Lectures and seminars BFC Flow Cytometry User Group Meeting: A Workflow for Longitudinal Biomarker Discovery in Vascular Surgery
Welcome to an online seminar with Dr Kathryn Hally, Senior Lecturer and group Lead, Surgical Immunology Research Group, University of Otago, New Zealand. Dr Kathryn Hally is a translational research scientist with expertise in immunology, cardiology, and surgery and extensive experience in high-parameter flow cytometry.
Speaker biography
Dr Kathryn Hally is a Senior Lecturer at the University of Otago in Wellington, New Zealand. She is a translational research scientist with expertise in immunology, cardiology, and surgery, and with extensive experience in high-parameter flow cytometry. She leads a multidisciplinary research group – the Surgical Immunology Research Group (SIRG) – that focuses on in-depth characterization of circulating human immune cells. The overarching goal of SIRG is to discover and validate cytometry-based immune biomarkers – particularly circulating innate immune cell phenotypes – that may serve as early predictors of adverse postoperative recovery.
Content
In the past year, our research group has sought to overcome a key limitation in biomarker discovery: the inability to accurately characterise certain immune cell subsets and activation markers following cryopreservation, requiring a workflow based on real-time staining and acquisition of fresh samples. We sought to develop a streamlined, real-time workflow for phenotyping all circulating immune cells that is suitable for longitudinal biomarker discovery in human disease.
In this talk, I will discuss our optimization of four spectral flow cytometry panels to comprehensively phenotype T, B, and Natural Killer cells (28 markers), monocytes and dendritic cells (23 markers), neutrophils (20 markers,) and platelets (7 markers). All panels were optimized on freshly isolated cells that were minimally handled to avoid artificial platelet and erythrocyte aggregation. I will highlight our optimisation that underpinned the final workflow, which included refining our cell isolation protocols, testing alternative staining reagents, and use of sequential staining to preserve marker expression in these high-parameter panels. To ensure reproducibility across real-time experiments, we show that cryopreserved cells, including granulocytes, are effective batch controls. Our protocol offers a solution for capturing immune dynamics in real time, and we currently utilize these panels to study the immune response to major vascular surgery. Finally, I will show some of our preliminary data from vascular surgery patients. This marks the beginning of our research into understanding how perturbations in this immune response predispose patients to adverse postoperative recovery.