While inter-site CVs were relatively higher in site-stained populations (Figure 4F), suggesting a contribution of test thawing and control methods towards experimental variability, these were still 15% for many populations, apart from basophils, a rare cell type relatively. Open in another window Figure 4: Last harmonized pipeline and protocol evaluated across sites predicated on PBMCs.Major cell populations were gated, and general phenotypic distributions were visualized using tSNE (A-B). by manual gating and computerized methods (Astrolabe). Outcomes: Typical coefficients of variant (CVs) across sites for every cell population had been reported and in comparison to a earlier multisite CyTOF research. We reached an inter-site CV of under 20% for some cell subsets, nearly the same as a published research previously. Conclusions: These outcomes establish the capability to reproduce CyTOF data across sites in multi middle clinical trials, and high light the need for quality control methods also, like the usage of spike-in control examples, for monitoring variability with this assay. Intro The Cancer Defense Monitoring and Evaluation Centers – Cancer Immunologic Data Commons (CIMAC-CIDC) Network was launched in September 2017 through the National Cancer Institute (NCI) Cancer MoonshotSM Initiative. Its goal is to identify biomarkers for optimizing immunotherapeutic strategies for cancer patients. The four CIMACs are composed of the Icahn School of Medicine at Mount Sinai (MSSM), the Dana-Farber Cancer Institute (DFCI), MD Anderson Cancer Center (MDACC), and Stanford University (https://cimac-network.org/). To accomplish its goal, the CIMACs offer a variety of harmonized immune-monitoring and genomic assays, which are applied to study responses to immunotherapies across multiple clinical trials. The sites have adopted cytometry by time-of-flight (CyTOF) as the assay of choice for high-dimensional single-cell immune monitoring and biomarker discovery of CHMFL-ABL/KIT-155 cells circulating in the blood. Mass cytometry largely avoids the issues of overlap between detection channels, as seen in high-dimensional flow cytometry, and thus allows the routine use of over 40 simultaneous markers to perform high-throughput single-cell analyses (1C3). Since the first publication of comprehensive immunological data generated by CyTOF (4), most translational studies adopting mass cytometry have been performed in a single lab at a single site (5). Few studies have tested machine-to-machine variation, although in recent years the use CHMFL-ABL/KIT-155 of normalization beads to account for intra-instrument day-to-day variation has become common practice CHMFL-ABL/KIT-155 (6,7).At least two multicenter studies have been reported (8), and one multicenter CyTOF assessment compared staining and instrument performance consistency (9). While challenging to coordinate, multicenter immune-monitoring studies have been successfully performed using fluorescent flow cytometry (10C12). To ensure consistency in sample collection and processing and reproducibility of the data across the Network, the CIMACs have validated and harmonized sample collection protocols, immune-phenotyping panels, and staining and acquisition protocols across sites. A reference panel for all major immune cell populations and best practices for tracking batch variability have been previously reported by our group (13). The data presented in this publication represent the completion of iterative efforts to integrate laboratory-specific mass cytometry protocols across CIMAC sites, followed by a multi-site experiment designed to evaluate reproducibility using the harmonized protocol. Our aim here was to at least match the level of concordance reached using immune monitoring by flow cytometry (12). The experiment utilized a combination of healthy donor PBMCs Rabbit Polyclonal to A26C2/3 and lyophilized reference standards and was designed to evaluate both intra-site and inter-site reproducibility. The harmonization process also focused on identification of variables that impact assay performance, including those attributable to sample preparation, staining step, or those associated with mass cytometry data acquisition. We demonstrated that cross-site harmonization is feasible by means of integration of laboratory-specific protocols, schematically shown in figure 1, allowing identification and improvement of variables relevant to assay performance. We observed that, in CyTOF analysis, experimental rigor, gating strategies, and data reporting play an important role in data comparability. In addition, we present the metrics (coefficient of variation (CV) of cell frequencies, variance component analysis) that proved to be essential in validating the performance of the assay across different sites. Finally, we further discussed the need for internal and cross-site controls in each experiment for continued monitoring ofconcordance between sites. The harmonized CyTOF protocol is provided at https://cimac-network.org/ for access and potential application by the larger scientific community. Open in a separate window Figure 1: Schematic of material used for second round of cross-site harmonization CHMFL-ABL/KIT-155 study.All sites stained cells with lyophilized 14-marker.