title: An automated method for determining the cytoadhesion of Plasmodium falciparum-infected erythrocytes to immobilized cells.
abstract: BACKGROUND
  Plasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing an automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin.
  
  
  METHODS
  Chinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO cells, was quantified by microscopy of Giemsa-stained chamber slides. In the automated assay, binding was quantified spectrophotometrically in microtiter plates after cell lysis using tetramethylbenzidine as peroxidase-catalysed substrate. The relevance of the method for binding studies was assessed using: i) binding of P. falciparum-infected erythrocytes to CHO cells over-expressing chondroitin sulfate A and ii) CHO cells transfected with CD36. Binding of infected erythrocytes including field isolates to primary endothelial cells was also performed. Data was analysed using linear regression and Bland-Altman plots.
  
  
  RESULTS
  The manual and automated quantification showed strong, positive correlation (r(2) = 0.959, p <0.001) and with similar detection limit and precision. The automated assay showed the expected dose-dependent reduction in binding to CHO cells when blocking with soluble chondroitin sulfate A or anti-CD36 antibody. Quantification of binding to endothelial cells showed clear distinction between selected vs. non-selected parasite lines. Importantly, the assay was sufficiently sensitive to detect adhesion of field isolates to endothelial cells.
  
  
  CONCLUSIONS
  The assay is simple and in a reproducible manner quantifies erythrocyte adhesion to several types of immobilized cells.
authors.affiliation: Centre for Medical Parasitology at Department of Clinical Microbiology, Copenhagen University Hospital Department Clinical Microbiology, 7602, Ole Maaløesvej 26, 2200, Copenhagen N, Denmark. casperhempel@gmail.com.
authors.forename: Casper
authors.initials: C
authors.lastname: Hempel
authors.affiliation: Centre for Medical Parasitology at Department of Clinical Microbiology, Copenhagen University Hospital Department Clinical Microbiology, 7602, Ole Maaløesvej 26, 2200, Copenhagen N, Denmark. idamarie.boisen@gmail.com.|Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. idamarie.boisen@gmail.com.
authors.forename: Ida M
authors.initials: IM
authors.lastname: Boisen
authors.affiliation: Institute of Medical Microbiology and Infectious Disease Epidemiology, Medical Faculty, University of Leipzig, Leipzig, Germany. drefunshile@yahoo.com.|Department of Medical Microbiology, Federal Teaching Hospital/Ebonyi State University, Abakaliki, Nigeria. drefunshile@yahoo.com.
authors.forename: Akinwale
authors.initials: A
authors.lastname: Efunshile
authors.affiliation: Centre for Medical Parasitology at Department of Clinical Microbiology, Copenhagen University Hospital Department Clinical Microbiology, 7602, Ole Maaløesvej 26, 2200, Copenhagen N, Denmark. joergen.kurtzhals@regionh.dk.|Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. joergen.kurtzhals@regionh.dk.
authors.forename: Jørgen A L
authors.initials: JA
authors.lastname: Kurtzhals
authors.affiliation: Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark. staalsoe@sund.ku.dk.
authors.forename: Trine
authors.initials: T
authors.lastname: Staalsø
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D002452:Cell Count / Q000379:methods*; D002478:Cells, Cultured; D018914:Cells, Immobilized; D018920:Coculture Techniques; D006224:Cricetinae; D003412:Cricetulus; D042783:Endothelial Cells / Q000469:parasitology; D004912:Erythrocytes / Q000469:parasitology*; D006801:Humans; D010274:Parasitology / Q000379:methods*; D010963:Plasmodium falciparum / Q000472:pathogenicity*