Presenters

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.

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. 

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.

This session features five short talks by sponsors of the event.

Come join an exciting panel discussion on the various careers that flow cytometry has to offer, whether you are looking for a change or wanting to break into a part of the industry. Learn about some challenges that you may face or what you may need to learn to stand out from the crowd.

This session features five short talks highlighting different data analysis approaches used in real projects. Speakers will focus on what worked for them, why they made those choices, and what they learned along the way, followed by an interactive Q&A with the audience.

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.