Specialized Workshop Th2 - Thursday Sep. 30, 2021: 8am - 5pm, ET - Lecture List

"Cytometry Innovation Day: Novel Techniques & Advanced Approaches in Cytometry"

Part 1: Is Spectral Cytometry the next generation of High Dimensional Cytometry?

Part 2: Current Regulatory Challenges in Flow Cytometry Validation

Part 3: Advancements of Mass Cytometry and Imaging Cytometry in Clinical Applications

Part 4: Evolving Science & Innovation: Multivariate Analytical Techniques & Multiparameter Flow Cytometry

 

Lecture DETAILS of Th1 – Cytometry Innovation Day

Part 1: Is Spectral Cytometry the next generation of High Dimensional Cytometry?
  • Lecture 1
    Applications of Spectral Cytometry in Clinical Assays: Recent Developments and Innovations in High Dimensional Cytometry
    - Dr. Michael Nathan Hedrick, Director Clinical Flow Cytometry, Bristol-Myers Squibb
    • Similarities and differences among
      • Conventional Flow Cytometry
      • Innovative Spectral Cytometry
        • Hardware
        • Detection
        • Analysis
    • 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
    • Increased the number of parameters that are identifiable
      • Careful panel design for spectral cytometry
      • Understanding
        • Instrument capabilities
        • 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
      • High throughput
      • Efficiency
      • Consistency
      • Reproducibility
    • 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
  •  

  • Lecture 2
    RecentAdvancements in High Dimensional Flow Cytometry - Traditional Hierarchical Gating vs Automated Gating vs Reduction Analysis
    - Dr. Vilma Decman, Director and Head Cellular Biomarkers, GlaxoSmithKline
    • Feasibility of automated data analyses in Clinical Trial settings for Spectral Flow Cytometry
      • Requirements and best industry practices for establishment of automated data analysis
      • Limitations in automated data analysis approaches
      • Comparison of traditional gating vs automated data analysis/validation
      • Reduction analysis
      • Different approaches evaluation
    • Advancing the Discussion on how to best assess large data sets coming from High Dimensional Flow Cytometry
      • Current Flow Cytometry applications
      • Accessibility of High parameter Flow Cytometry
    • Novel applications on High dimensional Flow Cytometry
      • Applications in clinic research
      • Examination of multiple targets on various cell subsets
      • Possibility to analyze limited sample size
      • Identification and enumeration of multiple cellular markers
      • Tracking of cellular markers across time and treatment
    • Case Studies: Application of High parameter flow cytometry instrument in Clinical Trials
  •  

  • Lecture 3
    Implementation of High Parameter Spectral Cytometry in the Clinics: Roche’s Experience
    - Dr. Enrique Gomez Alcaide, Translational Biomarker Leader, Roche
    • Role of High Dimensional Cytometry Panels in Clinical Trials
      • Spectral cytometry analyzers as enabling technologies available to meet current scientific needs
        • Measuring the complete spectrum
        • Take advantage of all spectral data to distinguish fluorochromes in multicolor experiments.
      • Expanded the capabilities to simultaneously investigate a larger number of parameters in each experiment
      • Strives to increase panel complexity
        • Tools to achieve effective panel design
    • Challenges in development and implementation High Parameter Spectral Cytometry
      • Multiparametric analysis at the single cell level
      • Additional advantages
        • Improved flexibility in reagent selection
        • Removal of autofluorescence
    • Data generation
      • Comparison of traditional Flow Cytometry vs Spectral Cytometry
      • Compensation versus spectral unmixing
    • Limitations in High Parameter experiments
      • Pairing fluorescent readouts to fit both the needs of the experiment and instrument hardware components
      • Improvements
        • Advanced mathematical unmixing models
    • Case Studies: Challenges in data analysis, Holistic vs specific project-oriented data analysis in clinical setting

 

Part 2: Current Regulatory Challenges in Flow Cytometry Validation
  • Lecture 4
    Recent Developments in Flow Cytometry Validation in a Bioanalytical Lab: Learning from Actual Case Studies
    - Dr. Steven Eck, Associate Director Integrated Bioanalysis, AstraZeneca
    • Lesson Learned in the Fit-For-Purpose (FFP) Validation of Flow Validation Assays
      • Immunophenotyping looking for correlative associations
    • Planned analytes
      • Prospective hypotheses testing
      • Considerations for Receptor Occupancy
      • Special considerations for Rare Events
        • Need to use entrance/exit criteria
    • Unplanned analytes
      • Strategies to evaluate fit-for-purpose for poorly characterized analytes present within well characterized assays
    • High Dimensional Data
      • Need to use study outcomes to identify additional analytes of interest and assessing their analytic performance
    • Case Studies: Recent real-life applications of the CLSI H62 and 2018-2020 White Paper in Bioanalysis recommendations to illustrate how to qualify and validate Flow Cytometry assays
  •  

