title: Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1.
abstract: BACKGROUND
  The need for improved malaria diagnostics has long been recognized.
  
  
  METHODS
  Human parasitized erythrocytes based on the principles of flow cytometry (FCM) method is described for the determination of parasitemia in Plasmodium falciparum cultures using the fluorescence activated cell sorter and DNA-binding fluorescent dye, YOYO-1. The same assay samples can be also evaluated both microscopically and by scintillation counting after use of (3)H-hypoxanthine-labeled parasites.
  
  
  RESULTS
  The counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were categorized into different populations according to their physical or chemical properties such as RNase treatment and compensation required optimization. The detection and quantitation limits in the assay were 0.003% and 0.008% parasitemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r(2) = 0.923-0.968) with the parasitemia measured by (3)H-hypoxanthine incorporation assay when parasites variants incubated with various antimalarial drugs. In addition, the low levels of parasitemia (7.9%-21.3%) detected by microscopy than by FCM may be related to a number of tiny schizonts externally attached to the erythrocyte membranes but were not definitely inside the erythrocyte that normally would never be included in microscopy counting.
  
  
  CONCLUSIONS
  On the basis of data reported herein, a rapid, high sensitivity, lower sampling error and reliable identification of human parasitized erythrocytes by the principles of FCM have been established. Published 2007 Wiley-Liss, Inc.
authors.affiliation: Department of Pharmacology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland 20307-5100, USA. qigui.li@na.amedd.army.mil
authors.forename: Qigui
authors.initials: Q
authors.lastname: Li
authors.forename: Lucia
authors.initials: L
authors.lastname: Gerena
authors.forename: Lisa
authors.initials: L
authors.lastname: Xie
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Dennis
authors.initials: D
authors.lastname: Kyle
authors.forename: Wilbur
authors.initials: W
authors.lastname: Milhous
mesh_terms: D000818:Animals; D001583:Benzoxazoles / Q000032:analysis*; D002138:Calibration; D004351:Drug Resistance; D004912:Erythrocytes / Q000166:cytology / Q000469:parasitology; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes / Q000032:analysis; D005976:Glutaral; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D008853:Microscopy; D018512:Parasitemia / Q000175:diagnosis* / Q000469:parasitology*; D010963:Plasmodium falciparum / Q000166:cytology* / Q000302:isolation & purification*; D011806:Quinolinium Compounds / Q000032:analysis*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
chemical_list: D001583:Benzoxazoles; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D005976:Glutaral