Specialized Workshop Th2Thursday September 29, 2022: 7am - 5:30pm - Topic List

"Flow Cytometry Bioanalysis - Rising of the 3rd Most Common/Important Technology in Bioanalytical Labs"

Importance of learning Flow, Mass, Imagine & Spectral Cytometry Bioanalysis and Major Applications in Biomarkers, Immunogenicity, Cell Therapy, Vaccine, Omics and Non-Liquid Matrices; Novel Techniques, Autogating Developments and Innovation in High Dimensional Cytometry
Social Networking Reception and Dinner is also Included - Flow Cytometry Day and Night!

(You can scroll down to see the details of each topic)

Part 1: Understanding the Cytometry Conventional/Novel Technologies and Main Applications

  • Topic 1:

    Importance of Conventional Flow Cytometry Bioanalysis for Clinical Trials – Past, Present, Future: Technology Introduction & Main Applications in Immunophenotyping, RO and PD Assays
    Dr. Erin Stevens, Associate Research Fellow / Lab Head - Early Clinical Development, Pfizer

  • Topic 2:

    Importance of Spectral Cytometry for Biomarker Development – Current Status and Future Developments: Technology Introduction & Primary Unique Features vs Conventional Flow Cytometry, Main Application in Drug Discovery & Development
    Dr. Michael Nathan Hedrick, Director Clinical Flow Cytometry, Bristol-Myers Squibb

  • Topic 3:

    Importance of Mass Cytometry for Bioanalytical Needs – Past, Present, Future: Technology Introduction, Primary Unique Features vs Conventional Flow Cytometry & Applications in Bioanalysis & Biomeasures
    Mr. Chad Stevens, Senior Scientist BioMedicine Design Department, Pfizer

  • Topic 4:

    Importance of Imaging Cytometry for Biomeasures – Past, Present, Future: Technology Introduction & Applications in Cell-based Biomeasure Assays
    Dr. Polina Goihberg, Discipline Lead BioMedicine Design Department, Pfizer

Part 2: Current Challenges with Validation & Sample Collection

  • Topic 5:

    Strategies for Rationalizing Fit-for-purpose Criteria for Flow Cytometry Biomarker Assay Validation (BAV): How to Determine what Parameters/Acceptance Criteria are really needed without overdoing or underdoing?
    Dr. Steven Eck, Director Translational Sciences, Clinical Immunology and Bioanalysis, AstraZeneca

  • Topic 6:

    Turning the CLSI H62 into Practise for Bioanalytical/Clinical Laboratories: First-hand experience in trying to apply the CLSI H62 to real situations with pros & cons
    Dr. Christèle Gonneau, Lead Scientist Flow Cytometry, LabCorp

  • Topic 7:

    Challenges and Strategies for Clinical Sample Collection in Flow Cytometry and Cell-Based Assays
    Dr. Anka Ehrhardt, Director Cell-Based Assays, Merck

Part 3: Specific Complex Applications

  • Topic 8:

    Flow Cytometry in the Monitoring of Immune Responses to Vaccines: Method Development challenges and Assay Optimization Strategies to Increases the Robustness and Accuracy of the Data
    Dr. Gabriela Franco Salinas , Associate Scientific Director Clinical Immunology, Janssen

  • Topic 9:

    Use of Model Antigens to Validate Antigen Specific Vaccine Flow Cytometry Assays
    Dr. Bruno Salaun, Senior Scientist, GlaxoSmithKline

  • Topic 10:

    Challenges and Value of Clinical Receptor Occupancy (RO) Assessments for Bispecific T Cell redirection by Flow Cytometry
    Dr. Katharine Grugan, Associate Scientific Director, Janssen

  • Topic 11:

    Challenges in the Development of Novel Flow Cytometry Assays for Bioanalysis of PD Markers: Macrophage Polarization and Phagocytosis Measurements
    Dr. Sarita Sehra, Senior Research Investigator Translational Sciences, Incyte

Part 4: Innovation in Cytometry

  • Topic 12:

    Can we use Cytometry in Tissues Bioanalysis? Current Challenges, Innovative Approaches and Technologies
    Dr. Yongliang "Steve" Sun, Associate Director Translational Sciences and Diagnostics, Bristol Myers Squibb

  • Topic 13:

    Recent Advancements in Fit-for-purpose Validation Requirements for High Parameter Cytometry Assays with/without Autogating
    Dr. Vilma Decman, Senior Director Head of Cellular Biomarkers, GlaxoSmithKline

  • Topic 14:

    Current Evolution of High Dimensional Flow Cytometry Data in Clinical Settings and needs for Autogating
    Dr. Enrique Gomez Alcaide, Strategy Area Leader Cellular Biomarkers & Bioanalytics, Roche

