NWFCS Third Annual Meeting
NWFCS Third Annual Meeting
Daniel T. Chiu is the A. Bruce Montgomery Professor of Chemistry, Endowed Professor of Analytical Chemistry, Washington Research Foundation Professor, and Professor of Bioengineering at the University of Washington, Seattle. He is also a member of the Cancer Consortium at the Fred Hutchinson Cancer Research Center. He obtained a B.A. in Neurobiology and a B.S. in Chemistry from the University of California at Berkeley in 1993, then a Ph.D in Chemistry from Stanford University in 1998. After completing postdoctoral research at Harvard University, he started in the Fall of 2000 as an Assistant Professor of Chemistry at the University of Washington.
He is the author of more than 200 publications and is the inventor on over 200 issued patents. He is the recipient of various awards and honors, including the American Chemical Society National Fresenius Award and Pittcon Achievement Award. He was named a Fellow of the AAAS, Keck Distinguished Young Scholar in Biomedical Research, and an Alfred P. Sloan fellow. His research field of interest is nanomaterials, microfluidics, and new instrumentations for ultra-sensitive bioanalytical measurements and digital assays.
We have developed a multi-parametric high-throughput and high-sensitivity flow-based method for counting single molecules, and applied this method to the analysis of individual extracellular vesicles and particles (EVPs). EVPs are promising biomarkers but they are highly heterogeneous and comprise a diverse set of surface proteins as well as intra-vesicular cargoes. Yet, current approaches to the study of EVPs lack the necessary sensitivity and precision to fully characterize and understand the make-up and the distribution of various EVP subpopulations that may be present. Digital flow cytometry (dFC) provides single-fluorophore sensitivity and enables multiparameter characterization of EVPs, including single-EVP phenotyping, the absolute quantitation of EVP concentrations, and biomarker copy numbers. dFC has a broad range of applications, from analysis of single EVPs such as exosomes or RNA-binding proteins to the characterization of therapeutic lipid nanoparticles, viruses, and proteins. dFC also provides absolute quantitation of non-EVP samples such as for the quality control of antibodies (Ab), including the concentration of individual and aggregated Ab-dye conjugates and the Ab-to- dye ratio.
Lisa is a Research Associate on the Experimental Immunology team at the Allen Institute for Immunology, where she studies the role of B cells in health and disease. Her work leverages high-dimensional spectral flow cytometry, ex vivo immune cell cultures and assays, and immunofluorescence imaging of tissues and primary cells. Lisa earned her BS in Biochemistry from the University of Puget Sound. Prior to joining the Institute, she supported clinical and preclinical research in Maternal and Fetal Medicine at Madigan Army Medical Center in Tacoma.
B cells are adaptive immune cells that support durable and effective protection against a wide range of pathogens via antigen presentation as well as differentiation into memory B cells and antibody-secreting plasma cells. Understanding the composition of the human B cell compartment is necessary to effectively isolate and analyze B cells subsets in immune monitoring and systems immunology studies. Here, we present a guide to comprehensively phenotype human B cell subsets using high dimensional spectral flow cytometry. In this guide, we outline the heterogenous B cell identities detectable in peripheral blood and describe their unique surface phenotypes and cellular functions. We further provide guidance for cell surface molecule staining and cytometry data acquisition, informed by factors such as antigenic density, post-translational modifications, cellular activation state and staining conditions. Finally, we offer subset gating strategies and antibody panels that supported our recent analyses of B cells in the context of healthy human aging and pre-clinical Rheumatoid Arthritis.
Katharine Schwedhelm is a research manager for the HIV Vaccine Trials Network (HVTN) Endpoints laboratory at Fred Hutchinson Cancer Center in Seattle, WA. She gained experience in flow cytometry and instrumentation at Benaroya Research Institute before moving to Fred Hutch, where her focus has been directed towards high parameter panel development to support HIV and COVID-19 vaccine studies and cytometer standardization and characterization. Katharine also supports the Cape Town HVTN laboratory, facilitating panel transfers and ensuring cross-lab assay and instrument concordance, and has been an instructor at the African Flow Cytometry Workshop.
