ISSUE HIGHLIGHTS ‐ March 2020

In this issue the review article by BEJ Spurgeon and KM Naseem (Spurgeon and Naseem, 2020) thoroughly discusses the technical requirements of platelet phosphoprotein network analysis, also applying the high-throughput technique of fluorescent cell barcoding. Phosphoprotein signaling is essential in driving the cell behavior and functions, and the measurement of the degree of phosphorilation of intracellular molecules, both in nucleated cells and platelets, provides information on the functional status of signal transduction pathways (Zimman et al., 2014). The “phospho-flow” techniques are being increasingly used in functional cell analysis since they offer many advantages over the more conventional western blotting assays, as Spurgeon and Naseem pointed out. The need of analyzing as many functional actors of the signaling network as possible, along with their respective positive and negative controls, has taken advantage from the application of the delicate fluorescent cell barcoding technique (Krutzik et al., 2011; Akkaya et al., 2016). Cell barcoding is a multiplex technique in which cells from various subjects or cells involved in different phenotypical or functional assays are pre-stained with multiple fluorescent tags and are then admixed to undergo the testing under the same working conditions in a simultaneous analysis batch while keeping the components of the cell mixture always distinguishable. This technique, once mastered, can accommodate up to 6 simultaneous subjects/assays (Spurgeon and Naseem, 2020), thus sparing reagent usage, working time and minimizing the analytical variability. In this review many valuable technical details for the optimization of phosphoprotein and barcoding assasy in functional platelet analysis are provided, that will be of help in extending the knowledge and the practice of these innovative techniques. A very special barcoding technique using palladium isotopes has been applied in the paper by GK Behbehani (Behbehani et al., 2020), where an extensive study of myelodysplastic syndromes (MDS) by 34-parameter mass cytometry is illustrated. Mass cytometry is a mature analytical technique that can be applied today outside its traditional research boundaries, as shown in many recent valuable papers on leukemia, myelodysplasia (Zeng et al., 2017; Das Gupta et al., 2017; Nissen et al., 2019; Wierz et al., 2018; Bandyopadhyay et al., 2019), immunological studies (Leipold et al., 2018) and even erythroid development (Thomson-Luque et al., 2018). Mass cytometry allows for the analysis of cell heterogeneity on a very large scale, theoretically measuring up to 100 different cell features simultaneously with metal isotope-tagged antibodies without any spectral overlap (Olsen et al., 2019). Such a huge array of analytical data requires however special softwares for the mapping of high-dimensional data and interpretation of the results (Amir et al., 2013, Levine et al., 2015; Bagwell et al., 2015), sometimes causing variations in the final results due to the features of their respective mathematical architecture (Amir et al., 2013; Levine et al., 2015; Li et al., 2019; Olsen et al., 2019). In the impressive study by Behbehani 20 barcoded samples were simultaneously analyzed for surface and intracellular markers in a single tube by mixing bone marrow samples from MDS patients and healthy controls, thus allowing a very consistent mapping of normal and abnormal cell subpopulations in a multidimensional space. This paper demonstrates that a mass cytometry approach may provide superior analytical sensitivity and specificity as compared to conventional flow cytometry in diagnostic applications requiring multidimensional data (Behbehani et al., 2020; Das Gupta et al., 2017), and also disclosed some hitherto undescribed phenotypic abnormalities related to MDS. The study by CB Bagwell (Bagwell et al., 2020) continues the standardization process of immune cell deep phenotyping promoted by the “Human ImmunoPhenotyping Consortium” some years ago (Maecker et al., 2012; Finak et al., 2016). The feasibility of multicenter studies on the deep phenotyping of functional immune cells by mass cytometry is brilliantly demonstrated here using a dried 30-marker array, enabling the quantitative and qualitative definition of 37 immune system cell subpopulations with good interlaboratory reproducibility. The added value of an integrated analysis system including instrumentation, dried isotope-tagged antibody mixtures and automated software for cell subclassification is emphasized here. An ever increasing role of single-cell high-dimensional analysis seems therefore likely also in the clinical setting, whenever intratumor Bruno Brando [Color figure can be viewed at wileyonlinelibrary.com] DOI: 10.1002/cyto.b. 21873