title: Re-evaluating acridine orange for rapid flow cytometric enumeration of parasitemia in malaria-infected rodents.
abstract: Methods facilitating research in malaria are of pivotal relevance. Flow cytometry offers the possibility of rapid enumeration of parasitemia. It relies on staining the parasite DNA to distinguish between infected and non-infected red blood cell (RBC) populations. Unfortunately, in rodents abundant reticulocyte RNA interferes with the application of the method. This results in time-consuming sample preparation protocols that offer no clear advantage over microscopic counting. We re-evaluated the use of the DNA/RNA discriminating vital fluorochrome acridine orange (AO) for rapid flow cytometric enumeration of parasitemia in rodents. Whole blood from rodents infected with Plasmodium berghei and Plasmodium yoelii was stained with AO and analyzed by flow cytometer. A newly developed two-channel (FL1/FL3) detection method was compared with conventional one-channel (FL1) detection and microscopic counting. The new AO two-channel detection method clearly discriminated between infected and non-infected RBC populations. It showed to be linear above parasitemias of 0.3%. Sample processing time amounted to approximately 5 min. It is shown that AO can be used for rapid, precise, and accurate enumeration of parasitemia in rodents. Due to its ease of handling the method might find widespread application in malaria research.
authors.affiliation: Division of Instruments for Research, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Sebastian Chakrit
authors.initials: SC
authors.lastname: Bhakdi
authors.forename: Panudda
authors.initials: P
authors.lastname: Sratongno
authors.forename: Pattamawan
authors.initials: P
authors.lastname: Chimma
authors.forename: Thanaporn
authors.initials: T
authors.lastname: Rungruang
authors.forename: Aporn
authors.initials: A
authors.lastname: Chuncharunee
authors.forename: Hartmut P H
authors.initials: HP
authors.lastname: Neumann
authors.forename: Prida
authors.initials: P
authors.lastname: Malasit
authors.forename: Kovit
authors.initials: K
authors.lastname: Pattanapanyasat
mesh_terms: D000165:Acridine Orange; D000818:Animals; D006224:Cricetinae; D004912:Erythrocytes / Q000469:parasitology*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D008288:Malaria / Q000175:diagnosis / Q000469:parasitology*; D008647:Mesocricetus; D051379:Mice; D008813:Mice, Inbred ICR; D018512:Parasitemia / Q000469:parasitology; D010962:Plasmodium berghei / Q000302:isolation & purification* / Q000469:parasitology; D010967:Plasmodium yoelii / Q000302:isolation & purification* / Q000469:parasitology; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D000165:Acridine Orange