NWFCS First Annual Meeting (2024)

Annual Meeting NW Flow Cytometry Society
March 6th, 2024, Fred Hutchinson Cancer Center

This talk focuses on the fundamentals of panel design. We’ll use examples in each step to demonstrate how to apply best practices of panel design to generate a theoretical panel. We will then discuss how to identify and resolve issues to ultimately create a successful, optimized panel.

Key topics :

• Step-by-step guidance and best practices for panels of any size

• Panel design tools to make the process easier

• How to determine if a panel is successful

CAR-T cell therapies have changed the therapeutic landscape for hematologic malignancies since the first approval by the FDA in 2017. However, certain hurdles remain, including cost, process optimization, and limited solid tumor infiltration, and represent opportunities for innovation and discovery.

The advent of spectral cytometry has greatly increased the number of parameters that can be measured simultaneously. Spectral approaches allow researchers to make novel biological connections that would have been impossible only years ago. Incorporating this technology into the CAR-T world will be critical for researchers to better face limitations encountered with cell therapy products such as the ones mentioned above.

Bristol Myers Squibb has leveraged spectral cytometry to answer several questions focused on CAR-T product development. Broadly, this talk will examine the evaluation and implementation of spectral cytometry to characterize cell therapy drug products. The development and use of in-house and external software and hardware tools will be reviewed. Additionally, the development lifecycle of two spectral flow panels will be discussed, leveraging work published by the Allen Institute for Immunology. The talk will conclude with insights made from utilizing a technique called Infinity Flow, a technique first described by the Headley laboratory at Fred Hutch Cancer Center (Seattle, WA USA).

The various steps of manual analysis has persistently presented a bottleneck in flow cytometry-based workflows due to its complexity, subjectivity, costliness, and time-consuming nature. We are leveraging open data, citizen science, and a massively online multiplayer game to overcome the different challenges that exist. Thousands of gamers produced millions of novel annotations and paved the way for the development of robust and universally applicable machine learning approaches to flow cytometry gating. Yet, the replacement of manual gating is merely one step in the necessary transformation of scientists' contribution from manual workflows to compute-enabled discovery. Further improvements to the infrastructure supporting the reuse of data is a critical requirement that will facilitate and scale ambitious research into areas that are presently economically prohibitive. Achieving this objective entails the acknowledgement, development, and extensive promotion of solutions to the remaining impediments to big data science, including leveraging the same citizen science infrastructure for standardized and automated cell population annotation.

Flow Cytometry, the tool of the trade of today’s immunologists, is a highly multi-parametric platform, capable of high-speed quantitative assessment of cells, at the single cell level. The success of high parameter Spectral flow cytometry in multiple major areas of development includes understanding receptor expression, the instrument, affinity reagents, new dyes and flurochromes, application development, software, and bioinformatics. As biology advances, the need for deep and broad profiling of cell populations is becoming even more important. Higher order flow cytometry panels on high-parameter instrumentation and instruments with configurations that minimize compensation are key components to success. The integration of flow cytometry to basic, clinical and translation biomedical research is significantly enhanced with the development of the new BD-Horizon dyes off the Blue, Green, Violet, UV and deep UV which have been integrated in BD High Parameter solution. Matching appropriate bright fluorochromes to dim markers, and the ability to spread markers across multiple lasers, can minimize the spectral overlap and maximize population resolution. This talk will spotlight how BD and our community of users are continuing to innovate flow cytometry by sharing some of the advancements and insights in our continued efforts to create tools of discovery to unlock the full potential of flow cytometry in your research.

Are you overwhelmed with the potential in your high-parameter cytometry data and unsure of which analytical tool is right for you?  In this talk, we will explore strategies to clarify your goals and make the tool selection process easier. From identifying key features required for your analysis, to understanding the capabilities and limitations of these tools, we will cover the essential aspects to help you get the most out of your cytometry data analysis.

Flow cytometry is a powerful tool for monitoring immune function. Over the years, it has increasingly become an indispensable component in the clinic trials setting, specifically in vaccine research, where it is used to measure cellular immune responses to candidate vaccine regimes. However, for flow cytometry to be an effective tool, the instruments must be thoroughly characterized and standardized. At the HIV Vaccine Trials Network (HVTN) cellular laboratory, each new flow cytometer must undergo 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 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.

Have you ever found yourself looking at cytometry results, wondering if something went wrong, but struggling to identify the issue? Have you seen someone following questionable practices and creating uninterpretable datasets? Let us go beyond the basic troubleshooting and discuss the true "crimes" of cytometry practice, from minor infractions to major offenses, that result in compromised datasets. Some of these horrendous deeds may deeply disturb you! But rest assured - using series of case studies featuring bad data, we will discuss the best ways to avoid bad practices across the experimental workflow, from panel design to staining practices, data acquisition, and analysis.

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.