  • Topic 15:

    Recent Advancements in Mass Cytometry (CyTOF) Panels and comparison with Conventional Flow Cytometry
    Ms. Amelia Au-Yeung, Principal Scientific Researcher, Genentech

Part 5: White Paper in Bioanalysis

  • 2022 White Paper on Cytometry Topics:

    Consensus & Conclusions on Cytometry Topics for 2022 White Paper

 

Topic DETAILS of Th2

Part 1: Understanding the Cytometry Conventional/Novel Technologies and Main Applications
  • Topic 1

    Importance of Conventional Flow Cytometry Bioanalysis for Clinical Trials – Past, Present, Future: Technology Introduction & Main Applications in Immunophenotyping, RO and PD Assays
    Dr. Erin Stevens, Associate Research Fellow / Lab Head - Early Clinical Development, Pfizer

    • Evolution of Flow Cytometry assay complexity
      • Advances in instrumentation
      • Initially assays evaluated only 1 or 2 parameters and explored major cell populations
      • T cell enumeration for diagnosis and treatment of AIDS
      • Established the importance of the Flow Cytometry platform for clinical assessments
      • Development of 8-20 parameter instruments in the 1990s
      • Identification of distinct cell populations that could be further interrogated to understand their role in disease.
    • Immunophenotyping Assays
      • Transitioned from detecting major populations to assays that interrogate specialized cells
    • Receptor Occupancy (RO) Assays
      • Assessed through direct or indirect detection of receptor-bound drug
      • Assessment of functional blockade of a targeted signaling pathway
    • Pharmacodynamic (PD) Assays
      • Developed to assess changes in immune cell activation or depletion
    • Assessments of T cell activation or exhaustion
      • Cell depletion or normalization of immune cell profiles
      • Establishing proof of mechanism for drug candidates
      • Optimizing clinical dosing strategies
    • Case Studies: Main applications in Immunophenotyping, RO and PD Assays
  •  

  • Topic 2

    Importance of Spectral Cytometry for Biomarker Development – Current Status and Future Developments: Technology Introduction & Primary Unique Features vs Conventional Flow Cytometry, Main Application in Drug Discovery & Development
    Dr. Michael Nathan Hedrick, Director Clinical Flow Cytometry, Bristol-Myers Squibb

    • Differences between Conventional Flow Cytometry and Spectral Cytometry
      • Hardware
      • Detection
      • Analysis
    • Differences between normal narrow bandwidth Flow Cytometry and Spectral Cytometry
      • Advantages of spectral over narrow bandwidth
      • Spectral Cytometry unique optical collection and analytical capabilities
      • Emission spectrum of every fluorescent molecule
      • Recorded as a spectral signature
      • Used as reference in multicolor applications
      • Discrimination of fluorescence by identifying differences in the overall spectral signatures
    • Emphasis on data collection and increased the number of parameters that are identifiable
      • Careful panel design for spectral cytometry
      • Instrument capabilities and Cell lineage sub populations
      • Expected antigen density, Antibody-conjugates and their properties
    • Novel use of Spectral Cytometry in Clinical Trials for generation of high quality and reproducible data
      • Target Engagement (TE), Receptor Occupancy (RO), Pharmacodynamics and Mechanism of Action (MoA)
    • Case Studies:: Novel applications of high dimensional Spectral Cytometric assays in Clinical Trials
  •  

  • Topic 3:

    Importance of Mass Cytometry for Bioanalytical Needs – Past, Present, Future: Technology Introduction, Primary Unique Features vs Conventional Flow Cytometry & Applications in Bioanalysis & Biomeasures
    Mr. Chad Stevens, Senior Scientist BioMedicine Design Department, Pfizer

    • How is Mass Cytometry utilized in Pharma
      • Pioneer in high dimensional single cell cytometry
      • Deep phenotyping
      • Biomarkers
      • Quantitative biomeasures
      • Clinical Applications
    • Receptor Quantification
      • Cornerstone quantitative biomeasure for biotherapeutic development
      • High number of analyzed parameters
      • Highly quantitative nature
      • Development a multiplexed receptor quantification assay
    • Immune-oncology
      • Simultaneously measuring targets on a wide range of cell populations.
      • Ability to inform modeling and simulation with multiplexing approaches
      • Supporting pre-clinical teams
    • Pushing the technology to thrive in an ever emerging field
      • Becoming more efficient in ability to multiplex outputs
    • Case Studies: Generation of high quality quantitative biomeasures

  • Topic 4:

