The Speaker

Jonathan M. Irish, PhD - Professor | University of Colorado

Jonathan M. Irish Ph.D is Professor at the University of Colorado working in immunology, cancer, and computational biology and Scientific Director of the CytoLab data science group.  The Irish lab uses bench and computational cytometry techniques to study how signaling controls cell identity in healthy tissues, cancer, the human brain, and immune disorders.   Jonathan trained in chemistry and biology at the University of Michigan, went to Stanford for training in cancer biology and immunology, started his independent lab at Vanderbilt in systems cancer immunology, and was recruited to the University of Colorado in 2024 to focus on brain tumors and neuroimmunology.  A research theme in the Irish lab is the study of human cells and tissue using advanced cytometry, including phospho-flow, high dimensional mass cytometry and spectral flow, and machine learning analysis.  Jonathan is active in ISAC, including previously as Chair of Leadership Development and Data Committees and now as Secretary and Chair of Governance and an active member of the FlowRepository and FCS 4.0 taskforces.

Summary

Traditional cytometry analysis uses a series of biaxial gates to explore data and identify cells.  This approach works best when measured immunophenotypes match known cell types from hierarchical models of cell identity.  However, gating schemes may not accurately represent immune and cancer cell types that diverge from expected protein expression profiles or that exist in hybrid states that are between or apart from known cell types. I will present a new version of the Marker Enrichment Modeling algorithm, Velociraptor (MEM 4.0), which addresses these issues by quantifying cell identity using multidimensional, continuous measurements.  This approach enables flexible searching for cells based on feature sets expressed as readable text labels. This new approach also efficiently learns cell identity from training data and can seek any number of defined cell identities in new testing datasets, including cytometry from different instruments or platforms (e.g., training in imaging mass cytometry and validation in spectral flow cytometry). Scoring cell identity on a continuous scale is especially useful for characterizing cells that deviate from expected expression profiles. Such cells are commonly observed in human blood and tissue samples and are prevalent in disease and following activation of cell signaling. Additional applications of Velociraptor include measuring known cell subsets without manual gating, quantifying shifts in heterogeneity over time, and registering cells between blood and tissue microenvironments to track and characterize rare, clinically significant cells.

Learning Objectives:

1. Learn multiple approaches to identify cells in cytometry data
2. Understand the pros and cons of hierarchical, binary models of cell identity
3. Learn about disease-associated ‘iconoclast’ cells observed in human tissue

Who Should Attend:

- Trainees interested in high dimensional cytometry and associated data analysis
- Clinical researchers who want to identify rare, clinically significant cells in human tissue
- Immunologists and cell biologists who want to distinguish healthy and malignant cells
- Shared resource leaders who want to automate routine cell identification
- Data Scientists who want to integrate multiple single cell data types
-Hematopathologists and researchers who use biaxial gating to analyze flow data


Keywords: Cell identity, gating, cancer, immunology, machine learning

CMLE Credit: 1.0

The Speaker

Katrien Quintelier - VIB-UGent Center for Inflammation Research | Data Mining and Modeling for Biomedicine

Katrien Quintelier is a [doctoral / postdoctoral] researcher working in the group of Prof. Yvan Saeys at the VIB-UGent center for Inflammation Research in Belgium. Her research lies at the intersection of computational biology and immunology, with a strong focus on the analysis of high-dimensional cytometry and CITE-seq data. Since Katrien is working with clinical data, her main research focus is on the preprocessing of the data including quality control, batch effect detection and correction.
 

Summary

This webinar will provide a comprehensive overview of the preprocessing pipeline for cytometry data. We will cover the required and optional steps to obtain high-quality, preprocessed data that is ready for downstream analysis, including compensation, transformation and doublet removal. Particular focus will be given to quality control: addressing strategies at the per-file level to filter out low-quality events, as well as across files to identify and manage batch effects.

Learning Objectives:

After attending this webinar, participants will be able to:

1. Understand the different preprocessing steps
2. Be aware of the different levels of quality control
3. Understand how batch effect correction works

Who Should Attend:

Anyone interested in cytometry data analysis.

CMLE Credit: 1.0

The Speakers

Katharine A. (Kathy) Muirhead, PhD - Chief Operating Officer, SciGro Inc.

Kathy Muirhead, PhD is Chief Operating Officer of SciGro, Inc., which she co-founded in 1996. In prior lives she served as Assistant Professor of Pathology at the University of Rochester, director of the first flow cytometry core facility at SmithKline Beckman R&D, and Senior V.P. of Research & Business Development at Zynaxis, Inc., a cell therapy start-up co-founded with colleagues from SmithKline. Dr. Muirhead has served as an ISAC Councilor, Associate Editor and reviewer for Cytometry A, and on CLSI subcommittees formulating guidelines for clinical immunophenotyping and validation of flow cytometric assays. While at the University of Rochester she co-founded the Annual Courses in Applications of Cytometry, a “for the community, by the community” educational series now nearing its 50^th anniversary. She currently serves on the ISAC Governance Committee and the Board of Directors of Cytometry Educational Associates, Inc.  

Paul K. Wallace, PhD - CSO, SciGro, Inc, Southwest Office, Roswell Comprehensive Cancer Center Professor Emeritus

Paul K. Wallace served from 2003 to 2021 as Director of the Flow and Image Cytometry Department at Roswell Park Comprehensive Cancer Center (RPCCC) in Buffalo, NY, where he is now Professor Emeritus. He is the current Educator-in-Chief and a Past President of the International Society for Advancement of Cytometry, an organization. Dr. Wallace is also Chief Scientific Officer of SciGro, Inc., a biomedical consulting group. He remains active in the International Clinical Cytometry Society, serves as Associate Editor of Clinical Cytometry B, and in 2018 received their Wallace H. Coulter Award for lifetime achievement in clinical cytometry.