Flow cytometry is a powerful tool for monitoring immune function at the single-cell level. Over the years, it has increasingly become an indispensable component in the clinical trials setting, including in vaccine research, where it is used to measure cellular immune responses to candidate vaccine regimes. As instrument capabilities and available reagents have continued to improve and expand, so too has the number of markers able to be measured. Development of a successful high parameter panel depends on a fully characterized, optimized, and standardized instrument, knowledge of the instrument spillover/spreading matrix (SSM), appropriate fluorochrome-marker pairings based on antigen expression, optimized reagent concentrations, thorough scrutiny of the resolution of each marker, and appropriate use of controls, such as fluorescence minus one (FMO) controls. At the HIV Vaccine Trials Network (HVTN) cellular laboratory, each cytometer has undergone a rigorous quality control procedure to ensure that it is functioning optimally, followed by establishing standardized target median fluorescence intensity (MFI) values using hard-dyed calibration beads for each detector. The result is that the flow cytometers can be used interchangeably, batch-to-batch variability is minimized, and most importantly, the data produced are consistent and of the highest quality. In response to efforts to combat the SARS-CoV-2 pandemic through a safe and efficacious vaccine, the HIV Vaccine Trials Network (HVTN)/COVID-19 Prevention Trials Network (CoVPN) developed and validated a 27-color intracellular cytokine staining (ICS) flow cytometry assay to characterize antigen-specific T-cell responses to both SARS-CoV-2 natural infection and vaccine candidates, with a focus on sensitive detection of both Th1 and Th2 responses. The approach to the validation was based on the FDA document (May 2018): Bioanalytical Method Validation Guidance for Industry and the International Conference on Harmonization (ICH) Q2(R1) guidance document (2005), with some modifications as the ICS assay measures functional cellular responses and differs in some important respects from a bioanalytical assay. Five assay parameters were validated for Th1 and Th2 response: specificity, LOD/LLOQ, precision, linearity, and accuracy. The assay validation was submitted to the FDA and following several rounds of revision, was approved for potential future testing for Phase 3 clinical trials. This achievement demonstrates the feasibility of a rigorous validation of a functional cellular assay for both Th1 and low-level Th2 responses and would not have been possible without rigorous adherence to the principles of panel design and quality-controlled instruments. While this staining panel was developed and validated in the context of COVID-19, the approach described here can be applied to other novel functional flow cytometric assays used to assess HIV, tuberculosis, and malaria in the context of infection and vaccination.
BD Biosciences - Aaron Tyznik, PhD
From Spectrum to Insight: Innovations Driving Higher-Dimensional Cell Analysis
Hear about the innovation and latest advancement in spectral cytometry available on the BD FACSDiscover™ S8 Cell Sorter and A8 Cell Analyzer. These instruments leverage full-spectrum detection across multiple lasers with Spectra FX™ Technology and our Fully Integrated Autoloader to improve throughput and reliability available on the A8 Cell Analyzer.
The advances have influenced fluorochrome development, highlighting improvements in brightness, spillover-spread, stability, and reliability. These next generation fluorochromes may be combined with others into multi-color dried reagent formats, contributing to reproducibility across experiments and instruments.
Finally, we will look at the integration of spectral flow cytometry with imaging capabilities, with BD CellView™ Image Technology enabling the extraction of image-derived parameters for subcellular classification. This convergence represents a transformative step expanding the dimensionality and depth of cytometric analysis.
Cytek Biosciences - Laura Johnston, PhD
Getting The Most Out Of Cytek Cloud And Our SpectroPanel™ Algorithm
Learn about the full suite of features in Cytek® Cloud, including Full Spectrum Viewer, Panel Builder with SpectroPanel™ automated panel design algorithm, Experiment Builder, and Quote Builder. We’ll provide an overview of the tools and highlight the newest features that can streamline your experiment workflows.
