No CMLE Credits offered

The Brightest Light in Flow Cytometry and My Career: A Tribute to Mario Roederer
Pratip K. Chattopadhyay, PhD - CEO, Founder Talon Biomarkers, CSO, terraFlow Bioinformatics
In 2003, when I joined Mario Roederer’s lab at the NIH, few could imagine a world full of high parameter flow cytometers. I certainly could not have envisioned the success I had under his wing, nor could I have predicted the career he inspired me to in government, academia, and industry. Even today, eight years removed from his lab, there isn’t a day that I don’t think of him or speak of some soft skill that I learned from him. And for most people in flow cytometry, a day doesn’t go by without touching or doing something that Mario developed, explained, or influenced. It’s not hard to celebrate Mario’s innovation in technology, and – though perhaps we don’t talk about them enough – it is easy to appreciate the important contributions he made in immunology. We’ll catalogue those, and discuss their impact, in this presentation and the other talks in the session. I will also share behind-the-scenes stories of our innovative and ground-breaking work together. And, most importantly, I hope to convey some of the lessons I learned watching him and describe their indelible impact on me. Mario influenced me as a scientist in so many ways – at many different wavelengths, one might say – my talk will aim to capture the brightness of that light.

Understanding memory T-cell differentiation: from ImmunoTechnology to Translational Immunology
Enrico Lugli, PhD - Laboratory of Translational Immunology and Flow Cytometry Core, IRCCS Humanitas Research Hospital
Following pathogen or tumor clearance, T cells survive giving rise to long-lived memory T cells, providing a faster and more powerful response upon secondary challenge. The memory T cell pool is highly diverse, comprising subsets of less differentiated cells with improved persistence capacity and differentiation potential, and subsets of terminally differentiated cells with immediacy of effector functions but limited persistence. The precursors, now commonly referred to as stem-like memory T cells, have been show to maintain immunological memory following acute and chronic infection, to exert the most potent anti-tumor functions following adoptive cell transfer and to generate long-lived effector progenies in response to cancer immunotherapy with checkpoint blockade. During this talk, I will review the discovery of stem-like memory T cells during my post-doctoral fellowship in the laboratory of Mario Roederer at the NIH, how this key event under Mario’s guidance shaped my subsequent career as an independent investigator and, above all, how it inspired a multitude of preclinical and clinical studies on the use of these cells for the benefit of patients with cancer and chronic viral infections. In the last part of the talk, I will mention our current contribution to enhance stem-like T cell responses in response to cancer immunotherapies. Acknowledgements - AIRC 5×1000 program UniCanVax 22757

A journey through science wonderland - My postdoctoral training in the Roederer lab
Thomas Liechti, PhD - National Institutes of Health
Yolanda Mahnke, PhD - President, FlowHowYouKnow, Scientific Advisor, FluoroFinder
I started my postdoctoral training in Mario Roederer’s lab at the National Institutes of Health (NIH) in 2017. Little did I know that the next 5 years would become one of the most rewarding times in my career. There I was, part of a group with some of the brightest minds in the field of immunology and cytometry and got to experience the sheer unlimited possibilities that the NIH has to offer. A cornerstone of the NIH is the highly collaborative environment. In Mario’s lab we lived up to these standards with the goal to democratize science. Mario has made it his mission to build sophisticated immunological tools, which enable the community to tackle complex scientific questions. Most of us, whether knowingly or unknowingly, tremendously benefited from those technological advancements and immunological discoveries. Besides his remarkable scientific contributions, Mario is heavily invested in mentorship, which has been critical for the professional progress of his trainees. It is my distinct pleasure to speak at this session in honor of Mario’s career. I will share my experience as a postdoctoral trainee at the NIH and how Mario’s mentorship impacted my career and my approach to science and mentorship.

