Standardization for Precision: Building Best Practices for Automated Cell Sample Preparation Across Multi-Site Flow Cytometry Labs

Presenters:
Lili Wang, PhD, Senior Scientist, National Institute of Standards and Technology
Tom Hayday, PhD, Co-founder and Chief Research Officer, IMU Biosciences
John Ferbas, PhD, Senior Director, Cytometry & Imaging Sciences, Amgen
Raffaello Cimbro, PhD, Director of Flow Cytometry, AstraZeneca

Problem Focus/Summary:

Standardization and reproducibility remain the cornerstone challenges in translating high-dimensional flow cytometry data into clinically meaningful outcomes. Recent multi-institutional efforts led by the NIST Flow Cytometry Consortium have accelerated consensus-building on pre-analytical variables, assay standardization and validation frameworks, and data quality benchmarks.

This tutorial will explore how automation and standards are converging to address these challenges—from sample handling to antibody cocktailing and washing—by minimizing operator-driven variability and enabling traceable, reproducible workflows.

This tutorial will explore:

Define and Apply Reference Process Standards
Integrate Automation into Standardized Workflows
Evaluate Cross-Site Validation Strategies

Keywords: Sample Prep, Protocol standardization, Cytometry Hardware, Experimental Design & controls

CMLE Credit: 1.5

CYTO Women: Career Pathways Panel Discussion

Panelists: 
Arielle Ginsberg, MSc, SCYM, CEO, terraFlow
Kathy Muirhead, PhD COO, SciGro, Inc.
Laura Ferrer-Font, PhD, Scientific Solutions Manager -Scientific Affairs, R&D, Waters Biosciences
Pratip Chattopadhyay, PhD, Founder and CEO, Talon Biomarkers

Unlock the secrets to a fulfilling career in cytometry! Whether you are a student, a postdoc, or a seasoned professional, this essential mentoring session is designed to help you navigate your next chapter within the flow cytometry field. Join distinguished leaders Arielle Ginsberg, Kathy Muirhead, Peter Mage, and Pratip Chattopadhyay as they share firsthand accounts of the career paths, pivots, and breakthroughs that shaped their journeys. Gain actionable strategies for long-term planning and participate in an open Q&A to get personalized advice on your professional development.

Don’t leave your future to chance—come learn how to turn your passion for cytometry into a strategic career roadmap!

Keywords: CYTO, CYTO Women, Career Development

CMLE Credit: 1.5

Fundamental Considerations for Quantitative Fluorescence Standardization

Presenters:
Vera Tang, PhD, Facility Manager & Adjunct Professor, University of Ottawa
Joshua Welsh, PhD, Staff Scientist, BD Biosciences

Problem Focus/Summary:
Standardization of flow cytometry data reporting is necessary for reproducibility irrespective of the sample type across flow cytometry platforms. Cross-platform standardization as a topic is becoming increasingly important in both clinical and academic research settings. Fluorescence quantification is a method used to standardize reporting of flow cytometry data, making intra- and inter-platform comparisons possible. These are published methods, but there are currently a multitude of tools available for fluorescence standardization. In this tutorial we will identify the different tiers of standardization and then provide practical guidance on the tools necessary to achieve these different levels of standardization.

Goals: The purpose of this tutorial is to provide insight and guidance towards selecting methods and materials for fluorescence quantification, aiming to address specific standardization goals, and provide options to achieve concordance in standardized data reporting.

What this Tutorial will not do: Cover pre-acquisition variables – sample isolation/processing, staining optimization, panel design.

Learning Objectives:
To understand the different tiers of flow cytometer assay standardization from longitudinal single instrument studies to cross-platform comparison studies.
To understand the nuance in definitions for fluorescence quantification, calibration, normalization, and standardization.
To understand the practical differences between each of the fluorescence calibration units available, including cost, consistency, and ergonomics.
To demonstrate the impact of different fluorescence units on concordance in comparing cross-platform data, i.e. ERF, MESF, and ABC.

Keywords: Calibration, Quantitative, Fundamental Concepts, Hardware, Standardization, Experimental Design

CMLE Credit: 1.5

Navigating SRL Recognition: How to Prepare Your Application

Presenters:
Jessica Prieto-Chavez, MSc, Co-Coordinator of the Cytometry Network of the Health Research Coordination, Mexican Social Security Institute (IMSS)

Abstract:
The ISAC SRL Recognition Program is a cornerstone initiative to promote quality, reproducibility, and sustainability in shared resource laboratories (SRLs). As participation grows worldwide, many applicants seek clearer guidance on how to prepare strong, well-documented submissions.

This tutorial provides practical, evidence-based insights drawn from real applicant experiences and reviewer feedback. Presenters will highlight common challenges encountered during the Recognition process, typical strengths and weaknesses observed in evaluation reports, and concrete strategies that have helped SRLs achieve success.

