Flow cytometry was originally developed by the United States Army during World War II for detection of airborne anthrax spores (Gucker et al., 1947
). The original cytometer passed an air stream through the machine to attempt to detect the bacteria. Improvements since that time have reduced the amount of sample required and increased the strength and number of lasers/filters which can be used to analyze cells. While high-end cytometry equipment remains comparatively expensive (Shapiro and Perlmutter, 2008
), there is an expanding understanding that to investigate infectious diseases these machines need to be used by endemic populations at or near the point of care. The information provided about cells by cytometers, cannot be discerned as easily or as quantitatively by other means. Because of the speed and amount of information it provides, cytometry is becoming particularly important for the study of malaria parasite growth and invasion because it overcomes the limitations of existing non-cytometric methods.
The study of malaria parasite infected cells historically relied on visualization of parasites in stained blood slides. It was not until the introduction of the Giemsa stain in 1904 (Giemsa, 1904
), that reliable microscopic examination of blood smears for diagnosis of circulating malaria parasites could be performed (Fleischer, 2004
). This rapidly became, and remains, the official gold standard for malaria diagnosis (Makler et al., 1998
). However, there are shortcomings in microscopic evaluation of malaria parasite growth and invasion particularly because of subjective inter-operator error. Significant levels of misdiagnosis have been demonstrated with microscopic detection of malaria (Li et al., 2007
) showing false positive rates as high as 36% and false negatives as high as 18% (Milne et al., 1994
). Factors such as microscopist training are only part of the problem, methods used in the creation and staining of slides from patient samples are also an issue. Therefore, there has been a long-standing need for improved methodology.
The use of radioactive hypoxanthine (HX) was developed in an attempt to reduce the subjective nature of microscopic assays of parasite growth (Desjardins et al., 1979
). This technique, which tracks the incorporation of tritiated HX into DNA as it is synthesized, could be used to perform high-throughput assays and was a large improvement compared with slide counting. Although widely used, this method also has several challenges including the need for radioactivity and it cannot differentiate when the H3-
purine is incorporated by human cells or by the parasites, which can lead to a higher background. In addition, HX uptake cannot measure parasitemia because its incorporation is dependent on DNA synthesis which only occurs in the later stages of the parasite life cycle (Yayon et al., 1983
). DNA synthesis in turn is dependent on the growth rate of the parasite strain being observed.
Other methods for monitoring malaria growth have used plate readers to detect the presence of indicators of DNA quantity or enzyme activity in ELISA based assays. Detecting DNA levels with these methods involved the lysis of parasite cultures after exposure to drugs of interest and then comparing the total DNA content within each sample well using fluorescent DNA stains such as PicoGreen (Corbett et al., 2004
; Quashie et al., 2006
). The detection of parasite enzyme activity on the other hand has focused on the parasites’ lactate dehydrogenase (pLDH) which metabolizes 3-acetyl pyridine NAD (APAD) faster than human erythrocyte native LDH. However, field studies showed a low degree of concordance between this method and standard microscopy based determinations of parasitemia (Knobloch and Henk, 1995
; Jelinek et al., 1996
). Alterations have been made to improve the specificity of this assay using monoclonal antibodies (which are in limited supply) against LDH (called the DELI assay : double-site enzyme-linked LDH immunodetection) which showed results similar to Hypoxanthiene uptake (Moreno et al., 2001
). By switching focus to the parasite’s histidine-rich protein 2 (HRP2), researchers were able to maintain a good correlation with hypoxanthine uptake (Desakorn et al., 1997
). However, this new assay, while more readily available and therefore useful in the field, requires a longer incubation time (72 hr). All of these assays have the advantage of being able to be performed in the field using patient blood samples (ex vivo
) to test for drug resistance or invasion inhibition while the patient is still nearby. However, these assays also have the same shortfall swhich are that they cannot be stage specific, once the enzymes are expressed there is no way to detect parasite death, and for parasites which are quiescent, these assays are uninformative.
Flow cytometry based assays address all of the above challenges presented by microscopy, HX uptake, total DNA content detection, and both enzyme and antibody based assays and have become crucial to the study of malaria because of the objective, high content/moderate throughput assays that can be performed. As the cost of cytometers decreases, they will play a larger role in parasite evaluation in malaria endemic countries. Cytometry will also contribute to epidemiologic assessments and direct evaluation of patient’s malaria infection status, and drug resistance parasite status. Evaluation of drug/antibody efficacy and growth inhibition by flow cytometry will be crucial for the development of new antimalarial drugs and for erythrocyte stage vaccine candidates. The purpose of this review is to serve as a primer of new malaria cytometry users and as a reference to experienced users by presenting the historical background of its uses in malaria, current methods and their pitfalls, as well as future possibilities for the field.