  • Lecture 5
    The Evaluation of Accuracy for Flow Cytometry Applications in Regulated Laboratories
    - Dr. Christèle Gonneau, Lead Scientist Flow Cytometry, Covance
    • Various approaches taken to date to assess Accuracy for Flow Cytometry Assays Validation used in a Regulated Environment
      • QC material target ranges
      • Proficiency testing
      • Lab concordance
      • Longitudinal data
    • Regulatory feedback on the importance of Accuracy Evaluation
      • Importance of device/technique accuracy in a regulatory environment
      • Importance to understand how accurate a device is if the results are to be interpretable
      • Importance, in the absence of a reference standard, to define an intermediate understanding of accuracy
      • Official definition of accuracy of a bioanalytical method: "Accuracy is defined as the closeness of a result to the true value"
    • Nature of the determinations for flow cytometric assays
      • Quasi-quantitative assays
      • Impossibility to validate accuracy in the traditional manner
      • Alternative approaches for accuracy
    • Case Studies: State-of-the-art alternative approaches to calculate accuracy for Flow Cytometry Assays used in Regulated Lab
  •  

  • Lecture 6
    LLOQ & Sensitivity Determination for Flow Cytometry Assays Validation: Lesson Learned from New Case Studies
    - Dr. Ulrike Sommer, Senior Principal Scientist Translational Medicine/Biomarker Development, Novartis
    • Guidance for
      • High Sensitive Flow Cytometry assays
      • Design
      • Development
      • LOB, LOD, LLOQ
    • Sensitivity for rare event analysis/high-sensitive flow cytometry assays
      • Determination
      • Validation
    • Strategies for LLOQ evaluation in Flow Cytometry
      • Staggered approach
      • Impact of minimal number if events and LLOQ
      • Data interpretation for establishing the assay sensitivity/LLOQ
    • Current industry best practices and approaches to measure and validate LLOQ in Flow Cytometry
      • Complexity of LLOQ measurement been overcome
      • Consideration on when LLOQ assessment should be included inthe Flow Cytometry validation as in flow-based RO assays
    • Case Studies: Clinical data analysis considering the assay sensitivity and LLOQ determination

 

Part 3: Advancements of Mass Cytometry and Imaging Cytometry in Clinical Applications
  • Lecture 7
    Imaging Cytometry in Drug Development: Quantitative analysis of Target Distribution, Target Engagement, and Intracellular Trafficking
    - Dr. Polina Goihberg, Discipline Lead BioMedicine Design Department, Pfizer
    • Developing best practices for quantifying antibody-induced Target Internalization for the PKPD modeling
      • Multiparameter approaches for measuring
        • Target internalization
        • Proximal signaling
    • In depth discussion on
      • Current Gaps in technology
        • Advantages and disadvantages of Image Cytometry providing complementary information to conventional Flow Cytometry
      • Unmet needs in bioanalysis that can be addressed by expanding Imaging Cytometry capabilities
        • Limitations of traditional Flow Cytometry: Low sample stability, Need of mid-high sample volumes, Not real images of Analyzed cells, Spacial distribution of a marker within a cell or cell surface, Cellular interactions
      • Recent evolution in Imaging Cytometry
        • Combining single cell imaging with multi-parameter high-throughput single cell analysis
        • Imagining technologies, Electronics, Digital computing
    • Case Studies: Fit-for-purpose assays with Quantitative Imaging Cytometry for drug discovery
  •  

  • Lecture 8
    Novel and Innovative Application of Mass Cytometry in Clinical Biomarkers
    - Dr. Jose Estevam, Head Clinical Biomarkers, Takeda
    • High Complexity Mass Cytometry in Clinical
      • Novel data from exploring Mechanism of Action (MoA) of drugs
      • Clinical bioamrkers application
      • Discovery and clinical validation of new prognostic biomarkers of disease progression by Mass Cytometry
      • Advancements in Mass Cytometry in Clinical Research
        • Current strategies Robustness and Reproducibility
        • Rigorously applying this platform to clinical samples
    • Importance in optimizing Mass Cytometry workflows
      • Sample handling
      • Optimization of staining conditions for maximum cell recovery and staining
    • Possibility to examine of up to 50 parameters
      • Multiplexed staining and metal-isotope-tagged antibodies
      • Performance beyond fluorescence-based detection
        • Avoiding the interference of spectral overlap of fluorescent agents
      • In depth characterization of cell populations
      • Identification of number of functionally distinct cell subsets
      • Identification of rare immune cell subsets
    • Case Studies: Clinical sample testing and monitoring Mass Cytometry data reproducibility in in clinical development
  •  

