In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei

L Hein-Kristensen, L Wiese, J A L Kurtzhals, T Staalsoe

Abstract

Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However, preliminary studies showed concerns when dealing with Plasmodium berghei-infected blood samples with high numbers of reticulocytes. In hyperparasitemic or chronic P. berghei infection, enhanced erythropoietic activity results in high numbers of circulating immature reticulocytes. We show that even though the protocol offered good discrimination in newly infected animals, discrimination between infected erythrocytes and uninfected reticulocytes became difficult in animals with hyperparasitemia or chronic infections maintained with subcurative treatment. Discrimination was especially hampered by increased nucleic acid content in immature uninfected reticulocytes. Our data confirms that though flow cytometry is a promising analytical tool in malaria research, care should still be taken when analysing samples from anemic or chronically infected animals.

Original languageEnglish
JournalExperimental Parasitology
Volume123
Issue number2
Pages (from-to)152-7
Number of pages6
ISSN0014-4894
DOIs
Publication statusPublished - Oct 2009

Keywords

  • Acridine Orange/standards
  • Animals
  • Blood Preservation/methods
  • Female
  • Flow Cytometry/standards
  • Fluorescent Dyes/standards
  • Malaria/blood
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Parasitemia/parasitology
  • Plasmodium berghei/growth & development
  • Rats
  • Rats, Sprague-Dawley
  • Rats, Wistar
  • Reticulocyte Count/standards
  • Reticulocytes/parasitology
  • Time Factors

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