title: Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing.
abstract: BACKGROUND
  Malaria pigment (haemozoin, Hz) has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurate and its adaptation to common bench top flow cytometers might allow several applications. These can range from the ex-vivo and in-vitro detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs) to assess antimalarial activity.
  
  
  METHODS
  A standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and Plasmodium falciparum Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with Plasmodium berghei ANKA or P. berghei NK65 and Hz-containing leukocytes were analysed using CD11b and Gr1 expression. Parasitized RBC from infected mice were identified using anti-Ter119 and SYBR green I and were analysed for depolarized Side Scatter. A highly depolarizing RBC population was monitored in an in-vitro culture incubated with chloroquine or quinine.
  
  
  RESULTS
  A flow cytometer can be easily adapted to detect depolarized Side-Scatter and thus, intracellular Hz. The detection and counting of Hz containing leukocytes in fresh human or mouse blood, as well as in leukocytes from in-vitro experiments was rapid and easy. Analysis of CD14/CD16 and CD11b/Gr1 monocyte expression in human or mouse blood, in a mixed populations of Hz-containing and non-containing monocytes, appears to show distinct patterns in both types of cells. Hz-containing pRBC and different maturation stages could be detected in blood from infected mice. The analysis of a highly depolarizing population that contained mature pRBC allowed to assess the effect of chloroquine and quinine after only 2 and 4 hours, respectively.
  
  
  CONCLUSIONS
  A simple modification of a flow cytometer allows for rapid and reliable detection and quantification of Hz-containing leukocytes and the analysis of differential surface marker expression in the same sample of Hz-containing versus non-Hz-containing leukocytes. Importantly, it distinguishes different maturation stages of parasitized RBC and may be the basis of a rapid no-added-reagent drug sensitivity assay.
authors: Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Lisbon, Hospital Universitário de Santa Maria, Av, Prof, Egas Moniz, P-1649-028 Lisboa, Portugal.
authors: Rosangela
authors: R
authors: Frita
authors: Maria
authors: M
authors: Rebelo
authors: Ana
authors: A
authors: Pamplona
authors: Ana M
authors: AM
authors: Vigario
authors: Maria M
authors: MM
authors: Mota
authors: Martin P
authors: MP
authors: Grobusch
authors: Thomas
authors: T
authors: Hänscheid
mesh_terms: D000818:Animals; D039481:CD11b Antigen / Q000097:blood; D002738:Chloroquine / Q000378:metabolism; D004906:Erythrocyte Count / Q000295:instrumentation / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry* / Q000469:parasitology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006420:Hemeproteins / Q000032:analysis*; D006801:Humans; D007958:Leukocyte Count / Q000295:instrumentation / Q000379:methods; D007962:Leukocytes / Q000737:chemistry*; D018950:Lipopolysaccharide Receptors / Q000097:blood; D008288:Malaria / Q000097:blood / Q000175:diagnosis / Q000469:parasitology / Q000473:pathology*; D051379:Mice; D010962:Plasmodium berghei; D010963:Plasmodium falciparum; D011803:Quinine / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000097:blood; D017452:Receptors, IgG / Q000097:blood; D012680:Sensitivity and Specificity
chemical_list: D039481:CD11b Antigen; D006420:Hemeproteins; D018950:Lipopolysaccharide Receptors; D011956:Receptors, Cell Surface; D017452:Receptors, IgG; C551746:granulocyte receptor 1, mouse; C020753:hemozoin; D002738:Chloroquine; D011803:Quinine