    Importance of Imaging Cytometry for Biomeasures – Past, Present, Future: Technology Introduction & Applications in Cell-based Biomeasure Assays
    Dr. Polina Goihberg, Discipline Lead BioMedicine Design Department, Pfizer

    • Imaging Cytometry capabilities for quantitative bioanalysis
      • Combing the throughput of flow cytometry with the capabilities of fluorescence light microcopy
      • Visualizing samples
      • Interrogating numerous characteristics of cell morphology intensity and spatial features of the signals generated by fluorescence markers
      • Generation of abundant data on cell phenotype and biological functions
      • Imaging cytometry platform
    • Advancing Imaging Cytometry applications
      • Target membrane kinetics
      • Target Engagement (TE)
    • Drug discovery and pre-clinical development
      • Drug subcellular trafficking
      • Endosomal escape
      • Transgene Expression
    • Discussion of continuous challenges
      • Increasing throughput
      • Cross-platform comparison
    • Case Studies: New applications for Biologics and Gene Therapies

Part 2: Current Challenges with Validation & Sample Collection
  • Topic 5:

    Strategies for Rationalizing Fit-for-purpose Criteria for Flow Cytometry Biomarker Assay Validation (BAV): How to Determine what Parameters/Acceptance Criteria are really needed without overdoing or underdoing?
    Dr. Steven Eck,Director Translational Sciences, Clinical Immunology and Bioanalysis, AstraZeneca

    • Establishing analytic performance criteria that are fit-for-purpose in Flow Cytometry
      • Keeping with WRIB’s philosophy of teaching through real world examples
    • Challenging a simple Flow Cytometry Panel by assessing both
      • Targeted Cell Depletion
      • Receptor Occupancy (RO)
    • Focus on the validation process including:
      • Defining and documenting the purpose of the testing
      • Selecting development and validation experiments
      • Using guidance from White Papers in Bioanalysis and CLSI H62 Guidance on analytic validation of assays performed by Flow Cytometry
    • Justifying Fit for Purpose nature of
      • Precision criteria that are in line with broad based recommendations
      • Situations that merit consideration of alternate (more/less stringent) performance criteria from the assay
    • Leveraging the iterative process to refine specific validation criteria
      • Assessment of fit-for-purpose performance on an ongoing basis
    • Case Studies: Determine what Parameters/Acceptance Criteria are needed for Flow Cytometry Fit for Purpose Validation

  • Topic 6:

    Turning the CLSI H62 into Practise for Bioanalytical/Clinical Laboratories: First-hand experience in trying to apply the CLSI H62 to real situations with pros & cons
    Dr. Christèle Gonneau, Lead Scientist Flow Cytometry, LabCorp

    • Meeting Minimal Criteria for Validation
      • Precision
      • Sample Stability
      • How to achieve minimal requirements with real life samples
      • BMA for highly sensitive assays such as MRD applications
      • Biological variability of the validation samples
    • LLOQ Bioanalytical challenges
      • Adapting experimental design to validation samples
      • Challenges to use contrived samples using cell lines or frozen BMMCs/PBMCs
      • Unpredictability of the cellularity of the samples and the short stability for some assays
    • Use of intended Matrix & Disease State indication challenges
      • Obtaining appropriate validation samples
      • Validation samples considered as rare matrices
      • Logistical issues in obtaining rare matrices samples
      • Issues with viability or hemodilution of the samples.
    • Case Studies: First-hand experience of hemato malignancy validations

  • Topic 7:

    Challenges and Strategies for Clinical Sample Collection in Flow Cytometry and Cell-Based Assays
    Dr. Anka Ehrhardt, Director Cell-Based Assays, Merck

    • In dept understanding of the challenges and limitations with Clinical Sample Collection for Flow Cytometry
      • Is there a Universal Sample?
      • Strategic Collection & Use of Clinical Samples
    • Know before you go
      • Optimization for transport and storage
    • Colorful Caps
      • Optimization for Target Cell Population
    • Alive or dead
      • Optimization for Processing and Analysis
    • Alive or dead
      • Collection strategies for Rare Disease clinical samples
      • Importance of coordination of clinical sample use
      • Harmonizing sample collection among multiple sites
    • Case Studies: Clinical Sample Collection in Flow Cytometry and Cell-Based Assays

 

Part 3: Specific Complex Applications
  • Topic 8:

    Flow Cytometry in the Monitoring of Immune Responses to Vaccines: Method Development challenges and Assay Optimization Strategies to Increases the Robustness and Accuracy of the Data
    Dr. Gabriela Franco Salinas , Associate Scientific Director Clinical Immunology, Janssen

