Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1.
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.
Li
Qigui
Q
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
Gerena
Lucia
L
Xie
Lisa
L
Zhang
Jing
J
Kyle
Dennis
D
Milhous
Wilbur
W
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
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