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Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.

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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 ...