Highly detailed scrutiny of cellular phenotypes and functions within very complex and heterogeneous types of cells in biological systems such as leukocytes of the peripheral blood is still technically challenging but rewarding too. From one perspective, it appears as if adding more and more colors just because it is nowadays technically feasible is a virtue of its own sake. These developments have been fostered by the availability of more and improved light sources including LEDs, lasers, improved detecting systems, increased number of different (commercially) available fluorochromes, improved computing power, and more. The first published optimized immunophenotype panel (OMIP) (1), consisted of 16 different cell phenotype/function markers and was based on 15-color flow cytometry (2). One may rightly question if these technically driven advances are done because they became possible or if they are the driving forces to gather new knowledge about nature.

Indeed, it turns out that getting into the in-depth details of all subtypes is essential, particularly with regard to the potentials of cell therapy. In recent years, we began to understand the complex interrelationships and regulatory effects of (mostly of highly infrequent) cell subsets. This includes the regulatory T cells and their various phenotypically and functionally different subsets. Now, Murdoch and colleagues from Duke University, Durham, North Carolina, USA (this issue, page 281) developed a nine-color OMIP to further characterize the various sub-types of regulatory T-cells in cryopreserved samples. This panel partially varies from the recently published OMIP-004 (3), and also attempts to further understand the kinetics of regulatory T-cells subsets in health and disease.

Critical for use of specific in vitro modified or even generated immunomodulatory cells is that these obtained cell products exert the functions that are expected from them. But additionally, it is important to have a measure of the heterogeneity within the cell population that is intended to be introduced into the patient in future therapeutic applications. Functionally important for NK cells is their ability to kill cancer cells. Zamai and colleagues from Urbino and L'Aquila in Italy (this issue page 294) investigated NK cells generated in vitro from hematopoietic progenitor cells with respect to this function as compared to native cells of similar phenotype. The authors find that these in vitro generated NK cells resemble “natural” activated NK cells and are highly cytotoxic as well as resistant to apoptosis. Population heterogeneity quantitation, which is important as quality control for cell therapy, is easily done by flow cytometry and demonstrates the importance of a detailed knowledge of NK cell development and phenotypes.

Polychromatic flow cytometry is also, in addition to the above mentioned applications, useful for understanding the mode of action of drugs in humans. One such example was published recently by us on the modulating effect of methylprednisiolone on the immune response to heart surgery in children (2). Another important example is the work presented by Roat and colleagues from Modena and Milano in Italy (this issue page 303). The research group developed a polychromatic eight-color panel for peripheral blood mononuclear cells (PBMCs), to test differences in the efficacy of two standard immunosuppresiva used in transplant patients. The report shows marked differences in the immunomodulatory effect of both drugs in liver transplant patients with respect to maintenance or elevation of the number of circulating regulatory T cells and naïve T cells allowing us to decide which drug is preferable. These findings will also have implications for other organ transplantations and possibly for autoimmune diseases as well.

Essential for scrutinizing cells systems and stem cells (5) by (polychromatic) flow cytometry are monoclonal antibodies as well as functional assays. This is reviewed by Greve and colleagues from Münster and Düsseldorf in Germany (this issue, page 284) for circulating tumor cells, CTCs, and an upgrade of the present knowledge of phenotypes including methodological aspects is presented. The authors demonstrate that CTCs are not only still an issue of controversy but they are also relatively heterogeneous in nature depending on the tumor entity they have been identified in or isolated from.

Finally, critical for (stem) cell identification is the availability of a library of specific markers, mostly monoclonal antibodies. Whereas for men and mice a plethora of monoclonal antibodies for hundreds of surface and intracellular antigens exists, this is not the case for other mammalian and vertebrate models. This renders the possibilities for (stem) cell research in large animals very limited and clearly more antibodies against cell surface antigens are desperately needed. Production of monoclonals specific for species-related antigens is tedious, time consuming and expensive. Therefore, De Schauwer and colleagues from Gent University in Belgium (this issue, page 312) approached their object of interest, mesenchymal stem cells (MSC) of the horse, from the discovery standpoint. The authors screened 30 commercially available monoclonals for their binding of equine MSC. Surprisingly, eight antibodies exhibited specific binding of antigens on these MSCs and may be used in future studies to better characterize MSCs in heterogeneous samples. Unfortunately, the authors could not find any commercial antibody against CD34 that did also bind equine stem cells. Even so, their study is very promising as it contains the hope that screening for antigens of other equine cell types as well as cell subtypes in other mammals may end up with cross-reactive commercial antibodies useful for cellular research.

Polychromatic cytometry enables for discoveries in unexplored complex cell systems, the cytome. But because biological systems are dynamic by nature, kinetic cytome measurement seems to be obviously more representative of complex cellular behaviors than one snap-shot. This is in particular the case when cells systems are stimulated by viruses or treated with drugs, among others. Gondois-Rey and colleagues from Marseille, France (this issue, page 332) demonstrate their work on ex vivo infection of PBMCs with influenza virus how complex the temporal changes in cell systems can become when analyzed by 12-color polychromatic panels. To quantify and visualize these novel multidimensional changes (and for the cytometry community), not yet common bioinformatics and data analysis tools have to be used or developed, as traditional two and three dimensional data display only insufficiently describe such data (6). The findings of the authors excellently demonstrate how complex changes within cells systems are. By the use of mixed cell systems like PBMCs not only direct activations but also indirect (mediated) effects of a stimulation can be observed in concert. Heat map displays render good at-a-glance representations of the actual situation and may be considered as diagnostic tools to determine temporal aspects of infection in future clinical diagnostic setups.

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