Summary
Cytometrists have long been interested in the biology of proliferation, both in normal growth and in tumors. Flow cytometric methods to measure proliferation fall into two main categories: those that assess DNA content or cell cycle status, and those that track cell division by dye dilution.

Cell cycle analysis is a well-established application. S-phase fraction, for example, indicates the proportion of cells actively synthesizing DNA at a given time. Combining DNA binding dyes with phase-specific markers allows discrimination between cells with identical DNA content that are actively cycling (G1) or quiescent (G0). However, these methods don’t reveal how many times a cell has divided in response to stimulation, how much a particular subset has expanded, or what fraction of the starting population has participated in the response.

In contrast, dye dilution allows more direct monitoring of cell division. Cells are labeled with bright, stable, non-toxic fluorescent dyes that bind to proteins or membranes and are distributed approximately evenly between daughter cells at each division. The resulting fluorescence intensity profile reflects the proportion of responder cells in the starting population and the number of cell divisions undergone by each responder during the assay period. This approach has been widely applied to in vitro proliferation studies, antigen-specific cell enumeration, and the identification of quiescent stem cell populations.

After a brief overview of DNA-based methods, this presentation will focus on dye dilution methods for proliferation monitoring, highlighting dye-specific considerations, analysis strategies, critical controls, technical challenges, and key applications. 

Learning Objectives:
After attending this webinar, participants will be able to:
1.  Compare approaches for monitoring proliferation by DNA cell cycle analysis and dye dilution, including the strengths and limitations of each.
2.  Select appropriate dyes and establish the necessary experimental controls and analysis strategies for reliable dye dilution assays.
3.  Describe how dye dilution can be used to quantify low frequency populations of interest, including antigen-specific T cells or quiescent/stem-like tumor cells.

Who Should Attend:
SRL Staff, Biologists interested in measuring proliferation, Cancer biologists, Immunologists, Individuals doing infectious disease & vaccine research, Individuals involved in R&D and manufacturing of cellular therapeutics 

CMLE Credit: 1.0

Authors

Kylie M. Price
Chief Technology Officer & Hugh Green Fellow
Hugh Green Technology Centre
The Malaghan Institute of Medical Research

Katharine A. (Kathy) Muirhead, Ph.D.
Chief Operating Officer, SciGro, Inc.

Paul K. Wallace, Ph.D.
CSO, SciGro, Inc., Southwest Office
Roswell Park Comprehensive Cancer Center, Professor Emeritus 

SUMMARY

Lesson 3: Monitoring Cell Division by Dye Dilution 

Lesson 3 describes the use of dye dilution to monitor cell division, with an emphasis on multicolor methods for detecting differential responses in complex cell populations without the need for manual isolation and counting.

Module 3B, the second of four modules, focuses on methods for quantifying extent of cell division based on dye dilution profiles. These include:

1. Describing qualitative trend(s) in antigen expression across generations.
2. Simple quantitation using standard cytometric software (proliferative fraction, stimulation index).
3. Proliferation profile deconvolution using specialized modeling software (% responders, responder expansion)

Anyone with a basic understanding of flow cytometry who is unfamiliar with (or wishes to review) dye dilution-based methods for monitoring cell proliferation by flow cytometry is encouraged to attend.

CMLE Credit: 1.0

A Basic Introduction to the Science of Cell Sorting By Matthew Goff

About the Presenter

Matthew Goff
Senior Product Manager, Flow Cytometry
Beckman Coulter Life Science

Matthew began his career in flow cytometry doing graduate research at Virginia Tech followed by his role as a core lab manager at Eastern Virginia Medical School. After leaving EVMS for industry, Matthew stayed close to flow cytometry supporting researchers and industrial scientists in flow cytometry with reagents, software, and hardware products. In 2021 he became a commercial product manager at Beckman Coulter Life Sciences where his work is focused on sustaining the CytoFLEX Platform and launching new products. 

Webinar Summary
This webinar introduces scientists with an interest in learning more about flow cytometry an introduction to the mechanics of cell sorting, best practices when sorting cells and contemporary discussion topic associated with cell sorting.

CMLE Credit: 1.0

Author

Zosia Maciorowski - Flow Cytometry Core Facility at the Curie Institute, Paris (Retired)

Zosia Maciorowski was responsible for the Flow Cytometry Core Facility at the Curie Institute in Paris, France for 28 years, from which she recently retired. Originally from Montreal, she graduated from McGill University and worked in the UK, Canada and the US, where she was finally introduced to flow cytometry in the 80’s, specializing in solid tumor preparation and cell cycle analysis. She served as chair of the ISAC Membership Services Committee and the Education Committee and is currently chair of the Live Education Delivery Subcommittee of the Education Committee which organizes workshops around the world, particularly in resource limited areas where there is little access to cytometry education.

Summary

This introductory course covers the basics of flow cytometry, starting with a short overview of light scatter, fluorescence, fluorochromes and their characteristics. We then walk through the inside of a basic flow cytometer with detail on the fluidics, optics and electronic components. The effects of fluorochrome spectral overlap and spillover are discussed. Finally, we look at how the new spectral cytometers function and how they differ from conventional cytometers.

At the end of this course, you will understand the basics of light scatter and fluorescence and how conventional and spectral flow cytometers function. You will have learned how factors such as instrument setup and spectral spillover can affect flow cytometric measurements. Hopefully this understanding of how a flow cytometer works will help you to better plan experiments and ensure quality results on your instrument.

This basic course is an initiation to flow cytometry and is aimed at students and scientists with no prior knowledge.

*ISAC is pleased to offer this course complimentary to the cytometry community.

CMLE Credit: 1.0

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