The malaria pigment, or haemozoin (Hz), is gaining increasing attention, as has been reviewed recently [1, 2]: (i) Hz production is an important drug target, (ii) Hz appears to have immunomodulatory properties, (iii) detection of Hz-containing leukocytes allows diagnosis of malaria, and (iv) Hz-containing leukocytes appear to be associated with disease severity. However, one important drawback in this area of research is the fact that counting of Hz-containing leukocytes or the detection of Hz-containing parasitized red-blood cells (pRBCs) is based on microscopy [3, 4]. This is not only cumbersome, but introduces a significant statistical error if the number of Hz-containing leukocytes is low . An alternative to this is based on the detection of depolarized Side-Scatter by flow cytometry . Hz is birefringent and as a consequence rotates the plane of polarized light, a process called depolarization. LASER light, commonly used in flow cytometers, produces polarized light. Thus, by placing a polarization filter orthogonally (90° rotated) to the plane of the LASER light in front of a second SSC detector allows to detect depolarized light and consequently Hz.
Depolarized SSC detection is incorporated into the Cell-Dyn® haematology analysers (Abbott, Santa Clara, CA, USA) to differentiate eosinophils from granulocytes. As a result of this, these instruments detect Hz-containing leukocytes as well as Hz in parasitized red blood cells (pRBC) without need for modifications, [7, 8]. Unfortunately, the software analysis and analysis algorithms of the analysers cannot be accessed without the intervention of the manufacturer and thus, studies depended on counting the events (cells) on the instrument's screen or the printout, with consequent data loss and no option for further analysis of the raw data . However, if a simple modification of common flow cytometers created a reliable method for detection and analysis of Hz in leukocytes or pRBC it might open novel approaches for diagnostic applications and research.
For example, flow cytometric counting of Hz-containing leukocytes may be a better marker for disease severity than microscopy based counts [5, 10]. Detection and functional analysis of Hz-containing leukocytes may also help to elucidate further aspects on the immunomodulatory properties of Hz. In fact, most in-vitro studies use concentrations of Hz that maximize uptake by the majority of monocytes in a given cell population and compare them with a control population that was not exposed to Hz . However, the situation may be different in vivo, where both populations coexist. For example, a recent large study, based on microscopy, reported that the median percentage of circulating Hz-containing monocytes was only 2-5% .
Interestingly, monocytes are a heterogeneous cell population, even in peripheral blood [12, 13], mainly based on the CD14 and CD16 expression: classical "inflammatory" monocytes (CD14+/CD16-), "intermediate" monocytes (CD14+/CD16+) and "resident, pro-inflammatory" monocytes (CD14dim/CD16+). They also seem to differ functionally . Recent research showed differences in the ratio of these populations in malarious children with different types of malaria , as well as differences in surface marker expression and parasite inhibitory action in acute uncomplicated malaria . However, little is known whether Hz phagocytosis contributes to these findings in vivo. In fact, the interplay of Hz-containing and non-Hz-containing subpopulations might produce different biological results in vivo, when compared to those in vitro models, where nearly all monocytes contain Hz.
Another important area for flow cytometric detection of Hz could be the reliable detection of pRBC harbouring different Hz content. This may be useful for sensitivity testing and drug development, because the Hz-content increases proportional to the maturation of the parasite .
This paper describes a simple way to modify a standard bench-top flow cytometer to allow depolarized Side-Scatter measurements. The method was then tested for its usefulness to detect Hz-containing leukocytes and erythrocytes and examples for potential applications are presented.