FlowJo - Timothy Crawford, PhD
FlowJo™ Version 11: The next generation of cytometry data analyses solutions. An introduction and orientation.
FlowJo™ version 11 is the latest installment of cytometry data analysis tools from the BDB Informatics group (Formerly FlowJo). With a single immersive workbench window, built in quality control, dimensionality reduction and clustering algorithms, plus a new graph gallery and integrated charting capabilities, FlowJo v11 aims to simplify, enable and enhance your interrogation of traditional and next-generation imaging-enabled cytometry data. Please join us for an introduction to the FlowJo v11 platform.
Standard BioTools - Connie Inlay PhD
Advancing clinical research with new mass cytometry workflows
The complex nature of biological systems requires deep interrogation at the single cell level. Mass cytometry can simultaneously resolve phenotypic and functional markers, enabling insights into disease mechanisms and identification of therapeutic targets and predictive biomarkers. We leveraged modular, ready to use Flex-Fit CyTOF panels with TeMal sample barcoding, to comprehensively profile both immune and non-immune cells from tumor tissue and PBMCs from endometrial cancer patients in a single tube. This workflow can be adapted to research personalized treatment strategies for patients with endometrial cancer, and overall showcases mass cytometry as a powerful tool for comprehensive immune profiling in cancer research.
Ozzette - Arpan Neupane
Confidently unmix Spectral data using Ozette Resolve™
Spectral cytometry has revolutionized the field by unlocking unprecedented possibilities for high-dimensional single-cell analysis. The reliability of these insights hinges on the critical process of spectral unmixing. To ensure raw measurements are accurately translated into meaningful signals, high-quality unmixing requires both single-color controls and precise spectral estimates derived from them. To this end, we have developed Ozette Resolve™, an end-to-end, adaptive single-cell unmixing solution which accounts for cell level autofluorescence and places Quality Control (QC) of single-color controls at its core. By moving beyond the traditional reliance on “bright” signals and providing a robust toolkit for identifying true positives, Ozette Resolve enhances data quality and empowers researchers to transition from raw data to confident biological discovery with greater speed and precision.
Cytek® Biosciences, Global Customer Education Manager, US
Julie Hill has a master’s degree in Toxicology from the University of Washington in Seattle. With over 20 years of flow cytometry experience, Julie has worked in research and clinical settings in academia, government, and biotechnology laboratories. She spent over five years at the NIH in the Vaccine Research Center (NIAID) working on projects to advance high parameter flow cytometry. In 2019, Julie joined Cytek® Biosciences where she worked for over five years as a Technical Application Specialist (Pacific Northwest) and she managed technical applications in the western US and Canada. For almost two years, Julie has worked in Cytek’s Global Customer Education Group, managing the US team.
Garrett Collett is currently a scientist at Bristol Myers Squibb, where he applies flow cytometry and related immunophenotyping approaches in an industry drug-development setting. He currently focuses on cell therapy, developing assays and strategies for characterizing CAR-T starting material and drug products for both autologous and allogeneic programs. With several years of hands-on experience in flow cytometry, Garrett offers insight into how cytometry skills translate into biopharma environments, as well as what day-to-day scientific work looks like outside academia.
Julian Reading is a long-time cytometrist at the Allen Institute, where he supports large-scale, high-throughput biological research using advanced flow cytometry and cell-sorting technologies. Trained in academic research, Julian has built a career centered on enabling science across diverse teams rather than running a single independent lab. With many years of experience in flow cytometry, he brings perspective on careers in research institutes, shared scientific infrastructure, and collaborative, team-based science.
Thane Mittelstaedt is based at the University of Washington, where his work integrates flow cytometry into academic immunology research and training. His career path reflects a more traditional academic trajectory, including formal scientific training and research appointments, while maintaining a strong focus on advanced cytometry methods. With extensive experience using flow cytometry as a core experimental tool, Thane represents academic-based careers that blend research, mentorship, and technical expertise.
