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

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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.

Introduction

Experimental rodent models for malaria have been instrumental in understanding the pathogenesis of malaria and identifying key components of the disease infecting hundreds of millions of people worldwide each year. These models require fast and accurate tools for determining the parasitized fraction of red blood cells (RBC)1. The tedious and observer dependent counting of Giemsa-stained blood smears has long been the golden standard for parasite enumeration but an automated alternative would greatly facilitate scientific work.

Flow cytometry for determining the proportion of infected RBCs utilizes the fact that mature erythrocytes lack a nucleus and therefore normally do not contain nucleic acids. Infected RBC contain DNA and RNA from Plasmodium, and they are therefore easily discriminated from the non-infected population using the great variety of nucleic acid staining dyes (Barkan et al., 2000, Bianco et al., 1986, Bhakdi et al., 2007, Howard et al., 1979, Jimenez-Diaz et al., 2005). This has been done most successfully with cultured human RBC infected with Plasmodium falciparum where only small numbers of reticulocytes containing small amounts of ribosomal RNA can interfere with the signal (Bianco et al., 1986, Whaun et al., 1983, Hare and Bahler, 1986). Similarly some nucleic acid staining dyes, especially acridine orange, have also been successfully utilized in microscope-based differential fluorescent techniques to determine parasitemia (Keiser et al., 2002, Xu and Chaudhuri, 2005).

Low specificity of the dyes used and inconsistency of the results have posed problems for using flow cytometry in experimental rodent malaria models i.e., rats or mice infected with Plasmodium berghei, Plasmodium yoelii or Plasmodium chabaudi. Here, high numbers of reticulocytes are frequently seen in particular in samples from chronically ill animals, decreasing the sensitivity of the method. This can be overcome by digesting reticulocyte RNA with RNAse (Barkan et al. 2000) or by the use of a specific DNA-dye, such as Hoechst 33258. The latter has been used successfully with P. berghei-infected murine samples (Howard et al., 1979) but requires a time consuming staining procedure and the use of an ultraviolet laser, which is not part of most common flow cytometres.

Recently a new method using acridine orange staining and two-channel flow cytometry has been shown to clearly discriminate between infected and uninfected RBC populations (Bhakdi et al., 2007).

In preliminary studies of incorporating this method into our work it became clear that the discrimination between uninfected and infected RBC could be difficult when reticulocyte counts were elevated (Hein-Kristensen et al., unpublished). For our work we use P. berghei, which preferably invades reticulocytes (Cromer et al., 2006). This rodent parasite is used extensively for rodent models of cerebral malaria and hyperparasitemia (Li et al., 2001). In both cases infection leads to enhanced erythropoietic activity in the animals, leading to increasingly more immature RBC becoming infected thus aggravating the severe anemia and the reticulocytosis (Chang and Stevenson, 2004).

In order to assess the practical application of the acridine orange technique in the facilitation of scientific work with rodent malaria models, we conducted a study of the method with special focus on samples from animals with hyperparasitemia or high numbers of reticulocytes or both.

Section snippets

Animals and reagents

C57BL/6j and Balb/c mice were obtained from Taconic Laboratories (Lille Skensved, Denmark), SPRD rats were obtained from Charles River Laboratories (Sulzfeld, Germany) and DA rats from Harlan (Horst, The Netherlands). All animals were kept on pelleted diet and sterile tap water ad libitum under standard conditions at a room temperature of 25 °C. All experiments were approved by the National Board of Animal Studies and followed the international guidelines for animals in experimental studies.

Results and discussion

The FL3/FL1 protocol made it possible to distinguish between the non-infected and infected RBC populations. Infected samples plotted in an easily recognizable pattern as showed in Fig. 1. The percentage of parasitized red blood cells was determined by gating the population. The infected red blood cells and the uninfected reticulocytes often plotted very close making the need for strict gating obvious. The method also offered the possibility to identify other types of blood cells (Fig. 1).

The

Application and limitations

Flow cytometry has the potential of becoming the main analytical tool for parasite counting in experimental malaria. The newly described protocol is a fast and promising method for parasitic enumeration, which greatly reduces the time spend on microscopy. The present paper shows that the method is, however, most accurate when dealing with newly infected animals. When dealing with samples from anemic animals with enhanced erythropoietic activity and suffering from a prolonged infection, the

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