title: A simple protocol for platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes in a resource poor setting.
abstract: P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules (1). By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes (2) and restrict their environment to a more favorable low oxygen pressure (3). As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood. Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria (4,5). The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor (6). Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)(7,8) has also been associated with severe disease (9). One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps (10). Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM (11). In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian (12) and Mozambican patients (13), (although not in Malian (14)). With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay (15). Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.
authors.affiliation: Malawi-Liverpool-Wellcome Trust Clinical Research Programme.
authors.forename: Dumizulu L
authors.initials: DL
authors.lastname: Tembo
authors.forename: Jacqui
authors.initials: J
authors.lastname: Montgomery
authors.forename: Alister G
authors.initials: AG
authors.lastname: Craig
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Wassmer
mesh_terms: D001792:Blood Platelets / Q000166:cytology / Q000469:parasitology*; D002448:Cell Adhesion / Q000502:physiology; D004912:Erythrocytes / Q000469:parasitology* / Q000473:pathology*; D006801:Humans; D016778:Malaria, Falciparum / Q000097:blood* / Q000469:parasitology / Q000473:pathology; D008856:Microscopy, Fluorescence / Q000379:methods; D010963:Plasmodium falciparum / Q000166:cytology*; D015539:Platelet Activation; D010973:Platelet Adhesiveness; D053657:Platelet-Rich Plasma / Q000166:cytology / Q000469:parasitology*; D013194:Staining and Labeling / Q000379:methods