Cytofluorograf Detection of Plasmodium yoelii, Trypanosoma gambiense, and Trypanosoma equiperdum by Laser Excited Fluorescence of Stained Rodent Blood

Abstract

Samples of rat blood infected with Plasmodium yoelii (3% parasitized erythrocytes), Trypanosoma gambiense, or Trypanosoma equiperdum (greater than 50 trypanosomes per microscope field at 400 X) were fixed with 0.5% glutaraldehyde in phosphate buffered saline, then stained with acridine orange (AO) at 10(-4), 10(-5), or 10(-6) M for 0 to 15 min at 5 C or 25 C and/or ethidium bromide (EB) at 0.05 mg/ml for 20 min at 25 C. Stained cells were analyzed with a laser Cytofluorograf (Bio/Physics Systems, Inc.) to determine if parasites could be detected and differentiated from blood cells by their fluorescent characteristics. Samples of uninfected rat blood with and without leukocytes and P. yoelii-infected blood without leukocytes were treated similarly. In addition, suspensions of T. gambiense and T. equiperdum without all blood cells were stained with AO or EB and analyzed with the Cytofluorograf, as were mixed suspensions of both trypanosome species. EB- but not AO-stained P. yoelii-infected erythrocytes had fluorescent characteristics different from most blood cells. Neither AO- nor EB-stained T. gambiense or T. equiperdum could be differentiated from host blood cells or from each other. The results are discussed with respect to the use of laser flow systems in the detection and analysis of bloodstream dwelling protozoan parasites.

Methodology and application of flow cytometry for investigation of human malaria parasites

“…In 1977 (Jackson et al, 1977) the first experiments were performed to investigate malaria parasites using flow cytometry by staining cells with ethidium bromide and acridine orange. Other early experiments were carried out on parasites which infect other mammals such as Plasmodium vinckei with (DiOC 1- 3 + Hoechst 33342) (Jacobberger et al, 1984) and Plasmodium berghei ( P. berghei , DRAQ5)(Billker et al, 2004).…”

Analysis of malaria parasite‐infected blood by flow cytometry

“…Reports are lirxiiled to the counting and classification of WBC (1,12) and reticulocytes (23). Applications of flow cytometry in malaria research have also been published (5,7,8,15). Only one report, however, was concerned with the analysis of growth stages within RBC (5).…”

Flow cytometric enumeration of parasitaemia and haematologic changes in trypanosoma-infected mice

“…Because of these constraints, the dynamics of blood parasite and blood cells changes is not accessible, whereas it could reveal key informations on host-pathogen interactions. Prompted by three pioneering studies (Martin et al, 1972;Jackson et al, 1977;Mills and Valli, 1978), a methodological approach combining everyday microsampling of tail blood and its analysis by flow cytometry was developed. The technical 0001-706X/$ -see front matter © 2008 Elsevier B.V. All rights reserved.…”