Light depolarization measurements in malaria: A new job for an old friend

The use of flow cytometry in malaria research has increased over the last decade. Most approaches use nucleic acid stains to detect parasite DNA and RNA and require complex multi-color, multi-parameter analysis to reliably detect infected red blood cells (iRBCs). We recently described a novel and simpler approach to parasite detection based on flow cytometric measurement of scattered light depolarization caused by hemozoin (Hz), a pigment formed by parasite digestion of hemoglobin in iRBCs. Depolarization measurement by flow cytometry was described in 1987; however, patent issues restricted its use to a single manufacturer's hematology analyzers until 2009. Although we recently demonstrated that depolarization measurement of Hz, easily implemented on a bench top flow cytometer (Cyflow), provided useful information for malaria work, doubts regarding its application and utility remain in both the flow cytometry and malaria communities, at least in part because instrument manufacturers do not offer the option of measuring depolarized scatter. Under such circumstances, providing other researchers with guidance as to how to do this seemed to offer the most expeditious way to resolve the issue. We accordingly examined how several commercially available flow cytometers (CyFlow SL, MoFLo, Attune and Accuri C6) could be modified to detect depolarization due to the presence of free Hz on solution, or of Hz in leukocytes or erythrocytes from rodent or human blood. All were readily adapted, with substantially equivalent results obtained with lasers emitting over a wide wavelength range. Other instruments now available may also be modifiable for Hz measurement. Cytometric detection of Hz using depolarization is useful to study different aspects of malaria. Adding additional parameters, such as DNA content and base composition and RNA content, can demonstrably provide improved accuracy and sensitivity of parasite detection and characterization, allowing malaria researchers and eventually clinicians to benefit from cytometric technology.