The most common objects of investigation for cytometric analysis, may it be image or flow cytometry, are leukocytes and cells or cell constituents isolated from cells or tissues. However, there are more than just leukocytes to be looked at in the world of cytometry.
Erythrocytes, as the transport vehicles for oxygen, are of central relevance in several red blood cell-related diseases. This includes anemia resulting from various acquired or congenital diseases, erythrocytosis, and erythrocyte deformation in sickle cell anemia or spherocytosis, among others. These diseases may lead to altered erythrocyte count, reduced hemoglobin content, shape alterations, or alterations of the cells' elasticity. All of these changes can be analyzed quantitatively on the single erythrocyte level using the cytometry approach. Possibly, the most advanced and challenging methods are those that take advantage of novel optical techniques and perform cytometry in vivo (1). By innovative in vivo flow cytometry technology, many of the abovementioned erythrocyte characteristics can be obtained (2) but it is still a glimpse into the future for most researchers. The image cytometry-based technology developed by Tomaiuolo and colleagues from Napoli, Italy (this issue, page 1040) is presently more accessible. The authors compared two flow-based image cytometry methods to measure the morphology of a large number of erythrocytes flowing through a large (50 μm, unbound) or small (4.7–10 μm, confined) capillary and images being captured by a high-speed camera. The authors report that their technique provides results comparable to standard technologies such as Coulter Counter with regard to erythrocyte volume, area and volume distribution. In addition, it renders additional information not available by standard methods such as deformation in small capillaries or sphericity index.
Philipp and colleagues from Kiel, Germany (this issue, page 1048) addressed the question of how to isolate viable Malaria parasite (Plasmodium falciparum) &!hyphen;infected erythrocytes by fluorescence activated cell sorting. In the past, many researchers developed flow cytometry methods to analyze different stages of development of Plasmodium spec. within the erythrocyte. The majority of these methods rely on staining the DNA of the parasite by a DNA-specific fluorochrome and as an example, the dye YOYO-1 can clearly distinguish different stages of development (3). However, not all of these dyes are suitable for life sorting because labeling, among others, may need erythrocyte permeability. Philipp and colleagues tested three different DNA dyes and reported that Vybrant DyeCycle Violet, a fluorochrome specific to the violet spectral range, provides best results with regard to sensitive detection and viable sorting of erythrocytes harboring immature parasites. Test results for in vitro maturation and reinfection rate of these sorted early developmental stage parasites is identical to that of unsorted controls. This new improved method provides an important tool for the investigation of P. falciparum maturation and for therapeutic drug development to fight malaria.
In bacteriology, a small but scientifically strong and active cytometry community is pushing the limits to improve the analysis and functional characterization of mixed bacterial communities (see as example: Ref. 4). In clinical reality, mixed infections by different bacterial strains are the usual situation. Rüger and colleagues from Magdeburg and Köthen, Germany (this issue, page 1055) raised the question regarding how to discriminate two different bacterial strains in mixtures and assess viability for both cell types by flow cytometry. To this end, they developed a three color assay for discriminating Staphylococcus aureus and Burkholderia cepacia, two clinically relevant strains. They combined staining regimens of SYBR Green I to label both cell types, propidium iodide for dead cell discrimination and fluorescence labeled wheat germ agglutinin to discriminate between Gram negative and positive bacteria. This novel method is validated by the authors and allows unequivocal discrimination of both strains to quantitate their function by using an elaborate gating strategy. Such assays are important steps forward towards analysis of population dynamics of mixed bio-communities for drug discovery in medicine but also as a research tool in additional scientific areas.
In plant biology, the relevance of flow cytometry was, and is, to rapidly determine genome size of plants in comparison with each other and for measuring their stage of development (5). In five different Fabaceae spec., Rewers and Sliwinska from Bydgoszcz, Poland (this issue, page 1067) raised the question, which calculation method is optimal for quantitatively reporting successful plant development based on flow cytometric measurement of genome size heterogeneity? The standard reporting is the ratio of tetraploid (4C, proliferating) to diploid (2C) nuclei. Probably and not surprisingly, the authors found that result in the maturing plant embryo, but much more pronounced in the cotyledons, a substantial proportion of the cells are highly endopolyploid with up to 64C or even 128C, depending on the species analyzed and its stage of development. Therefore, the authors propose a novel reporter for development, namely, the >2C/2C nuclei ratio which correlates better with the biological reality.
Cytometry is probably not the most common tool in agronomy and food industry but there are some important questions where it can provide answers. In the quality control of raw milk, quantitation of somatic cells is of importance to determine the health status of the cow. Inflammations or infections will substantially increase the concentration of these cells. A major problem herein is that the milk contains various particles of noncellular origin (lipid vesicles, debris) that are similar in size to somatic cells. Grenvall and colleagues from Lund, Denmark, and Seoul, Korea (this issue, page 1076) developed a new microfluidic method to clean up the milk from debris. The authors demonstrate that cleaning up the milk by acoustophoretic preprocessing removes undesired events, effectively allowing precise cell counting by Coulter Counter or flow cytometry. This cleaning procedure is less stressful for the cells than traditional methods such as centrifugation or chemical treatment.
Summarizing, this end-of-the-year issue provides a plethora of innovations in flow and image cytometry outside the well-known immunological paths. In my view, cross-pollination between these fields is highly relevant for steadily improving the abilities of cytometry to cover the whole area of biology. And more will come!