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

Abstract

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. V C 2015 International Society for Advancement of Cytometry Key terms Key terms: polarization; depolarized side scatter; hemozoin; flow cytometry; malaria; light depolarization MALARIA remains one of the most important parasitic diseases, killing around 700,000 people each year (1). Reliable detection of parasites and identification of the species are crucial for clinical diagnosis, as well as for many research applications. Both may require differentiation of asexual and sexual forms, determination of different maturation stages, and reliable quantification of parasite density (numbers per unit volume of blood or percentage of infected red blood cells). Antimalarial drug development and assessment of resistance to drugs may additionally require evaluation of parasite metabolic state and viability. Cytometry could potentially provide an alternative to microscopy of Giemsa stained smears (2), the accepted method for diagnosis for over a century.Malaria parasites growing inside infected ...

Efficient monitoring of the blood-stage infection in a malaria rodent model by the rotating-crystal magneto-optical method

“…Flow cytometric analysis was also performed for measurement series A and C ( Figs 3 and 4 , respectively). In these experiments two different parameters were assessed simultaneously: i) fluorescence of GFP expressing parasites 13 and ii) depolarized side scattering (DSS) properties of the hemozoin-containing red blood cells 17 18 . As an inherent advantage of flow cytometry, parasite counts can be determined in both cases directly.…”

“…The need for a universally applicable and automated method for diagnostics has motivated extensive research on malaria pigment (hemozoin), a natural biomarker of the infection. In particular, various magnetic and/or optical detection schemes targeting hemozoin have been proposed 17 18 19 20 21 .…”

Malaria diagnosis for malaria elimination

“…A number of recent studies have reported on strategies to exploit physical and/or electromagnetic features of hemozoin crystals to detect infection by malaria parasites [54,[80][81][82][83][84][85][86]. Generally, the LOD of these methods is between 1 and 30 iRBCs/ml and, therefore, not as sensitive as NAA strategies.…”

“…Further, hemozoin-containing monocytes observed by microscopy may provide insight regarding the total body parasite burden, as these cells do not sequester in P. falciparum infections [49,50]. Flow cytometric devices may also contribute new approaches for malaria diagnosis with emphasis on field deployment, however, further development of instrumentation is required [51][52][53][54][55]; limitations of speciesspecific detection must be acknowledged for flow cytometry.…”

Transdermal Diagnosis of Malaria Using Vapor Nanobubbles

“…An LOD can be obtained from serially diluted cultures or samples ( 3 ). In rodent models, detection of Hz tends to be much easier ( 4 ). Moreover, in Plasmodium falciparum infections, only immature forms have been observed, with little or no detectable Hz ( 5 ).…”