Development and validation of a high-parameter mass cytometry workflow to decipher immunomodulatory changes in celiac disease.

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.

Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes.

BACKGROUND: Cryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays. Many factors can affect the quality of PBMCs, and careful consideration and validation of an appropriate PBMC isolation and cryopreservation method is important for well-designed clinical studies. A major point of divergence in PBMC isolation protocols is the collection of blood, either directly into vacutainers pre-filled with density gradient medium or the use of conical tubes containing a porous barrier to separate the density gradient medium from blood. To address potential differences in sample outcome, we isolated, cryopreserved, and compared PBMCs using parallel protocols differing only in the use of one of two common tube types for isolation. METHODS: Whole blood was processed in parallel using both Cell Preparation Tubes™ (CPT, BD Biosciences) and Lymphoprep™ Tubes (Axis-Shield) and assessed for yield and viability prior to cryopreservation. After thawing, samples were further examined by flow cytometry for cell yield, cell viability, frequency of 10 cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production. RESULTS: No significant differences in cell recovery, viability, frequency of immune cell subsets, or T cell functionality between PBMC samples isolated using CPT or Lymphoprep tubes were identified. CONCLUSION: CPT and Lymphoprep tubes are effective and comparable methods for PBMC isolation for immunological studies.