title: Simultaneous determination of phagocytosis of Plasmodium falciparum-parasitized and non-parasitized red blood cells by flow cytometry.
abstract: BACKGROUND
  Severe falciparum malaria anaemia (SMA) is a frequent cause of mortality in children and pregnant women. The most important determinant of SMA appears to be the loss of non-parasitized red blood cells (np-RBCs) in excess of loss of parasitized (p-) RBCs at schizogony. Based on data from acute SMA where excretion of haemoglobin in urine and increased plasma haemoglobin represented respectively less than 1% and 0.5% of total Hb loss, phagocytosis appears to be the predominant mechanism of removal of np- and p-RBC.Estimates indicate that np-RBCs are cleared in approximately 10-fold excess compared to p-RBCs. An even larger removal of np-RBCs has been described in vivax malaria anaemia. Estimates were based on two single studies both performed on neurosyphilitic patients who underwent malaria therapy. As the share of np-RBC removal is likely to vary between wide limits, it is important to assess the contribution of both np- and p-RBC populations to overall RBC loss, and disclose the mechanism of such variability. As available methods do not discriminate between the removal of np- vs p-RBCs, the purpose of this study was to set up a system allowing the simultaneous determination of phagocytosis of p- and np-RBC in the same sample.
  
  
  RESULTS
  Phagocytosis of p- and np-RBCs was quantified in the same sample using double-labelled target cells and the human phagocytic cell-line THP-1, pre-activated by TNF and IFNγ to enhance their phagocytic activity. Target RBCs were double-labelled with fluorescent carboxyfluorescein-succinimidyl ester (CF-SE) and the DNA label ethidium bromide (EB). EB, a DNA label, allowed to discriminate p-RBCs that contain parasitic DNA from the np-RBCs devoid of DNA. FACS analysis of THP-1 cells fed with double-labelled RBCs showed that p- and np-RBCs were phagocytosed in different proportions in relation to parasitaemia.
  
  
  CONCLUSIONS
  The assay allowed the analysis of phagocytosis rapidly and with low subjective error, and the differentiation between phagocytosed p- and np-RBCs in the same sample. The presented method may help to analyse the factors or conditions that modulate the share of np-RBC removal in vitro and in vivo and lead to a better understanding of the pathogenesis of SMA.
authors.affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Gallo
authors.forename: Oleksii A
authors.initials: OA
authors.lastname: Skorokhod
authors.forename: Evelin
authors.initials: E
authors.lastname: Schwarzer
authors.forename: Paolo
authors.initials: P
authors.lastname: Arese
mesh_terms: D000328:Adult; D000740:Anemia / Q000097:blood / Q000209:etiology / Q000469:parasitology; D002460:Cell Line; D002648:Child; D004912:Erythrocytes / Q000276:immunology* / Q000469:parasitology*; D004996:Ethidium; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006790:Host-Parasite Interactions / Q000276:immunology; D006801:Humans; D016778:Malaria, Falciparum / Q000097:blood / Q000150:complications / Q000276:immunology* / Q000469:parasitology*; D009000:Monocytes / Q000276:immunology / Q000469:parasitology; D010587:Phagocytosis; D010963:Plasmodium falciparum / Q000254:growth & development / Q000276:immunology*; D011247:Pregnancy; D015597:Pregnancy Complications, Parasitic / Q000097:blood / Q000276:immunology / Q000469:parasitology
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; C024098:6-carboxyfluorescein; D004996:Ethidium