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Multiplex Immunofluorescence (mIF) 101: The Fundamentals

Webinar Series: November 9, November 30, December 2, December 9, 2021

Join us for this four-part webinar series, featuring members of the JEDI (Joint Effort to Develop Multiplex Immunofluorescence Standards) Council for Multiplex IF/IHC Global Standardization, a multi-disciplinary taskforce comprising globally >30 members of immunologists, oncologists, pathologists, computational biologists, technicians and imaging platform managers in the field of immuno-oncology. Founders: Bernie Fox, PhD; Houssein Abdul Sater, MD; Jaime Rodriguez-Canales, MD; Michael Surace, PhD; and Joe Yeong Poh Sheng, MBBS, PhD, M.Med.

Image Optimization: Building a Personalized Image Analysis Workflow

November 9, 2021 | 1 PM PT, 4 PM ET, 10 PM CET

This webinar is part one of a four-part series will provide a fundamentals training on Image Analysis for Multiplex Immunofluorescence (mIF).

mIF imaging of tissue sections with the Akoya PhenoImager™ platform (formerly Phenoptics™) enables robust quantification of immune subsets with spatial context. Dr. Jensen and Dr. Egelston are tumor immunologists with years of experience in the quantification and interpretation of mIF images from a variety of immuno-oncology projects. In this webinar, they will demonstrate how building an appropriate image analysis workflow can unravel tissue microenvironment heterogeneity and complexity with meaningful results that achieve the goals of your research needs. Drs. Jensen and Egelston will discuss standard approaches to extract and maximize data outputs from mIF images. They will go over commonly used software choices for image analysis and walk through common image analysis workflows, including tissue segmentation, cell segmentation, and cell phenotyping. This webinar will present a clear path for going from a raw image to quantified data of cell subsets identified by your mIF panel.

In this talk, attendees will:

  • Gain an understanding of the types of software options and software workflows that are used for mIF.
  • Learn the basic approaches towards analyzing mIF data, including tissue segmentation, cell segmentation, and cell phenotyping.
  • Gain insight into how you may tailor your image analysis towards the goals of your unique research project.

Speakers

Colt A. Egelston, PhD

Assistant Research Professor, Beckman Research Institute, City of Hope National Medical Center

Shawn Jensen, PhD

Senior Scientist, Earle A Chiles Research Institute – Portland Providence Medical Center

Michael Surace, PhD

Associate Director, Translational Medicine Oncology, AstraZeneca

Michael S. Nelson, MS

Laboratory for Optical and Computational Instrumentation (LOCI) Team, University of Wisconsin

Trevor D. Mckee, PhD

Director of Image Analysis, HistoWiz

Data Presentation and Analysis – Validating and Extracting Per-cell Statistics out of Your Multiplexed Images

November 30, 2021 | 1 PM PT, 4 PM ET, 10 PM CET

This webinar is part two of a four-part series will provide a fundamentals training on Data Analysis and Presentation for Multiplex Immunofluorescence (mIF).

Dr. Trevor McKee will discuss key considerations for quality control of cell segmentation when analyzing single- and multi-plex immunostaining datasets which can impact downstream analysis. He will demonstrate some examples using Imaging Mass Cytometry and PhenoCycler™ (formerly CODEX®) datasets. Michael S. Nelson will present a framework for analyzing multiplex images, utilizing QuPath for segmentation and CytoMAP for unsupervised clustering and/or neighborhood analysis. The results from CytoMAP will then be visualized within QuPath – overlaid upon the original image to provide context – and finally exported into heatmaps, t-SNE plots and other summary graphics  to provide meaningful high level interpretations of the data.

In this talk, attendees will:

  • Learn advantages and disadvantages to certain methods of analyzing and presenting multiplex results.
  • Learn strategies for the objective comparison of different segmentation strategies, and how to choose the right approach for your particular data.
  • Learn how to implement a quality control process within the context of cellular segmentation, in order to validate the results of your image analysis methods for downstream use of data and results.
  • Learn to identify and troubleshoot issues in the data analysis pipeline that can impact downstream analysis, including the identification of biologically impossible marker combinations.

Speakers

Trevor D. Mckee, PhD

Director of Image Analysis, HistoWiz

Michael S. Nelson, MS

Laboratory for Optical and Computational Instrumentation (LOCI) Team, University of Wisconsin

Joe Yeong Poh Sheng, MBBS, PhD, M.Med

Group Leader in the Institute of Molecular and Cell Biology

Colt A. Egelston, PhD

Assistant Research Professor, Beckman Research Institute, City of Hope National Medical Center

Michael Surace, PhD

Associate Director, Translational Medicine Oncology, AstraZeneca

Intro to the Tumor Immune Microenvironment and Panel Design that Leads to Bigger Outcomes

December 2, 2021 | 1 PM PT, 4 PM ET, 10 PM CET

This webinar is part three of a four part series will provide a fundamentals training on Panel Design in Multiplex Immunofluorescence (mIF).

