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Updated: Jun 21, 2026
Sample Preparation for Mass Cytometry Analysis
Published on: April 29, 2017
Immune Profiling Mass Cytometry Assay Harmonization: Multicenter Experience from CIMAC-CIDC
Bita Sahaf1, Mina Pichavant2, Brian H Lee3
1Stanford Cancer Institute, Stanford Medicine, Stanford University, California. bsahaf@stanford.edu.
Insights
Harmonizing standard operating procedures and using control samples enabled reproducible cytometry by time of flight (CyTOF) data across multiple Cancer Immune Monitoring and Analysis Centers (CIMAC) laboratories for cancer immunotherapy clinical trials.
Area of Science:
- Immunology
- Biomarker Discovery
- Clinical Trials
Background:
- The Cancer Immune Monitoring and Analysis Centers - Cancer Immunologic Data Commons (CIMAC-CIDC) Network utilizes advanced assays to identify biomarkers for cancer immunotherapy response.
- Cytometry by time of flight (CyTOF) is a key technology employed across all CIMAC laboratories for these studies.
- Ensuring data comparability across different sites is crucial for multicenter clinical trials.
Purpose of the Study:
- To establish a multistep cross-site harmonization process for CyTOF data generation within the CIMAC-CIDC Network.
- To ensure the ability to generate comparable CyTOF data across all participating CIMAC laboratories.
- To validate harmonized standard operating procedures (SOPs) and assay protocols, including a new vendor-introduced acquisition protocol.
Main Methods:
- Harmonization of standard operating procedures (SOPs) across all CIMAC sites.
- Testing of a new vendor-provided acquisition protocol (wide-bore injector) across sites.
- Cross-site assay harmonization experiments using shared cryopreserved and lyophilized peripheral blood mononuclear cells (PBMCs) and a standardized antibody cocktail.
- Centralized data analysis using both manual gating and automated methods (Astrolabe).
Main Results:
- Achieved an inter-site coefficient of variation (CV) below 20% for most cell subsets, comparable to previous multisite CyTOF studies.
- Demonstrated successful harmonization of CyTOF assay performance across multiple CIMAC laboratories.
- Validated the effectiveness of harmonized SOPs and the use of shared control materials.
Conclusions:
- The implemented cross-site harmonization procedures enable reproducible CyTOF data generation in multicenter clinical trials.
- Quality control measures, including the use of spike-in control samples, are essential for managing assay variability.
- These findings support the reliable application of CyTOF for biomarker discovery in cancer immunotherapy research across distributed sites.
Purpose:
The Cancer Immune Monitoring and Analysis Centers - Cancer Immunologic Data Commons (CIMAC-CIDC) Network is supported by the NCI to identify biomarkers of response to cancer immunotherapies across clinical trials using state-of-the-art assays. A primary platform for CIMAC-CIDC studies is cytometry by time of flight (CyTOF), performed at all CIMAC laboratories. To ensure the ability to generate comparable CyTOF data across labs, a multistep cross-site harmonization effort was undertaken.
Experimental Design:
We first harmonized standard operating procedures (SOPs) across the CIMAC sites. Because of a new acquisition protocol comparing original narrow- or new wide-bore injector introduced by the vendor (Fluidigm), we also tested this protocol across sites before finalizing the harmonized SOP. We then performed cross-site assay harmonization experiments using five shared cryopreserved and one lyophilized internal control peripheral blood mononuclear cell (PBMC) with a shared lyophilized antibody cocktail consisting of 14 isotype-tagged antibodies previously validated, plus additional liquid antibodies. These reagents and samples were distributed to the CIMAC sites and the data were centrally analyzed by manual gating and automated methods (Astrolabe).
Results:
Average coefficients of variation (CV) across sites for each cell population were reported and compared with a previous multisite CyTOF study. We reached an intersite CV of under 20% for most cell subsets, very similar to a previously published study.
Conclusions:
These results establish the ability to reproduce CyTOF data across sites in multicenter clinical trials, and also highlight the importance of quality control procedures, such as the use of spike-in control samples, for tracking variability in this assay.
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