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Updated: Nov 21, 2025
Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry
Published on: June 26, 2018
Development and validation of a high-parameter mass cytometry workflow to decipher immunomodulatory changes in celiac
Jose Estevam1, Peter Krutzik2, Jason Vander Tuig2
1Clinical Biomarker Innovation & Development, Takeda Pharmaceuticals Inc. Co, Cambridge, Massachusetts, USA.
Insights
A new 33-marker mass cytometry panel was developed and validated to analyze immune cells in celiac disease (CeD) patients. This robust workflow aids in understanding immune responses in clinical trials.
Area of Science:
- Immunology
- Biotechnology
- Medical Diagnostics
Background:
- Time-of-flight mass cytometry (CyTOF) offers high-dimensional immune system analysis.
- Understanding immune cell dynamics in celiac disease (CeD) is crucial for clinical trials.
Purpose of the Study:
- To develop and validate a 33-marker mass cytometry panel for gastrointestinal (GI) trafficking peripheral blood mononuclear cells (PBMCs) in CeD patients.
- To establish a reliable workflow for high-dimensional immune cell analysis in clinical trial samples.
Main Methods:
- Development of a 33-marker CyTOF panel, including optimization of sample processing and antibody specificity.
- Validation using a fit-for-purpose approach, assessing analytical parameters like precision and stability.
- Application to peripheral blood mononuclear cells from celiac disease patients.
Main Results:
- A validated 33-marker mass cytometry panel for CeD patient PBMCs was successfully established.
- The workflow demonstrated robustness and reproducibility for high-dimensional immune cell analysis.
- Assay validation confirmed reliability for intra-assay, inter-assay, and inter-operator precision.
Conclusions:
- This validated mass cytometry workflow provides a robust tool for characterizing immune cells in CeD patients.
- The panel enables detailed analysis of immune responses in clinical trials, aiding in a better understanding of celiac disease pathogenesis.
- The established workflow supports reproducible, high-dimensional immune profiling of peripheral blood samples.
Abstract:
The advent of time-of-flight mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system to simultaneous interrogate a multitude of parameters and gain a better understanding of immunologic data from clinical trial samples. Here we describe the development and validation of a 33-marker mass cytometry workflow for measuring gastrointestinal (GI) trafficking peripheral blood mononuclear cells (PBMCs) in patients with celiac disease (CeD). This panel builds upon identification of well-characterized immune cells and expands to include markers modulated in response to gluten challenge in patients with CeD. The CeD panel was optimized and validated according to accepted industry practice for validation of flow cytometry assays and builds upon established sample processing workflows for mass cytometry studies. Several critical parameters were evaluated during the assay development phase of this study including optimization of the sample processing steps, antibody specificity, and ensuring the panel as a whole performed to expectation. The panel was then validated using a fit-for-purpose approach tailored to the intended use of the data in the clinical trial. Validation included assessment of analytical parameters essential to understanding the reliability and robustness of the CeD panel such as intra-assay precision, inter-assay precision, inter-operator precision and sample processing stability. Together, this validated mass cytometry workstream provides robust and reproducible high-dimensional analysis of human peripheral blood immune cells to characterize patient samples from clinical trials.
