Flow cytometry is a powerful tool for clinical research, with its ability to measure multiple parameters on the same sample, collect information from millions of cells, and identify rare cell populations. However, transitioning a flow cytometry assay from the bench to the bedside can be challenging. Often, what works for a research project is not suitable for clinical studies and further development or planning is necessary for successful assay implementation.
In this article, we explore 3 critical considerations for moving from exploratory to clinical flow cytometry assays and share a case study highlighting the need for careful planning and creative solutions when implementing these assays in clinical trials.
Consideration #1: Subjectivity
Flow cytometry analysis can be subjective, and standardization is challenging since multiple, parameters are measured simultaneously, and internal controls are not always available. It is important to determine up front how panels will be set up, how data will be gated and analyzed, and how scoring will be determined. Building reference controls and employing a tertiary analyst to review data across an entire study can also help to minimize subjectivity.
Consideration #2: Robustness and Reproducibility
While the level of assay validation required will vary depending on how the resulting data will be used, any clinical flow cytometry assay should be robust and reproducible. There are two approaches to achieving the necessary robustness and reproducibility:
Setting up and standardizing multiple labs, which requires standardization of equipment and standard operating procedures (SOPs) and operator training across all these labs.
Testing at a central lab, which offers the most control but may require sample stabilization.
Achieving reproducibility where the results of one lab can be replicated by another lab with a high degree of consistency — involves eliminating as many sources of variation as possible. Variability in sample preparation, sample processing, instrument settings, reagents, antibodies, operators, and data analysis can all impact results. As such, generating SOPs and controls along with providing training and proficiency training are essential to standardization across labs.
Consideration #3: Logistics
Flow cytometry requires cells from either fresh, stabilized/fixed or cryo-preserved samples, so it is crucial to understand how long the cells of interest are intact and how storage and transit of samples may impact results. In the case of non-fixed cells, determining changes in cell viability of relevant populations can be challenging as cells die at different rates and activated cells tend to be more labile.
Sample stabilization offers the opportunity to prolong the stability window for flow cytometry analysis. There are multiple approaches to sample stabilization, which vary in ease of use and marker compatibility (see Figure 1).
When considering stabilization, feasibility studies are needed to evaluate whether and how the fixative modulates staining of each antibody. Moreover, the use of fixatives can increase site burden and complexity depending on the optimal fixative. Creating custom kits to simplify sample collection and stabilization may be necessary for ensuring consistency and compliance.
At Precision for Medicine, we provide an end-to-end solution, from custom kit creation and deployment to sample collection, logistics, and storage, to support flow cytometry studies (see Figure 2).
Case Study: Developing a clinical receptor occupancy (RO) assay for a novel monoclonal antibody
A sponsor with a first-in-class monoclonal antibody (MAb) targeting a co-stimulatory molecule that is highly expressed on CD4 and CD8 cells sought to develop a receptor occupancy assay to monitor target engagement and changes in receptor levels. Three challenges had to be addressed during the course of assay development:
Need to assess multiple cell types, since early data suggested that the expression of the target receptor differed by cell type and receptor engagement could also have different PK and a different response. To address these considerations, Precision for Medicine developed a flow panel with 10 colors to gate the various cell types.
Dispersed study geography, with clinical sites spread across the US, New Zealand and Australia. Based on the complexity of the flow assay and analysis, it was initially decided to run the study out of a single lab to optimize data quality. However, titration of the mAb into whole blood showed that samples would need to be tested within 48 hours of collection, making it infeasible to reliably transit to a single lab. Isolation of peripheral blood mononuclear cells (PBMCs) was tested as a potential solution since these cells could be cryo-preserved, but this approach adversely impacted RO data. Instead, Precision for Medicine tested multiple fixatives for stabilizing whole blood:
Cyto-Chex™ BCT tubes were not compatible with all markers in the panel
Smart Tube™ Proteomic fixative produced results that correlated with intra-assay, inter-assay, and inter-operator data from fresh whole blood at three different drug concentrations, though the total dynamic range was somewhat constrained (see Figure 3). Stability testing showed at least 120 days of stability.
Since stabilized samples could be stored long-term at −80 °C, they could be shipped to a single lab and run in a batch to control data consistency.
Implementation at clinical sites arose as a challenge due to the selection of Smart Tube™ Proteomic fixative, which comes in a 100 mL bottle rather than in a vacutainer. Precision’s solution involved building a custom sample collection kit with pre-aliquots of fixative and training each site to ensure they followed strict SOPs for using the fixative and storing stabilized samples.
Involving a translational clinical research organization and central lab service provider in assay planning, design, development, and implementation will help address the challenges associated with transitioning an exploratory assay to the clinical environment.
Pharma industry veteran and expert at biomarker-driven clinical trial design and execution. Leader of biomarker and drug development programs for pharmaceutical and diagnostics companies, as well as the National Institutes of Health. Spearheaded the discovery of pharmacodynamic biomarkers and novel targets for inflammatory disease therapy.
Precision for Medicine is part of the Precision Medicine Group, an integrated team of experts that extends Precision for Medicine’s therapeutic development capabilities beyond approval and into launch strategies, marketing communication, and payer insights. As one company, the Precision Medicine Group helps pharmaceutical and life-sciences clients conquer product development and commercialization challenges in a rapidly evolving environment.