Shafi J, Kai OK, Marsh K, Raza A: Nonimmune IgM, but not IgG binds to the surface of Plasmodium falciparum-infected erythrocytes and correlates with rosetting and severe malaria. Am J Trop Med Hyg. 2002, 66: 692-699.
Pain A, Ferguson DJ, Kai O, Urban BC, Lowe B, Marsh K, Roberts DJ: Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria. Proc Natl Acad Sci USA. 2001, 98: 1805-1810.
Chotivanich K, Sritabal J, Udomsangpetch R, Newton P, Stepniewska KA, Ruangveerayuth R, Looareesuwan S, Roberts DJ, White NJ: Platelet-induced autoagglutination of Plasmodium falciparum-infected red blood cells and disease severity in Thailand. J Infect Dis. 2004, 189: 1052-1055.
Arman M, Raza A, Tempest LJ, Lyke KE, Thera MA, Kone A, Plowe CV, Doumbo OK, Rowe JA: Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes is associated with high parasitemia but not severe clinical manifestations of malaria in African children. Am J Trop Med Hyg. 2007, 77: 943-946.
Biswas AK, Hafiz A, Banerjee B, Kim KS, Datta K, Chitnis CE: Plasmodium falciparum uses gC1qR/HABP1/p32 as a receptor to bind to vascular endothelium and for platelet-mediated clumping. PLoS Pathog. 2007, 3: 1271-1280.
Wassmer SC, Taylor T, Maclennan CA, Kanjala M, Mukaka M, Molyneux ME, Grau GE: Platelet-induced clumping of Plasmodium falciparum-infected erythrocytes from Malawian patients with cerebral malaria-possible modulation in vivo by thrombocytopenia. J Infect Dis. 2008, 197: 72-78.
Lambros C, Vanderberg JP: Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol. 1979, 65: 418-420.
Rowe JA, Scragg IG, Kwiatkowski D, Ferguson DJ, Carucci DJ, Newbold CI: Implications of mycoplasma contamination in Plasmodium falciparum cultures and methods for its detection and eradication. Mol Biochem Parasitol. 1998, 92: 177-180.
The Broad Institute of MIT and Harvard. [http://broad.mit.edu]
Ockenhouse CF, Magowan C, Chulay JD: Activation of monocytes and platelets by monoclonal antibodies or malaria-infected erythrocytes binding to the CD36 surface receptor in vitro. J Clin Invest. 1989, 84: 468-475.
Tandon NN, Ockenhouse CF, Greco NJ, Jamieson GA: Adhesive functions of platelets lacking glycoprotein IV (CD36). Blood. 1991, 78: 2809-2813.
Cooke BM, Nash GB: Plasmodium falciparum: characterization of adhesion of flowing parasitized red blood cells to platelets. Exp Parasitol. 1995, 80: 116-123.
Wassmer SC, Lepolard C, Traore B, Pouvelle B, Gysin J, Grau GE: Platelets reorient Plasmodium falciparum-infected erythrocyte cytoadhesion to activated endothelial cells. J Infect Dis. 2004, 189: 180-189.
Rowe JA: Rosetting of Plasmodium falciparum infected erythrocytes. D Phil thesis. 1994, Oxford University
Rowe JA, Obiero J, Marsh K, Raza A: Short report: Positive correlation between rosetting and parasitemia in Plasmodium falciparum clinical isolates. Am J Trop Med Hyg. 2002, 66: 458-460.
Perrota PL, Snyder EL: Platelet storage and transfusion. Platelets. Edited by: Michelson AD. 2007, Academic Press, 1265-1295. 2
McNicol A: Platelet preparation and estimation of functional responses. Platelets: a practical approach. Edited by: Watson SP, Authi KS. 1996, Oxford: Oxford University Press, 1-26.
May JA, Heptinstall S: Effects of anticoagulants used during blood collection on human platelet function. Methods Mol Biol. Edited by: Gibbins JM, Mahaut-Smith MP. 2004, New Jersey: Humana Press, 272: 3-11.
Treutiger CJ, Carlson J, Scholander C, Wahlgren M: The time course of cytoadhesion, immunoglobulin binding, rosette formation, and serum-induced agglutination of Plasmodium falciparum-infected erythrocytes. Am J Trop Med Hyg. 1998, 59: 202-207.