A tale of excellence, in a government/industry partnership
Bob Balderas - VP Biological Sciences, BD Distinguished Fellow
In 2015, a platform technology program was established under a CRADA between The National Institute of Allergy and Infectious Diseases (NIAID) and Becton, Dickinson and Company (BD), to optimize advanced multicolor flow cytometry for immune discovery in preclinical and clinical evaluation of novel vaccine candidates, as well as to develop tools and approaches for in-depth analysis of immune responses elicited by vaccination or natural infection. The program was established to advance the state of the art for flow cytometry, thereby advancing the state of cellular systems analysis through an integrated solution, effectively marrying and optimizing a new generation of software, reagents, and instruments to advance early vaccine development. A broad systems approach to flow cytometry was created to cover software, reagents, and instrumentation to enable extremely high content, high throughput flow cytometry. Specific project goals were mutually developed for an integrated solution to generate more considerably broader, biologically relevant information from rare cells and/or small samples. To accomplish these goals, two project teams were established across NIH and BD. A cadence of monthly meetings were held to facilitate the project, technologies were shared, a base instrument was developed, ideas were integrated into many subassemblies and the eventual creation of a 50 parameter, conventional flow cytometer. Success of this project was attributed to both teams, but today, we wanted to share the experience that we had with Mario and his team. It was truly an opportunity to learn, to share, to watch a mentor invent, to critique and complete what at that time was the first HP flow cytometer in our community.

CMLE Credit: 1.0

Cytometry Beyond Cells of the Blood

Sharath Tippur Narayana Iyengar, PhD - Postdoctoral Research Scholar, Purdue University
J. Paul Robinson, PhD - Distinguished Professor of Cytometry, The SVM Professor of Cytomics, Professor, Weldon School of Biomedical Engineering, Director, Purdue University Cytometry Laboratories

This tutorial will provide an overview of alternative applications where flow cytometry has been used in the past, and can have opportunities for the future. There will be two presentations, one focusing on microbial and marine organisms and one focusing on a large variety of applications such as cell cycling, sperm sorting, milk analysis, functional analysis, and other applications, all of which have strong potential in the flow cytometry domain. The goal will be to open up for discussion from the audience on experiences that might be useful for the audience. We plan on publishing a technical note from the results of this tutorial session.

CMLE Credit: 1.5

Before you click ‘Run’: what truly matters for successful high-dimensional data analysis? 

Anna Belkina, MD, PhD - Boston University Chobanian & Avedisian School of Medicine
Sarah Bonte, PhD - Ghent University
Givanna Putri, PhD - Walter and Eliza Hall Institute of Medical Research

When working with high-dimensional cytometry data, the instinct is often to dive straight into clustering and dimensionality reduction — but is that the best approach? Often, datasets are simply fed into automated pipelines for visualization and pattern discovery without careful consideration of how experimental design, data quality, preprocessing, and technical variations shape the results. Beyond just choosing an algorithm, in this tutorial, we will discuss how to structure a robust workflow, critically interpret outputs, and distinguish meaningful patterns from artifacts. We will also explore how automated methods can introduce bias and lead to misleading conclusions, as well as highlight a few flawed approaches that are common but can compromise analysis. Through practical examples and shared experiences, we aim to provide a framework for making informed decisions in high-dimensional data analysis.

CMLE Credit: 1.0

The Critical Aspects of Aerosol Containment Testing in Flow Cytometry – What are the Essential Requirements for Safety?

Kristen Reifel, PhD - Staff Scientist, Flow Cytometry Core, Vaccine Research Center, NIAID/NIH
Evan Jellison, PhD - Associate Professor/Director of Flow Cytometry, UCONN Health, University of Connecticut School of Medicine
Catherine Carswell-Crumpton - R&D Scientist Engineer II/ Director Flow Core, FACS Core, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University
Michael, Solga, MS, SCYM (ASCP) - Director, Flow Cytometry Core Facility, University of Virginia School of Medicine

Flow cytometers, especially droplet-based cell sorters, pose significant safety risks for operators and laboratory workers. In particular, high concentrations of aerosols can be generated within the sort collection area of cell sorters during instrument failures, such as a partial nozzle obstruction, that cause the stream to deviate. Protecting operators and laboratory workers by ensuring containment of these aerosols is essential when working with potentially infectious or hazardous samples. Currently, aerosol containment is accomplished through the generation of continuous negative airflow within the sort collection area using an aerosol evacuation system that is either an external vacuum unit or is integrated into a primary containment device such as a Class II biosafety cabinet. These aerosol evacuation systems are not generally certified or tested after installation. Thus, members of the ISAC Biosafety Committee developed a test that can be used to determine whether the system adequately contains and evacuates aerosols. This test is broadly compatible across cell sorter models and laboratory facilities to enable safety testing for all. It can also be used to evaluate any instrument that poses a risk of aerosol generation by providing a method to visualize aerosol release.