Andrew Konecny is a cytometry scientist at Fred Hutchinson Cancer Center and the University of Washington, where his work spans experimental design, high-parameter flow cytometry, and rigorous downstream data analysis. He has extensive experience analyzing complex immunophenotyping datasets in translational and clinical research settings, with an emphasis on reproducibility, population definition, and biological interpretation. Through his research and collaborative work, Andrew brings a practical, methodologically grounded perspective on how analytical choices shape conclusions drawn from cytometry data
Anna Kus is a senior cytometry analyst at Benaroya Research Institute, where she leads data analysis for high-dimensional flow and mass cytometry studies across autoimmune and translational research programs. Her work integrates traditional gating strategies with modern computational approaches, including dimensionality reduction, clustering, and longitudinal data analysis. With deep hands-on experience moving data from raw files to publication-ready results, Anna brings a grounded perspective on best practices, common pitfalls, and decision points in cytometry data analysis workflows.
Hugh MacMillan is an applied mathematician at Fred Hutchinson Cancer Center working on quantitative analyses of high-dimensional single-cell data, specifically CyTOF and spectral flow cytometry. He offers a computational scientist's perspective on coping with batch effects and integrating large-cohort flow cytometry studies. Hugh has contributed to the development and application of analytical frameworks that address scaling, normalization, and interpretation challenges inherent to these data.
Sam Klebanoff is an immunologist at Benaroya Research Institute and the University of Washington whose research relies heavily on advanced flow cytometry for immune profiling and functional analysis. Across multiple publications, his work integrates high-parameter cytometry with thoughtful analytical strategies to interrogate immune states in human disease. Sam brings a biologically driven perspective on flow cytometry data analysis, emphasizing how analytical approaches can enable—or obscure—meaningful immunological interpretation.
Peter Morawski is a cytometry expert at Benaroya Research Institute with extensive experience in flow cytometry data analysis, visualization, and interpretation across diverse human immunology studies. His work spans both hands-on analysis and strategic guidance, with a focus on extracting robust, biologically meaningful insights from complex datasets. Peter brings a broad, experience-driven perspective on analytical decision-making in flow cytometry, informed by years of collaborative work across projects and teams.
Dr. Evan Newell is an immunologist who develops and employs new technologies for accurately identifying specific biological signatures of human health and disease, including cancer and infectious diseases. He and his team work with blood and tissue samples, using mass cytometry and other single-cell analysis methods to better understand how the specificities of immune T cells influence their roles in clinically productive responses against pathogens or cancers.
High dimensional flow cytometry approaches, including mass cytometry and increasingly powerful spectral flow cytometry panels, have enabled detailed profiling of human T cell responses across diverse contexts of cancer and infectious disease. In this talk, I will describe how my laboratory is applying these tools to use T cell phenotypes as sensitive readouts of immune status in human studies and clinical trials. I’ll also talk about efforts to coordinate immune profiling across studies using broadly applicable panels and large-scale analysis approaches. In addition, I’ll describe how the Fred Hutch Innovation Lab is contributing to collaborative efforts to generate data in parallel using additional modalities such as single-cell and spatial multi-omics as well as TCR sequencing and plasma proteomics.
Rafael Villasmil - National Eye Institute
Integrating Nano-Cytometry: A Core Lab’s Perspective on the CytoFlex Nano
Abstract TBD
Rafael Villasmil has led the Flow Cytometry Core Facility at the National Eye Institute for nearly 20 years, overseeing cutting-edge cytometry platforms and supporting research in immunology, ocular disease, and clinical applications. An experienced educator and trainer, Rafael specializes in best practices for flow cytometry and quantitative single-cell analysis. Currently, Rafael is pursuing a Doctorate in Bioinformatics and Computational Biology at George Mason University, integrating computational approaches with empirical methods to advance health research from the lab to clinical applications.