The design and development of a multiplex IF panel must take into account several factors, with the scientific question being paramount. The scientific question should leverage the spatial and multimarker information in the multiplex IF image, or else the value of the panel may not justify the effort of development. In this webinar, pathologists and scientists involved in all aspect of mIF panel development will present and discuss considerations around the selection of lineage and activation markers, caveats of colocalizing epitopes, and the importance of an image analysis and analytical plan.

Addressing a complex tumor microenvironment can be challenging. The knowledge gained through multiplex discovery tools as MxIF requires a basic understanding of various disciplines, proper planning, and fine coordination with various stakeholders including the clinician, immunologist, pathologist, technician. In this talk, Drs. Sater and Surace will overview some of the basic immune microenvironment concepts and present an approach that will help with the design of a multiplex IF panel capable of properly answering your biology questions.

In this talk, attendees will:

  • Learn the structure of a complete multiplex IF team.
  • Learn how to plan the design phase of a multiplex IF panel with consideration toward common and expected technical challenges.
  • Learn how to design a multiplex IF panel to address specific hypotheses as well as for general immunophenotyping.

Speakers

Houssein Abdul Sater, MD

Lead Physician Scientist, Hematology Oncology, Cleveland Clinic Martin Health

Michael Surace, PhD

Associate Director, Translational Medicine Oncology, AstraZeneca

J. C. Villasboas, MD

Consultant, Division of Hematology, Department of Internal Medicine, Mayo Clinic

Jaime Rodriguez-Canales, MD, FEBP

Director, Translational Pathology, AstraZeneca

Joe Yeong Poh Sheng, MBBS, PhD, M.Med

Group Leader in the Institute of Molecular and Cell Biology

Imaging not Based on Imagination: What, How and Where?

December 9, 2021 | 1 PM PT, 4 PM ET, 10 PM CET

This webinar is part four of a four-part series will provide a fundamentals training on Staining and Imaging in Multiplex Immunofluorescence (mIF).

Multiplexed immunofluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides allow the identification of multiple cell phenotypes while retaining spatial and morphological context. Immunophenotyping analysis reliably depicts the immune landscape of cancer tissues that has been demonstrated to influence cancer development and progression as well as to have an impact on therapy responsiveness and resistance. The multiplexed immunolabelling workflow includes simultaneous staining of multiple biomarkers within a single paraffin tissue section, multispectral imaging followed by spectral unmixing and digital pathology. Typically, 6–8-plex assays are based on tyramide signal amplification (TSA), using Opal fluorophores and imaging is performed on Akoya’s PhenoImager HT instrument (formerly Vectra® Polaris™).

In this webinar, Dr. Carlos Andrea, as an experience practising pathologist, will discuss the multiplex Immunofluorescence assay development and validation, and image acquisition, that will enable analysis of cancer morphology and cell phenotypes in routine sections. Dr. Joe Yeong, as immuno-pathologist will discuss the panel design, regions/images selection from cancer immunology and immuno-oncology points of views.  On behalf of JEDI council for multiplex IHC/IF global standardization taskforce, He will also discuss the recent publication on “THE PATHOLOGIST” about multiplex IF 101, to provide multidisciplinary KOLs opinions to help labs to build their mIF capacity and to pave the way for – “Every histolab can multiplex”.

Learning objectives:

  • FFPE tissue sections of normal and tumor tissue can be used for (i) the initial optimization of staining conditions for each single primary antibody and (ii) the following setup and validation of the multiplex immunolabelling protocols.
  • An overview of the multiplex immunolabelling protocol validation workflow.
  • Panel Design Considerations: Staining interference, cross-talking reactions (bleed through), umbrella effect (e.g. blockage of molecules detection).

Speakers

Carlos de Andrea, MD, PhD

Anatomical Pathologist, University of Navarra, Spain

Joe Yeong Poh Sheng, MBBS, PhD, M.Med

Group Leader in the Institute of Molecular and Cell Biology

Trevor D. Mckee, PhD

Director of Image Analysis, HistoWiz

Carmen Ballesteros-Merino, PhD

Research Scientist, Earle A. Chiles Research Institute-Providence Cancer Center

Michael Surace, PhD

Associate Director, Translational Medicine Oncology, AstraZeneca