In this tutorial, we will review the key aspects of the updated aerosol containment test including critical steps, recommendations for commercially available aerosol sampler cassettes, interpretation of results, troubleshooting recommendations, and other potential applications of the test. We will also discuss how to incorporate this test into laboratory protocols and risk mitigation plans. Attendees of all backgrounds will be able to ask questions and participate in a panel discussion where experts from the ISAC Biosafety Committee will discuss how they have implemented aerosol containment testing and other risk mitigation measures in their own laboratories.

CMLE Credit: 1.0

Thinking outside the plot: a new era of imaging cytometry 

Daniel Vocelle, PhD - Incoming Director of Flow Cytometry Shared Resources, St Judes Children's Research Hospital
Jochen Behrends, PhD - Head of Core Facility Fluorescence Cytometry, Research Center Borstel, Leibniz Lung Center
Henry Hui, PhD - Research Associate, Translational Cancer Pathology Laboratory, Associate Lecturer, Pathology / Medical Science and Genetics, School of Biomedical Sciences, University of Western Australia
Jessica Back, PhD - Associate Director, Microscopy, Imaging, and Cytometry Resources Core, Assistant Professor, Department of Oncology, Wayne State University School of Medicine
Aja Rieger, PhD - Former Manager of the Faculty of Medicine and Dentistry Flow Cytometry Facility, University of Alberta

Imaging cytometry has advanced rapidly over the last 5 years, yet many researchers still overlook its expanding role in shared resource laboratories (SRLs). Imaging flow cytometry (IFC) brings together the statistical strength of flow cytometry with detailed visualization similar to microscopy, enabling high-throughput single-cell analysis. There are currently at least ten imaging flow cytometric platforms on the market that capture both quantitative and morphological data. This tutorial will highlight the starting points of imaging cytometry, key technological advancements, compare instrument options, and showcase emerging applications such as rare cell detection, immune profiling, and microbial analysis. Attendees will gain insights into practical considerations - like cost, complexity, and data management - so they can confidently integrate imaging cytometry into their work.

CMLE Credit: 1.0

Debunking Myths About Data Spread |

Diana L. Bonilla Escobar, PhD - Scientific Director, Cytek Biosciences
Dave Novo-Lake, PhD - De Novo Research

Spread assessment is critical to understand and troubleshoot data resolution in multicolor flow cytometry, being essential in panel design and assay optimization, as well as instrument optical performance evaluation. Spread manifest as dispersion in the distribution of a population and is caused by the stochastic nature of fluorescence emission and the extent of the spectral overlap between fluorochromes. A large amount of spread can severely impact resolution, especially for dimly expressed markers. In a multiparametric assay, the amount of spread can be influenced by multiple factors including the cytometer optical performance, the instrument setup, the choice of fluorochromes, the biology of the samples, the staining conditions, the extraction of autofluorescence, and the mathematical algorithm used to calculate the abundance of each fluorochrome in fully stained samples. In this tutorial, we will summarize the fundamentals of data spreading and evaluate in detail the impact of multiple factors in the extent of the spread. In addition, we will provide recommendations on how to mitigate spread to generate high quality multicolor flow cytometry assays and accurate result interpretation.

CMLE Credit: 1.0

Ensuring Rigor and Reproducibility in Flow Cytometry Experiments: Essential Record-Keeping Guidelines
Kewal Asosingh, PhD - Associate Staff Director Flow Cytometry, Cleveland Clinic
Michael Gregory, MS - Director, Caltech Flow Cytometry and Cell Sorting Facility

This tutorial is all about helping you understand the importance of keeping detailed records for flow cytometry experiments. For others to understand your work and be able to repeat your experiments successfully, it's crucial to keep track of all the technical details. In this step-by-step guide, we'll walk you through a complete flow cytometry experiment from experimental design to sample prep to acquisition and analysis. We'll highlight what you should note down and why it's important.

CMLE Credit: 1.0

Do you know your ABCs of PBMCs? (A Critical Evaluation of Pre-analytical Variables)

Sylvia Janetzki, MD - President, Zellnet Consulting, Inc.
Helen McGuire, PhD - Associate Professor, The University of Sydney
Karen Quadrini, PhD - Director, Clinical Biomarkers, Prothena Biosciences
Thomas Beadnell, PhD - Scientific Advisor, Eurofins Clinical Trial Solutions
Natalie Smith, B Sci (Hons I) - PhD Student, The University of Sydney
Vikas Singh, PhD - Research Scientist II, Eurofins Clinical Trial Solutions
Robin Walsh, MS - Advisor, ImmunoSafety, Eli Lilly and Co.