Through presentations and an interactive discussion, participants will learn how to:
- Interpret ISAC’s expectations at each application stage.
- Identify internal documentation and quality practices most valued by reviewers.
- Avoid frequent pitfalls that delay or weaken submissions.
- Apply lessons learned from recognized SRLs of different sizes and organizational structures.
- Recognize the tangible and long-term benefits of applying for SRL Recognition — including improved documentation, standardized procedures, and enhanced visibility within the community.

Ultimately, attendees will leave with a clearer understanding of how to plan, structure, and execute their SRL Recognition applications—while also strengthening their lab’s internal processes and culture of continuous improvement.

Learning Objectives:
Describe the structure and evaluation criteria of the ISAC SRL Recognition process.
Recognize common challenges and recurring feedback themes from reviewer reports.
Apply proven strategies and documentation practices that strengthen an SRL Recognition application.
Evaluate their own lab’s readiness and identify steps for sustained quality development beyond application submission.

Keywords: Management, Training and Education, SRL Recognition Program

CMLE Credit: 1.5

Business Intelligence for Flow Cytometry

Presenters:
Antonio Cosma, PhD, Head of the National Cytometry Platform, Luxembourg Institute of Health

Abstract:
The vast amount of data produced by cytometry, along with its accompanying metadata, necessitates the deployment of advanced and innovative tools. These tools must be adapted to manage data sourced from a multitude of origins. They must also be capable of generating visualizations that are specifically tailored for effective data analysis and sharing.  Business Intelligence (BI) addresses all these needs, but it is usually used in the business sector and not in a scientific environment. A critical, not fully recognized aspect of BI is the capability to transfer analytical capabilities to domain experts (i.e., cytometrists) rather than relying on generalized analysts who lack specialized knowledge of the data and the scientific context.

In this tutorial, I will initially lay the basis of data management with a special focus on cytometry. I will show how to organize files for instrument acquisition and introduce the concept of enriched FCS. I will then introduce the concepts of aggregation, joining, filtering, and levels of detail. Once the basis is established, I will proceed to the data preparation and visualization steps. At the end of the tutorial, I will showcase some well-known examples of data sharing already widely used by the cytometry community: (1) the OMIP Cytometry A database, (2) CPHEN Comprehensive Phenotypic Reports, and (3) HCDM CDmap database.

Attendees will learn the principles of BI applied to flow cytometry, enabling them to prepare data and create simple visualizations. The learning curve for BI software is relatively flat, and this introduction will allow participants to get started quickly with their own data.

Keywords: Instrument Monitoring, Bioinformatics, SRL, Shared Resource Laboratories, Operations and Finance, Management

CMLE Credit: 1.5

Excellence and Integrity in Cytometry Publishing: A Guide for Authors, Reviewers, and Associate Editors

Presenters:
Bartek Rajwa, PhD, Editor-in-Chief, Cytometry Part A

Introduction:
For over 40 years, Cytometry Part A – The Journal of Quantitative Cell Science has been the home for innovative research in single-cell analysis, publishing peer-reviewed studies on measurement, separation, manipulation, engineering, and modeling of cells. The journal also covers high-content screening and the molecular mechanisms underlying cellular function. As the field continues to evolve, Cytometry A remains committed to advancing quantitative cell biology and supporting the development of cutting-edge methods for cellular systems analysis.

At the same time, scientific publishing is undergoing rapid changes. The rise of open access, the growth of semi-predatory publishers, the demand for faster review cycles, and the popularity of preprints are reshaping how we share scientific knowledge. These shifts present both challenges and opportunities for Cytometry A. In response, we are expanding our editorial board, strengthening peer review, and broadening our scope to encompass emerging areas such as high-content screening and single-cell omics.

Learning objectives:
By participating in this tutorial, attendees will be able to:

1) Understand and navigate the peer review process for Cytometry Part A, including the workflow from submission to decision, time expectations for reviewers and Associate Editors, and how to craft constructive reviews that strengthen manuscripts.
2) Apply statistical rigor and data quality standards expected for publication, including proper analysis of cytometry data, avoiding common statistical pitfalls, and implementing FAIR data principles with appropriate use of RRIDs and data sharing practices.
3) Evaluate manuscript quality and publication readiness, understanding what distinguishes strong submissions from those likely to be rejected, technical requirements for figures and supplementary data, and how to assess scientific impact, novelty, and significance.
4) Recognize the role and value of OMIP (Optimized Multicolor Immunofluorescence Panel) publications, understanding how these standardized, validated panel descriptions advance reproducibility and serve as community resources for researchers designing multi-parameter cytometry experiments.