    • Fit for Purpose Validation of an Intracellular Cytokine Staining (ICS) assay
      • Detection of the production of Th1 and Th2 cytokines in response to Vaccination Antigens
    • Strategies for Antibody Panel Design
      • Designed to improve the detection of Th2 cytokines 
      • Antibodies to detect three different Th2 cytokines
      • Antibody against the activation marker CD40L included to reduce Th2 background signal 
    • Incubation times & Acquisition Parameters
      • Optimization to maximize the detection of Th2 cytokines
      • Method compromise for having an adequate Th1 response
    • Assay Precision
      • Characterization of assay precision across the expected range using PBMCs from healthy individuals and surrogate antigens
      • Precision calculation for single and combined readouts
      • Single Well Analysis vs Duplicate wells Analysis
      • Impact of different thresholds for positive events on assay precision
      • Impact of biological variability on the assay precision
    • Case Studies: Method Development challenges and Assay Optimization Strategies to Increases the Robustness and Accuracy of the Data for Monitoring of Immune Responses to Vaccines

  • Topic 9:

    Use of Model Antigens to Validate Antigen Specific Vaccine Flow Cytometry Assays
    Dr. Bruno Salaun, Senior Scientist, GlaxoSmithKline

    • Flow Cytometry Functional Assays in Vaccine clinical trials
      • Advanced application of Flow Cytometry as readout of Cellular Immunogenicity
    • Full Validation requirements for endpoints in registration studies
      • Intracellular Cytokine Staining assay (ICS)
      • Quantification of “positive” events from Peripheral Blood Mononuclear Cells (PBMC)
      • T cells expressing cytokines independently of antigen specificity
    • Antigen Specific T cells
      • Rare events and frequencies greatly variable
      • Need for high number of PBMC to capture relevant numbers of antigen specific T cells
      • Need for large volume of blood required to generate PBMC in sufficient numbers
    • Specific Validation Design for Rare Events
      • Overcoming issues due to insufficient blood volumes from standard vaccine clinical trials
      • Alternative use of PBMC from biobank studies (blood collection from healthy donors)
    • Use of Model Antigens
      • Model Antigens ability to stimulate PBMC
      • Generation of a wide range of responses from biobank samples
    • Case Studies: Use of model antigens to generate a wide range of responses from biobank study samples for validation of ICS assay

  • Topic 10:

    Challenges and Value of Clinical Receptor Occupancy (RO) Assessments for Bispecific T Cell redirection by Flow Cytometry
    Dr. Katharine Grugan, Associate Scientific Director, Janssen

    • Implementation of Receptor Occupancy (RO) of Bispecifics in Clinical Trials
    • Things to consider during method development/validation
      • Assay Format & Tube Type
      • Desired Stability
      • CRO selection
    • Focus on reaching Assay Sensitivity
      • Challenges with Sensitivity Determined in Validation vs Actual Clinical Samples
      • Strategies for Sensitivity evaluation in Flow Cytometry
      • Staggered approach and impact of minimal number if events
      • Data interpretation for establishing the assay sensitivity
    • Current industry best practices and approaches to sensitivity evaluation
      • Consideration on when LLOQ assessment should be included in the Flow Cytometry validation as in flow-based RO assays
    • What is the value of the Clinical RO data?
      • Ongoing need for implementation of RO Flow Cytometric assays in Clinical Trials
      • Applications of Flow Cytometry in early clinical trials
      • Flow cytometry as the method of choice for RO data generation
    • Case Studies: Flow Cytometry method development and validation for Clinical Receptor Occupancy (RO) Assessments for Bispecific T Cell redirection

  • Topic 11:

    Challenges in the Development of Novel Flow Cytometry Assays for Bioanalysis of PD Markers: Macrophage Polarization and Phagocytosis Measurements
    Dr. Sarita Sehra, Senior Research Investigator Translational Sciences, Incyte

    • Applications of Flow Cytometry in Tumor Growth, Phagocytosis, and Immune-suppressive Activity studies
      • Supporting a novel drug role drug in polarizing immunosuppressive macrophages to a proinflammatory phenotype
    • Orthogonal Flow cytometry & Confocal Microscopy analysis
      • Analysis of monocytes and dendritic cells of healthy donors
      • Studies of phagocytosis inhibition by both intermediate and classical monocytes
    • Cell-based Proliferation assay
      • Macrophage-mediated suppression of T-cell proliferation associated with increased interferon-γ production
    • Orthogonal Flow cytometry & NanoString analysis
      • Evaluation Marker expression on generated macrophages
    • Case Studies: Method development of Flow Cytometry Assays for Bioanalysis of PD Markers

 