Peripheral Blood Mononuclear Cells (PBMC) are a critical biospecimen in immune functional assays as well as phenotyping assays. Many variables can impact PBMC quality and therein impact the downstream analysis. Understanding these variables, the impact and the best practices surrounding them will provide researchers with a stronger foundation to improve data quality and consistency.

The PBMC tutorial will present current practices for PBMC isolation and processing, from whole blood collection to assay use preparation. Based upon pre-conference survey results, with input from the Flow Cytometry, ELISPOT, and live cell communities, standardization of processes and advancements to help improve consistency across studies, labs, and clinical trials remain important factors that need to be addressed. In addition, the community highlighted PBMC quality and variability deriving from preparations, freezing, and thawing to be critical factors impacting studies. The tutorial will highlight logistical hurdles for processing patient/donor samples, using case studies to demonstrate the impact variability has upon downstream functional and/or phenotypic analysis for various applications. Building upon these case studies, the tutorial will present important considerations for clinical trial implementation and will review the current best practices for improving quality in PBMC preparations.

An essential component to understanding PBMC quality is the establishment of effective PBMC quality assurance (QA) parameters. Responses from the pre-conference survey highlighted a need for improved guidance around PBMC QA parameters. The tutorial will highlight the current guidelines and best practices to help establish relevant PBMC QA parameters in your lab.

Taken together, the tutorial will establish a foundation for current best practices for obtaining high-quality PBMC and aims to be a launching point for the establishment of future guidance documentation. Following this tutorial there will be a workshop where we will aim to further define the key unmet needs within the field and establish the next steps for organizing committees and identifying action items to address and improve upon the current practices.

CMLE Credit: 1.0

Introduction to Image Cytometry and Quantitative Image Analysis
Dr. Dominic Jenner - Principle Scientist, Defence Science Technology Laboratory

The field of image cytometry is vast comprising many different techniques, technologies and analysis methodologies. This seminar will start with the exploration of the basics of image cytometry and the fields within its scope. This tutorial will focus on static imaging techniques/technologies such as wide field microscopy, confocal microscopy, high content imaging and spatial biology. It will provide those new to the field of image cytometry with a good knowledge base to start deciding which technologies/techniques fit their science the best. The latter half of the tutorial will present an introduction to image analysis and best practices for extracting quantitative single-cell data from image datasets.

CMLE Credit: 1.0

The road to successful high-parameter spectral cytometry experiments: guidelines for control optimization, handling of autofluorescence and quality control
Andrew Konency - Vaccine and Infectious Disease Division, Fred Hutch Cancer Center
Oliver Burton, PhD - Department of Pathology, University of Cambridge
Maria Jaimes, MD - Cytek Biosciences
Joanne Lannigan, MS - Flow Cytometry Support Services, LLC
Florian Mair, PhD - Managing Director, Flow Cytometry Core Facility, ETH Zurich

Spectral flow cytometry has expanded the boundaries of high-dimensional analysis, enabling deeper biological insights than ever before. However, the ever-increasing plexity of multiparameter panels introduces unique challenges in experiment design, quality control (QC), and data interpretation. To fully optimize an assay, distinct considerations are required to prepare appropriate control samples, including unstained controls, as well as a rigorous quality assessment strategy.

In this tutorial we will discuss common caveats and strategies that are needed for the successful setup of a high-dimensional spectral cytometry experiment. In addition to an overview of current best practices and practical tips and tricks, attendees will gain insight into: - Optimization of experimental procedures and single-stained controls - How to best manage autofluorescence - Effective use of control samples for multicolor data QC

By showing real-world examples, this tutorial will highlight both emerging solutions and well-documented approached to overcome typical challenges encountered with highly complex flow cytometry assays and will provide participants with actionable strategies to improve their experimental designs and analysis workflows. By the end of the session, attendees will be equipped with the knowledge to anticipate, troubleshoot and mitigate common pitfalls related to controls and autofluorescence, that will contribute to the generation of reproducible and high-quality spectral cytometry data.

CMLE Credit: 1.0