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• W3011982004 abstract "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 heterogeneity, subtle changes associated to clonal evolution and the definition of rare cell subpopulations may need a more in-depth evaluation. The present studies, however, do not overlook the technical limitations that mass cytometry still suffers in its way to a wider diagnostic and clinical application as compared with FCM, such as the questionable estimation of cell size/diameter, the lack of a side scatter parameter, the long sample preparation procedures, the relatively slow acquisition rate to minimize coincidences, the high background connected to defects in the purification of some metal isotope tags and the long post-analysis time needed to digitally clean the datafile and to define all the cell subsets. In the paper by TK Soh (Soh et al., 2020) a series of technical comparisons of the methods that can be of help in maximizing the cell yield of bone marrow samples for minimal residual disease (MRD) analysis in multiple myeloma are illustrated. The need to collect several millions of clean cell events as the “denominator” for cell frequency calculations is mandatory in high-resolution FCM, rare event and MRD analysis, to ensure appropriate sensitivity levels. The usage of multi-color protocols, the collection of very large cell datafiles and the establishment of adequate numbers of relevant events for the reliable definition of the lower limits of detection and quantitation are the technical cornerstones of all high-resolution FCM analyses (reviewed by Brando et al., 2019). In their comparison experiments Soh and coworkers point out that the widely used bulk lysis method may artifactually reduce the number of B cell precursors and mast cells, which may influence the judgement of the quality of bone marrow samples and the estimated degree of peripheral blood contamination (Flores-Montero et al., 2017). The study by AM Eckel highlights the unusual finding of CD33 expression on NK cells, which may be a confounding factor in the study of measurable/residual AML (Eckel et al., 2020). NK cells can express CD33 and CD117 during their early development (Eissens et al., 2012; de Mel et al., 2018), but in normal adults such expression is relatively rare, being restricted to about 16–18% of peripheral blood or bone marrow samples, and usually at low percentage levels on NK cells. Such an occurrence has not been however highlighted in recent studies, despite the usage of very comprehensive NK marker panels (Del Zotto et al., 2017; Liechti et al., 2019). Since NK cells may fall into the CD45 vs Side Scatter “blast” gate, the occasional CD33+ NK cells can be erroneously enumerated as blasts in AML MRD studies, especially if AML blasts are CD34-negative. Interestingly, the CD33-expressing NK cells are consistently CD56+ bright and negative for CD16, like cord blood NK cells. The paper by DR Sutherland and coworkers (Sutherland et al., 2020) refines the recent guidelines on the analysis of PNH clones in the red blood cell (RBC) populations (Sutherland et al., 2018), by focusing on the analysis of immature RBC. Nucleated RBC and reticulocytes can be detected by FCM either by intracellular nucleic acid staining with thiazole orange or by the analysis of surface CD71 (the transferrin receptor). The two analytical approaches are almost coincident, since >95% of CD71+ immature RBC contain reticular material (Thomson-Luque et al., 2018; Malleret et al., 2013). Immature RBC represent a minor fraction of steady-state peripheral blood erythrocytes, but can increase following hemorrhage or hemolysis, provided the bone marrow erythroid machinery is healthy and reactive. In PNH studies the analysis of immature RBC performed shortly after a hemolytic episode may offer useful information on the relative proportion of healthy vs PNH erythropoiesis (Höchsmann et al., 2011), which may be quite variable among patients and difficult to assess when transfusions or eculizumab are administered. When accurately analyzed, the proportion of immature RBC PNH clones approaches the one of WBC, indicating that erythroid and white cell precursors have a similar proliferative advantage in PNH (Ware et al., 1995). In the study by Sutherland the utility of immature RBC analysis to confirm or exclude in selected conditions the presence of the often artifactual type II RBC PNH clones is highlighted. In recent years the reported limited availability or the high cost of the FLuorescent AERolysin (FLAER) reagent in certain countries (Sutherland et al., 2018) have stimulated the development of non-FLAER-based staining protocols for PNH studies. Anti-CD157 has been validated as a replacement for FLAER, to be used along with CD24 and CD14 to determine neutrophil and monocyte PNH clones, respectively (Sutherland et al., 2014; Marinov et al., 2018). In the study by Y Zhang the prospective clinical usage of a 7-color PNH test combining CD157, CD24, CD14 and FLAER is described (Zhang et al., 2020). As expected, in such highly controlled analytical conditions CD157 has been demonstrated as an overall acceptable substitute for FLAER, thus confirming the utility of this antibody in case FLAER is unavailable. However, this study further confirms that CD157 can generate a sizable fraction of false PNH positives, for technical reasons related to an imprecise gating (Sutherland et al., 2014), for ethnical/genetic variants (Blaha et al., 2018; Sutherland et al., 2019) and also for pharmacological interferences in HBS Ag + patients taking anti-Hepatitis B medication (Sutherland et al., 2019). The recently validated high-sensitivity tests for PNH analysis are very accurate and robust in detecting and enumerating small RBC and WBC clones. However, the most frequent analytical errors in high-sensitivity PNH testing still reside in problems with assay specificity, that may sometimes generate false “minor PNH clones” for a number of reasons, as concluded in the study by Zhang and also as highlighted in international external quality assessment studies (Brando et al., 2019)." @default.
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• W3011982004 date "2020-03-01" @default.
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• W3011982004 title "ISSUE HIGHLIGHTS ‐ March 2020" @default.
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• W3011982004 doi "https://doi.org/10.1002/cyto.b.21873" @default.
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