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

TY - JOUR

T1 - In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei

AU - Hein-Kristensen, L

AU - Wiese, L

AU - Kurtzhals, J A L

AU - Staalsoe, T

N1 - Keywords: Acridine Orange; Animals; Blood Preservation; Female; Flow Cytometry; Fluorescent Dyes; Malaria; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Parasitemia; Plasmodium berghei; Rats; Rats, Sprague-Dawley; Rats, Wistar; Reticulocyte Count; Reticulocytes; Time Factors

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

U2 - 10.1016/j.exppara.2009.06.010

DO - 10.1016/j.exppara.2009.06.010

M3 - Journal article

C2 - 19545567

SN - 0014-4894

VL - 123

SP - 152

EP - 157

JO - Experimental Parasitology

JF - Experimental Parasitology

IS - 2

ER -