A Workflow For Longitudinal Biomarker Discovery In Vascular Translational Research

Accurate immune monitoring in human disease is critical for identifying biomarkers that can predict clinical outcomes and inform therapeutic strategies. Two key aspects for identifying promising biomarkers are 1) comprehensive analysis of all major peripheral immune cell subsets, and 2) tracking the immune trajectory over the course of an immunological insult. With specific regard to tracking immune responses, longitudinal studies are increasingly important for phenotyping cells and identifying markers affected by storage, which necessitate real-time staining and acquisition. 

To address the need for longitudinal immune biomarker discovery, we developed and applied a streamlined workflow to characterize circulating immune cells in real-time using cells harvested during major vascular surgery. Although often necessary, this type of surgery is frequently performed on high-risk, highly comorbid patients who are particularly vulnerable to adverse postoperative outcomes. This context presents an ideal model for evaluating immune biomarkers, as it involves a defined but variable immunological insult and there is an unmet need for predictive biomarkers to identify high-risk individuals.  

In this webinar, we present the optimization of four spectral flow cytometry panels to comprehensively phenotype T, B, and Natural Killer cells (26 markers), monocytes and dendritic cells (22 markers), neutrophils (19 markers), and platelets (5 markers). All panels were optimized on freshly isolated cells that were minimally handled to avoid artificial platelet and erythrocyte aggregation. We will highlight the final workflow, which includes refining our cell isolation protocols, testing alternative staining reagents, and the use of sequential staining to preserve marker expression in these high-parameter panels. To ensure reproducibility across real-time experiments, we will show that cryopreserved cells, including granulocytes, are effective batch controls. We will also showcase preliminary data where our platform is currently deployed to track the immune response. This work underscores the feasibility and power of real-time cytometry for longitudinal biomarker discovery in translational research.

Key topics discussed in this webinar will include:

• Development and optimization of high dimensional spectral flow cytometry panels for biomarker discovery
• Evaluation of immune cells in real time for longitudinal studies
• Using cryopreserved cells for effective batch controls

Who should attend:

• Researchers interested in biomarker discovery for immunological insults
• Scientists considering the use of spectral flow cytometry for translational research
• Anyone interested in flow cytometry applications

For Research Use Only. Not intended for use in diagnostic procedures.