Antibody batch with the brightest SI on stained cells could further help balance the lyophilisation effect. Reproducible results, a key aspect in multicenter trials, require minimal intra- and inter-assay variability. In order to reduce assay variation, we combined the use of lyoplates with strict SOPs for sample handling, rigorous instrument QC, and reproducible instrument setup. CFP and LFP showed minimal intra-assay variability, suggesting that experimental replicates are not an absolute requirement for flow cytometry analysis. Importantly, LFP allowed a more accurate 10457188 detection of Tregs. This is relevant as Tregs gating is notoriously difficult and subjective [24]. The high dimensional data generated by multi-parameter cell analysis need to be analysed in an unsupervised, multidimensional, and fast manner 16574785 to overcome the subjectivity and non-reproducibility of manual gating and analysis. In recent years, several computational tools for analysis of flow cytometry data have been developed by different research groups (see [25] for a review). Two broad categories of these tools have recently been evaluated by the FlowCAP project [26]:(1) Clustering algorithms for automated identification of cell populations (e.g., [19,27,28]) and (2) Binary sample classification pipelines for identification of immunophenotypic Deslorelin web differences between two groups of samples (e.g., [21,29,30]).Lyoplate Flow Cytometry for Biomarker DiscoveryFigure 3. Computational analysis identifies novel cell populations. A. A cellular hierarchy for the selected immunophenotypes. The edge-toedge width demonstrates the amount of predictive power (AUROC) gained by moving from one node to another. The color of the nodes demonstrates the predictive power of the cell population. This shows that IL-10, IFN-c, CD25, and Foxp3 are the most discriminative markers; CD25 and Foxp3 were previously unidentified with conventional manual analysis. B. Manual analysis confirms differences obtained by computational analysis. Frequencies of CD25+, Foxp3+, IL-10+, and IFN-c+ live T cells are increased in lyoplate based flow cytometry platform (LFP) compared to conventional flow cytometry platform (CFP) experiments. Each dot represents the average of experimental triplicates at two different time points. Paired t test or Wilcoxon signed rank test was performed, **P,0.01, ***P,0.001. doi:10.1371/journal.pone.0065485.gThe pipeline used in this work has been designed for identification of cell populations that correlate with an external variable (e.g., a clinical outcome). Detailed descriptions are available elsewhere [19,21,23]. Briefly, the pipeline can incorporate the background knowledge of the human experts into the gating process. Then, tens of thousands of immunophenotypes extracted from each sample are tested for correlation with the external variable (in this case, 6560 cell populations from every FCS file were correlated with the reagent type). Finally, the selected immunophenotypes are organized in a hierarchical structure based on their most common parent populations. These hierarchies not only provide intuitive data visualization, but also aid in adjusting the trade-off between the number of markers included in identification of a cell population of interest and the statistical significance of the correlation with the external variable. This information can also help in the use of high-dimensional Tetracosactrin site datasets to guide the design of low-dimensional panels: for example a Time-of-Flight mass.Antibody batch with the brightest SI on stained cells could further help balance the lyophilisation effect. Reproducible results, a key aspect in multicenter trials, require minimal intra- and inter-assay variability. In order to reduce assay variation, we combined the use of lyoplates with strict SOPs for sample handling, rigorous instrument QC, and reproducible instrument setup. CFP and LFP showed minimal intra-assay variability, suggesting that experimental replicates are not an absolute requirement for flow cytometry analysis. Importantly, LFP allowed a more accurate 10457188 detection of Tregs. This is relevant as Tregs gating is notoriously difficult and subjective [24]. The high dimensional data generated by multi-parameter cell analysis need to be analysed in an unsupervised, multidimensional, and fast manner 16574785 to overcome the subjectivity and non-reproducibility of manual gating and analysis. In recent years, several computational tools for analysis of flow cytometry data have been developed by different research groups (see [25] for a review). Two broad categories of these tools have recently been evaluated by the FlowCAP project [26]:(1) Clustering algorithms for automated identification of cell populations (e.g., [19,27,28]) and (2) Binary sample classification pipelines for identification of immunophenotypic differences between two groups of samples (e.g., [21,29,30]).Lyoplate Flow Cytometry for Biomarker DiscoveryFigure 3. Computational analysis identifies novel cell populations. A. A cellular hierarchy for the selected immunophenotypes. The edge-toedge width demonstrates the amount of predictive power (AUROC) gained by moving from one node to another. The color of the nodes demonstrates the predictive power of the cell population. This shows that IL-10, IFN-c, CD25, and Foxp3 are the most discriminative markers; CD25 and Foxp3 were previously unidentified with conventional manual analysis. B. Manual analysis confirms differences obtained by computational analysis. Frequencies of CD25+, Foxp3+, IL-10+, and IFN-c+ live T cells are increased in lyoplate based flow cytometry platform (LFP) compared to conventional flow cytometry platform (CFP) experiments. Each dot represents the average of experimental triplicates at two different time points. Paired t test or Wilcoxon signed rank test was performed, **P,0.01, ***P,0.001. doi:10.1371/journal.pone.0065485.gThe pipeline used in this work has been designed for identification of cell populations that correlate with an external variable (e.g., a clinical outcome). Detailed descriptions are available elsewhere [19,21,23]. Briefly, the pipeline can incorporate the background knowledge of the human experts into the gating process. Then, tens of thousands of immunophenotypes extracted from each sample are tested for correlation with the external variable (in this case, 6560 cell populations from every FCS file were correlated with the reagent type). Finally, the selected immunophenotypes are organized in a hierarchical structure based on their most common parent populations. These hierarchies not only provide intuitive data visualization, but also aid in adjusting the trade-off between the number of markers included in identification of a cell population of interest and the statistical significance of the correlation with the external variable. This information can also help in the use of high-dimensional datasets to guide the design of low-dimensional panels: for example a Time-of-Flight mass.
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