BACKGROUND: Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large-scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO-1 that are observed in infected erythrocytes compared with noninfected erythrocytes. METHODS: Samples of blood from Plasmodium yoelii-infected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO-1 in 96-well plate format. After acquisition, erythrocytes gated in logarithmic side/scatter plots were analyzed in bidimensional FL-2/YOYO-1 plots in comparison with unidimensional YOYO-1 analysis. RESULTS: The infected erythrocytes showed a characteristic pattern of staining different from that of noninfected erythrocytes. In routine evaluation, the limit of sensitivity was 0.01% and the measurements of parasitemia were linear at parasitemias above 0.1%. Interestingly, using this approach, infected reticulocytes could be differentiated from infected normocytes. CONCLUSIONS: The method described is robust, increases the specificity and sensitivity of detection in routine testing, and is especially well suited for detection of low parasitemias in murine models of malaria. (c) 2005 Wiley-Liss, Inc.
BACKGROUND: Microscopic analysis of blood smears is currently the most frequently used method to measure parasitemias in experiments of drug efficacy in murine models of malaria. However, it is subjective and labour intensive, which preclude its utilization in large-scale evaluation programs. Flow cytometry is an alternative method, but due to the limited specificity achieved with the currently available techniques, it has not been widely used in murine models of malaria during preclinical evaluation. We describe a new flow cytometric method based on the differences of autofluorescence and DNA content measured after staining with YOYO-1 that are observed in infected erythrocytes compared with noninfected erythrocytes. METHODS: Samples of blood from Plasmodium yoelii-infected animals were fixed with glutaraldehyde, incubated with RNAase, and stained with YOYO-1 in 96-well plate format. After acquisition, erythrocytes gated in logarithmic side/scatter plots were analyzed in bidimensional FL-2/YOYO-1 plots in comparison with unidimensional YOYO-1 analysis. RESULTS: The infected erythrocytes showed a characteristic pattern of staining different from that of noninfected erythrocytes. In routine evaluation, the limit of sensitivity was 0.01% and the measurements of parasitemia were linear at parasitemias above 0.1%. Interestingly, using this approach, infected reticulocytes could be differentiated from infected normocytes. CONCLUSIONS: The method described is robust, increases the specificity and sensitivity of detection in routine testing, and is especially well suited for detection of low parasitemias in murine models of malaria. (c) 2005 Wiley-Liss, Inc.
Authors: Robert H Barker; Sameer Urgaonkar; Ralph Mazitschek; Cassandra Celatka; Renato Skerlj; Joseph F Cortese; Erin Tyndall; Hanlan Liu; Mandy Cromwell; Amar Bir Sidhu; Jose E Guerrero-Bravo; Keila N Crespo-Llado; Adelfa E Serrano; Jing-Wen Lin; Chris J Janse; Shahid M Khan; Manoj Duraisingh; Bradley I Coleman; Inigo Angulo-Barturen; María Belén Jiménez-Díaz; Noemí Magán; Vanesa Gomez; Santiago Ferrer; María Santos Martínez; Sergio Wittlin; Petros Papastogiannidis; Thomas O'Shea; Jeffrey D Klinger; Mark Bree; Edward Lee; Mikaela Levine; Roger C Wiegand; Benito Munoz; Dyann F Wirth; Jon Clardy; Ian Bathurst; Edmund Sybertz Journal: Antimicrob Agents Chemother Date: 2011-03-21 Impact factor: 5.191
Authors: Aaron Nilsen; Alexis N LaCrue; Karen L White; Isaac P Forquer; Richard M Cross; Jutta Marfurt; Michael W Mather; Michael J Delves; David M Shackleford; Fabian E Saenz; Joanne M Morrisey; Jessica Steuten; Tina Mutka; Yuexin Li; Grennady Wirjanata; Eileen Ryan; Sandra Duffy; Jane Xu Kelly; Boni F Sebayang; Anne-Marie Zeeman; Rintis Noviyanti; Robert E Sinden; Clemens H M Kocken; Ric N Price; Vicky M Avery; Iñigo Angulo-Barturen; María Belén Jiménez-Díaz; Santiago Ferrer; Esperanza Herreros; Laura M Sanz; Francisco-Javier Gamo; Ian Bathurst; Jeremy N Burrows; Peter Siegl; R Kiplin Guy; Rolf W Winter; Akhil B Vaidya; Susan A Charman; Dennis E Kyle; Roman Manetsch; Michael K Riscoe Journal: Sci Transl Med Date: 2013-03-20 Impact factor: 17.956