Content: 
This tutorial will reintroduce Cytometry A to the ISAC community and explain how members can contribute as reviewers and Associate Editors. We'll examine the practical mechanics of peer review: manuscript evaluation workflow, timelines, time commitments, and how to write constructive, actionable reviews. We'll provide guidance on what distinguishes strong submissions from desk rejections, technical requirements for figures and data files, and when manuscripts are appropriate for Cytometry Part Aversus Part B.

A special focus will be placed on OMIP papers, which document fully validated, ready-to-use multi-parameter panels complete with reagent details, compensation strategies, and gating schemes. OMIPs serve as essential community resources that promote standardization, reproducibility, and facilitate the adoption of complex cytometry approaches. We'll discuss the criteria for OMIP submissions and how these papers differ from standard research articles.

We'll address critical research ethics issues: identifying and managing conflicts of interest, authorship disputes, image manipulation policies, and recognizing red flags for paper mills and data fraud that threaten the peer review system.

We'll also discuss the professional development benefits of serving as a reviewer or Associate Editor: career enhancement, networking opportunities, staying current with emerging methods, and contributing to the scientific community.

Statistical analysis and data representation remain critical areas where submissions often fall short. We'll review common statistical mistakes in cytometry data, including understanding of spectral unmixing, compositional data analysis issues, multiple testing corrections, and appropriate controls. We'll cover standards for reporting statistical methods, data visualization best practices, high-dimensional data representation, validation approaches for computational analyses, and transparency in parameter selection.

Finally, we'll explain how the journal supports FAIR data principles through policies on Research Resource Identifiers (RRIDs), code availability, and data deposition. Throughout the tutorial, we'll emphasize the journal's commitment to scientific impact, novelty, and significance, and welcome questions about any aspect of the submission, review, or editorial process."

Keywords: Publication, Peer Review, Cytometry Part A, Journal, OMIPs

CMLE Credit: 1.5

Solutions to SICS --Techniques and Technologies to mitigate Sorter Induced Cellular Stress

Presenters:
Peter Lopez, BS, Research Associate Professor, NYU Grossman School of Medicine

Abstract:
Purification of cell populations has been an important technique used in biological research since the early 1900's. Various purification techniques provide different levels of specificity and purification, ranging from filtration to purify cells based on size, to flow cytometric techniques delivering high purity of populations differentiated using dozens of phenotypic

markers. The advent of FACS , a flow cytometric purification technique, provided a mainstay purification technique which arguably changed the playing field for cellular purification and facilitated many critical discoveries in biological research. FACS, based on electrostatic droplet deflection, provides highly purified cell populations,. In some cases cells purified by FACS have downstream functional deficits, and depending on cell type may have issues with viability or proliferative capacity. The perturbed performance of purified cells has been described as SICS. This tutorial will review the various forms of SICS, and will then present methods and
technologies including optimization of traditional FACS as well as alternative flow cytometric approaches that can help mitigate SICS.

Learning Objectives:
1-- Learn the history of cell purification technologies, their strengths and weaknesses.
2-- Understand the definition of SICS-- metrics and manifestations.
3– Understand the specific SICS outcomes observed using FACS.
4-- Learn the latest techniques and new purification technologies that help mitigate SICS.

Keywords: Metabolomics, Cell Proliferation and Death, Cell Sorting, Sorting, Sorter, Cell Separation

CMLE Credit: 1.5

Panel Gains Without the Pains: Smarter Re-Optimization for High-Parameter Flow

Presenters:
Diana L. Bonilla Escobar, PhD, Scientific Director, Cytek Biosciences
Kamila Czechowska, PhD, Chief Diagnostic Product Development Officer, Metafora Biosystems
Megan McCausland, BSc, Scientific Advisor, Flow Cytometry, IQVIA Laboratories
Veronica Nash, PhD, Director of Flow Cytometry (Cellular Biomarkers), GSK

Abstract:
Assay optimization is essential for achieving reproducible and accurate results in multicolor flow cytometry. Recent advancements in cytometer hardware and fluorochrome chemistries, coupled with the ability to use an expanded number of fluorophores simultaneously, have enabled highly multiparametric assays. These capabilities create opportunities to re-optimize existing panels by adding new markers to gain deeper insights, without compromising resolution or data quality. However, expanding an optimized and/or validated panel is often challenging. Issues such as reagent availability, clone specificity, titer adjustments, and performance variability must be addressed, alongside technical problems like steric hindrance, reagent interactions, and increased spillover spreading. These challenges underscore the need for clear guidance when modifying complex panels. This tutorial will outline a structured approach for re-optimizing previously optimized and/or validated assays, including strategies for selecting optimal fluorochrome replacements or additions, evaluating reagent compatibility, and maintaining optimal resolution across all parameters in an expanded panel. We will walk through real case examples showing how updated panels were revalidated to ensure comparable marker performance, consistent population resolution, and robust identification of all relevant cell subsets after adding new markers. In these examples, careful panel redesign and iterative testing preserved data integrity even as dimensionality increased. Based on these findings, we will propose a practical workflow and a set of best-practice guidelines for panel expansion and re-optimization. This “panel expansion” workflow will serve as a template that participants can apply in their own labs – an increasingly important skill as new fluorochromes and next-generation instruments continue to push the boundaries of high-dimensional flow cytometry.