Part 4: Innovation in Cytometry
  • Topic 12:

    Can we use Cytometry in Tissues Bioanalysis? Current Challenges, Innovative Approaches and Technologies
    Dr. Yongliang "Steve" Sun, Associate Director Translational Sciences and Diagnostics, Bristol Myers Squibb

    • Challenges in Tissue Bioanalysis by Cytometry for Clinical Trials
      • Concerns in gaps between the peripheral blood and diseased sites
      • Blood is easy to access vs tissues
      • Tumor MicroEnvironment (TME) as ideal sample for Cytometry.
    • Conventional Flow Cytometry Application in Tissues Bioanalysis
      • Provides direct information for the diseased sites
      • Tissues need to be dissociated into single cell suspensions for cytometry analysis
      • Tissues have limited sample stability for cytometry analysis
      • The measurement of certain surface target markers might be compromised due to the digestion/dissociation step
    • ChipCytometry Application in Tissues Bioanalysis
      • Multiplexing markers quantitatively at the single-cell level
      • Using FFPE or frozen tissues
      • Providing spatial information
      • Challenges with Antigen retrieval affects the measurement of markers using FFPE sections
      • Throughput not high, only up to five markers can be stained simultaneously
      • Antibodies for FFPE tissues are not as broadly available as for fresh frozen tissues
    • Case Studies: Method development for tumor Flow Cytometry panel and tumor ChipCytometry panel for a clinical trial

  • Topic 13:

    Recent Advancements in Fit-for-purpose Validation Requirements for High Parameter Cytometry Assays with/without Autogating
    Dr. Vilma Decman, Senior Director Head of Cellular Biomarkers, GlaxoSmithKline

    • Advancing the Discussion on how to best assess large data sets coming from High Dimensional (HD) Flow Cytometry
      • Current HD Flow Cytometry applications
      • Accessibility of High parameter Flow Cytometry
      • Novel applications on High dimensional Flow Cytometry
    • Strategy for Fit-for-purpose Validation of High Dimensional Spectral Cytometry panels
      • Validation requirements for assay modifications
      • Importance of stability tracking of critical reagents
      • Lot-to-lot reagent comparisons
    • Novel approaches to batch-to-batch comparisons
    • Quality Control material
      • Dual purpose
      • Internal vs batch-to-batch controls
    • Validation Cut-off for rare events in high-dimensional Flow Cytometry panels
    • In depth discussion on how to do FFP validation of exploratory high-dimensional panels
      • Handling hundreds of reportables
      • How to best cover validation when working with limited number of events for certain populations
    • Case Studies: Application of High parameter flow cytometry instrument in Clinical Trials

  • Topic 14:

    Current Evolution of High Dimensional Flow Cytometry Data in Clinical Settings and needs for Autogating
    Dr. Enrique Gomez Alcaide, Strategy Area Leader Cellular Biomarkers & Bioanalytics, Roche

    • Deciphering the complexity for High Parameter Flow Cytometry
      • Current role of high dimensional Flow Cytometry Assays in Clinical Trials
      • Interpretation of the complexity of the immune response
      • New technical advances
      • Improvement of biomarker strategies
      • Discovery new biomarkers
    • Immunophenotyping complexity in Oncology programs
      • Handling data analysis from clinical programs
    • Challenges in Clinical trials setting
      • Benefits of implementing full Spectral Cytometry and the high dimensional assays
      • Novel Data from clinical settings
      • How to successfully handle central labs interactions
      • Need for deep collaborative effort to bring standard solutions
    • Case Studies: Developing and implementing high dimensional flow cytometry assays in clinical trials

  • Topic 15:

    Recent Advancements in Mass Cytometry (CyTOF) Panels and comparison with Conventional Flow Cytometry
    Ms. Amelia Au-Yeung, Principal Scientific Researcher, Genentech

    • Strategies in Mass Cytometry Panels development and validation
      • How to have more consistency
      • Increased control over a particular Mass Cytometry Panel
    • Current best industry practice for making Mass Cytometry Panels
      • Specific Panel design
      • Extensive panel validation
      • High number of parameters assessed
      • Thorough optimization of the staining
      • Impacts of signal spillover
    • Solutions to reduce panel variability
      • Developing custom panels to reduce variability
      • Comparison with Conventional Flow Cytometry
    • Panel Standardization for Sample Collection
      • Designing specific Mass Cytometry panels to meet program needs
      • Validating staining panel and staining samples
      • Optimization of staining conditions for maximum cell recovery and staining
    • Case Studies: Development of Mass Cytometry Panels and comparison with Conventional Flow Cytometry




Agenda at a Glance Agenda at a Glance