Workshop Abstracts
Spectral flow Workshop
Simon Monard (Walter and Eliza Hall Institute of Medical Research)
Alexis Gonzales (The University of Melbourne)
Intro to spectral flow cytometry:
- Why go full spectrum?
- How is it different from conventional flow cytometry
- Challenges and opportunities
- What do we mean “unmixing errors”
- Staining
Optimising reference controls:
- Including autofluorescent signatures
- How bright is too bright
- Group specific negatives
- Beads or cells.
Trouble shooting:
- Why does my data look weird?
- Is it staining or unmixing?
- Post acquisition compensation; A necessary evil?
- Examples of odd looking data and how it was “fixed”.
We will start the workshop with an overview of full spectrum cytometry, how it differs from conventional cytometry and its similarities. The experimental workflow will be demonstrated and the term “reference controls” will be introduced. The term “unmixing errors” is often heard. It is implied that due to a shortcoming of the instrument or the software the data doesn’t meet the users expectations. Unmixing errors are usually due to 1. Inappropriate reference controls, 2. autofluorescence, 3 Poorly designed experiments or 4. staining issues eg. failing to block unwanted staining.
We will explore reference control issues and when should one include one or more autofluorescence signatures as “colours” in the experiment and demonstrate how this works in practice.
Finally, we want to show some examples of experiments where the data looks terrible and how and if such data can be “fixed”. When is it permissible to alter compensation post unmixing and when is this falsifying data by distorting it to meet our expectations. We would like to discuss this last part with the audience.
Analysis of Spatial Transcriptomics and CITE-seq Data
A/Prof Alen Faiz (University of Technology Sydney)
The emergence of spatial transcriptomics and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) has enabled unprecedented characterization of cellular phenotypes and tissue organization. While CITE-seq provides high-resolution transcriptomic and protein-level characterization of individual cells, spatial transcriptomics preserves the tissue context necessary to understand cellular interactions and microenvironmental structure.
This workshop will focus on analytical strategies for spatial transcriptomic and CITE-seq datasets. Participants will be introduced to workflows for multimodal data preprocessing, quality control, dimensionality reduction, , and cell-type annotation using both transcriptomic and protein-derived information. The workshop will demonstrate methods for constructing robust cellular reference atlases from CITE-seq data and projecting these cell states onto spatial transcriptomic datasets.
Advanced analyses will include identification of spatially variable genes, characterization of cellular neighbourhoods and spatial co-localization analyses. Participants will also explore approaches for identifying disease-associated cellular niches, quantifying tissue architecture, and linking molecular programs to specific anatomical regions. Particular emphasis will be placed on integrating protein and transcriptomic information to improve cell-state resolution and enhance spatial mapping accuracy.
Hands-on examples will demonstrate the use of contemporary computational frameworks in R , including workflows implemented in Seurat and complementary spatial analysis programs. The workshop will highlight best practices for data integration, interpretation of results, and common analytical pitfalls encountered when working with large-scale single-cell and spatial datasets.
By the end of the workshop, participants will have gained practical experience in CITE-seq and spatial transcriptomic data, enabling them to identify cellular populations.
The Grey Zone: Interactive Case-Based Challenges in Ambiguous Lineage Leukaemias
Dr Mary Sartor, BSc, MSc, PhD
Dr Julie Curtin
Accurate classification of acute leukaemias with ambiguous lineage remains one of the greatest diagnostic challenges in flow cytometry. Overlapping immunophenotypic features, aberrant antigen expression, and evolving classification criteria often require the integration of flow cytometry with morphology, cytogenetics, molecular findings, and clinical context. These cases can challenge even the most experienced laboratory professionals and frequently benefit from multidisciplinary discussion.
This interactive workshop will present a series of six to eight cases that illustrate the diagnostic dilemmas encountered in ambiguous lineage leukaemias, including mixed phenotype acute leukaemia (MPAL) and other diagnostically challenging entities. Participants will be actively involved in the decision-making process through live audience polling using QR code technology. For each case, attendees will review the available data and vote on the most likely diagnosis or next diagnostic step via multiple-choice questions, with the aggregated results displayed in real time.
Following each poll, Dr Julie Curtin will lead a discussion exploring the rationale behind the correct interpretation, highlighting key immunophenotypic features, potential pitfalls, and practical approaches to challenging cases. A dedicated question-and-answer session will follow, allowing further exploration of complex issues raised during the workshop.
This session is designed for laboratory scientists, haematopathologists, clinicians, and trainees with an interest in diagnostic flow cytometry.
Viable CD34 testing principles
The Royal College of Pathologists of Australasia – Quality Assurance Programs
Workshop description
This workshop focuses on viable CD34 testing principles. The presentations will focus on ISHAGE guidelines, the importance of pre and post analytical variables, common pitfalls and quality matters. The session will include time for case scenarios and panel discussions.
Session 1: Technical component
- Speaker 1
- ISHAGE-based approach with viability
- Single-platform vs dual-platform considerations
- Gating strategies incorporating viability dyes
- Speaker 2
- Common pitfalls:
- Debris and non-specific binding
- Low event counts
- Compensation and fluorescence spillover
- Comparison of viability dyes:
- 7-AAD
- Propidium iodide
- Amine-reactive dyes
- Pros/cons in:
- Fresh samples
- Cryopreserved / thawed samples
- Speaker 3
- Finish with case scenarios/discussion
**Break**
Session 2: Quality + pre and post analytical variables
- Speaker 4
- Cryopreservation and thawing impact
- Result reporting:
- Units
- Significant figures
- Reporting
- Speaker 5
- ACS/national guideline on CD34 enumeration
- EQA
ACS Conference 2026 