Keywords: High Parameter, Data Analysis, Optimization

CMLE Credit: 1.5

Crimes Against Data Analysis

Presenters:
Sarah Bonte, PhD, Postdoctoral Researcher, VIB-Ghent University
Geoffrey Kraker, BSc, Senior Application Specialist, Dotmatics
Givanna Putri, PhD, Postdoctoral Researcher, Walter Eliza Hall Institute of Medical Research
Nicolas Loof, MSc, Informatics Solution Leader, BD/FlowJo

Abstract:

High parameter cytometry technologies enable simultaneous characterization of many cellular markers, offering unprecendented insights into complex biological systems. As panel sizes and complexities continue to expand with advances in instruments and reagents, our capacity to analyze data has not kept pace, making high-dimensional data increasingly challenging to interpret. While data analysis tools are becoming more sophisticated and accessible, whether through programming platforms or commercial software plugins, longstanding issues with data quality remain a significant barrier. These are further compounded by artifacts introduced during data processing, which can lead to erroneous interpretations. Yet, clear guidance on how to recognize, diagnose, and mitigate them is still lacking.

This tutorial will provide strategic and tactical approaches for identifying and troubleshooting common data quality issues and artifacts in cytometry data, showing both their causes and effects. Most commonly used algorithms for high-dimensional data analysis will be covered, along with guidelines for parameter settings for each.

Learning objectives:

Having an idea of the output of computational algorithms when there are no/minimal problems with data quality and no artifacts introduced during data analysis (""What it should look like"")
Recognizing artifacts introduced by data analysis and/or data quality issues (""What it looks like if you don't do it right"")
Knowing what to do to prevent these artifacts from occurring
Understanding parameter choices in commonly used computational algorithms for high-dimensional data analysis
Guidelines on how to pick the most optimal parameters for your data, and diagnose when you have picked the wrong ones

Keywords: CytoBytes, Clustering, High Parameter

CMLE Credit: 1.5

Challenging Sample Types in SRL Cores: From Biomedical to Environmental Cytometry

Presenters:
Nicole Poulton, PhD, Senior Research Scientist, Bigelow Laboratory for Ocean Sciences
Attila Bebes, PhD, Experimental Officer, University of Exeter
Raif Yuecel, PhD, Director, Centre for Life Sciences Technologies (CLST) and Research Director, Exeter Centre for Cytomics (EXCC), University of Exeter

Abstract:
In Shared Resource Laboratories (SRLs) we are often faced with challenging samples that can be difficult to prepare and analyze. In a biomedical research setting these could be fragile subcellular organelles, such as nuclei, mitochondria or peroxisomes, as well as debris-rich suspensions ranging from tissue digests to insect hemolymph. Environmental samples can vary from aquatic sources containing plankton, bacteria and viruses to complex sediment samples or snail mucin. Each of these samples presents unique challenges to the SRL cytometrist.

In this tutorial, we will share practical workflows and lessons learned from working with both biomedical and environmental samples within our respective SRLs. The session will offer general strategies, step-by-step guidance, and case studies covering examples such as tissue dissociation (mechanical and enzymatic), planktonic community profiling and genomic sizing of amphipods to help participants approach challenging samples with confidence and reproducibility.

This tutorial fits the Scientific Track on SRL Practices and Applications and is designed to provide attendees with evidence-based, didactic guidance on handling non-standard samples, combining best practices from clinical, research, and environmental cytometry.

Learning objectives:

Sample preparation strategies spanning organelles to sediments.
Instrument selection and setup to achieve optimal data quality.
Common artifacts and how to prevent or recognize them.

After attending this tutorial, participants should be able to:

Recognize the major physical and biological challenges associated with complex biomedical (e.g., tissue-derived, organelle, or primary cell) and environmental (e.g., aquatic, sedimentary, or microbial) samples.
Apply evidence-based preparation methods to minimize debris and aggregation, and to handle autofluorescence that may compromise cytometry data quality.
Optimize instrument configuration and quality control for non-standard particle sizes and signal ranges across cytometers (both conventional and spectral).
Implement validated workflows and safety practices to ensure reproducible results in SRL environments that deal with heterogeneous or high-risk samples.
Integrate lessons learned from biomedical and environmental workflows to expand SRL support capabilities and cross-disciplinary collaboration.

Keywords: Challenging Samples, Microbial Marine and Plant Cytometry, Microbial Cytometry, Plant Cytometry, Marine Cytometry

CMLE Credit: 1.5