It's not just leukocytes in cytometry

Abstract

most common objects of investigation for cytometricanalysis, may it be image or flow cytometry, are leukocytesand cells or cell constituents isolated from cells or tissues.However, there are more than just leukocytes to be looked atin the world of cytometry.Erythrocytes, as the transport vehicles for oxygen, are ofcentral relevance in several red blood cell-related diseases. Thisincludes anemia resulting from various acquired or congenitaldiseases, erythrocytosis, and erythrocyte deformation in sicklecell anemia or spherocytosis, among others. These diseasesmay lead to altered erythrocyte count, reduced hemoglobincontent, shape alterations, or alterations of the cells’ elasticity.All of these changes can be analyzed quantitatively on the sin-gle erythrocyte level using the cytometry approach. Possibly,the most advanced and challenging methods are those thattake advantage of novel optical techniques and perform cyto-metry in vivo (1). By innovative in vivo flow cytometry tech-nology, many of the abovementioned erythrocyte characteris-tics can be obtained (2) but it is still a glimpse into the futurefor most researchers. The image cytometry-based technologydeveloped by Tomaiuolo and colleagues from Napoli, Italy(this issue, page 1040) is presently more accessible. Theauthors compared two flow-based image cytometry methodsto measure the morphology of a large number of erythrocytesflowing through a large (50 lm, unbound) or small (4.7–10lm, confined) capillary and images being captured by ahigh-speed camera. The authors report that their techniqueprovides results comparable to standard technologies such asCoulter Counter with regard to erythrocyte volume, area andvolume distribution. In addition, it renders additionalinformation not available by standard methods such as defor-mation in small capillaries or sphericity index.Philipp and colleagues from Kiel, Germany (this issue,page 1048) addressed the question of how to isolate viableMalaria parasite (Plasmodium falciparum) infected erythro-cytes by fluorescence activated cell sorting. In the past, manyresearchers developed flow cytometry methods to analyze dif-ferent stages of development of Plasmodium spec. within theerythrocyte. The majority of these methods rely on stainingthe DNA of the parasite by a DNA-specific fluorochrome andas an example, the dye YOYO-1 can clearly distinguish differ-ent stages of development (3). However, not all of these dyesare suitable for life sorting because labeling, among others,may need erythrocyte permeability. Philipp and colleaguestested three different DNA dyes and reported that VybrantDyeCycle Violet, a fluorochrome specific to the violet spectralrange, provides best results with regard to sensitive detectionand viable sorting of erythrocytes harboring immature para-sites. Test results for in vitro maturation and reinfection rate ofthese sorted early developmenta l stage parasites is identical tothat of unsorted controls. This new improved method providesan important tool for the investigation of P. falciparum matura-tion and for therapeutic drug development to fight malaria.In bacteriology, a small but scientifically strong and activecytometry community is pushing the limits to improve theanalysis and functional characterizationof mixed bacterial com-munities (seeasexample: Ref. 4). In clinical reality, mixedinfec-tions by different bacterial strains are the usual situation. R€ugerand colleagues from Magdeburg and K€othen, Germany (thisissue, page 1055) raised the questionregarding how to discrimi-