  • Lecture 9
    Making Mass Cytometry "Clinical Trial Compatible"
    - Dr. Gregory Hopkins, Senior Associate Scientist II Cellular Analytics Team, Bluebird Bio
    • Present challenges in using Mass Cytometry in Clinical Trials
      • Challenges for Variability of the Assay over time due to length of Clinical Trial
      • Risk of confounding the analysis
      • Experiment to test variability
    • Adjustments in analysis strategy to minimize variability
      • Use of validated commercial kits to reduce assay variability
        • Maxpar Direct Immune Profiling Assay kit coupled with Pathsetter analysis (MDIPA kit)
      • Need around having commercially available solutions for Mass Cytometry
    • Importance of having more consistency and control over a particular Mass Cytometry Panel
      • Current best industry practice for making Mass Cytometry “clinical trial compatible”
      • Strategy for panel development and validation
        • Specific Panel design
        • Extensive panel validation
        • High number of parameters assessed
        • Thorough optimization of the staining
        • Impacts of signal spillover
    • Case Studies: Possible solutions for more Mass Cytometry custom panels to reduce variability and make them more clinical trial compatible
  •  

  • Lecture 10
    Advantages & Challenges in using Mass Cytometry for Quantitative Biomeasures
    - Mr. Chad Stevens, Senior Scientist BioMedicine Design Department, Pfizer
    • Receptor Quantification on Mass Cytometry
      • Method Development
      • Quantitative biomeasures
      • Increasing the number biomeasurements in preclinical settings
      • Enablingtranslational modeling
      • Enrichment of Activation and Proliferation Markers by Mass Cytometry
    • Panel Standardization for Remote 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
    • Strategies for Mass Cytometry applications in
      • Large pre-clinical
      • Exploratory clinical studies
    • Development and validation of high parameter Mass Cytometry
      • Application of Mass Cytometry platform for use in exploratory research programs
      • Measurement of multiple markers for simultaneous receptor occupancy assessment and high-parameter immune phenotyping
    • Case Studies: Gaps and challenges as well as future developments to fill these gaps. Application of Mass Cytometry in preclinical setting

 

Part 4: Evolving Science & Innovation: Multivariate Analytical Techniques & Multiparameter Flow Cytometry
  • Lecture 11
    Advanced Applications of Multivariate Analytical Techniques in Flow Cytometry Immunophenotyping Assays
    - Dr. Nathan Standifer, Associate Director, Head GxP New Modalities Bioanalysis, AstraZeneca
    • Current developments in Flow Cytometry-based Immunophenotyping Assays
      • Considerations for the quantification of dozens of discrete cell populations in a single sample
      • Leveraging the results to identify associations between changes in monitored cells and therapeutic intervention
    • Issues with Traditional Analysis Techniques
      • Multiple factors making traditional techniques inefficient and ineffective
        • Size of immunophenotyping datasets
        • Intrinsic heterogeneity in responses
        • Correlated lymphocyte population biology
    • Advantages of using Multivariate Analytical Techniques
      • Alternative means of assessing large immunophenotyping datasets
      • Reducing dimensionality
      • Assessing covariance
      • Identifying underlying variables associated with
        • Therapeutic dose
        • Clinical response
    • Case Study: Proposed framework of the utility of multivariate analysis to identify therapy- and dose-dependent pharmacodynamic changes associated with best objective response from Phase II study of immuno-oncology therapeutic molecules
  •  

  • Lecture 12
    Assessment of Steric Hindrance in Multiparameter Flow cytometry: How to tackle molecular competition in Clinical Biomarker Development?
    - Dr. Fabian Junker, Senior Scientist Protein Biomarker Development, Roche
    • Steric Hindrance
      • When large molecules bind to a bioanalytically relevant proteinon the cell surface of immune cells
    • Strategies to address Flow Cytometry
      • Reagent performance
      • Specificity
      • False-negative target assessment
    • Impact of Steric Hindrance in Flow Cytometry
      • Competition for overlapping epitopes
      • Detection antibodies fail to correctly bind to the cell surface target
      • Result in systematic under-estimation of target,
      • Incorrect assessment of immune cell activation status and TE
    • Importance of Assessing Steric Hindrance
      • Recent evidence from checkpoint-inhibitor experienced patients
      • Need to assess Steric hindrance methodologically to avoid false-negative reporting of PD-biomarkers
      • Similar effect can apply also to soluble/non-cell bound PD biomarkers
    • Ongoing need for implementation of high complex Flow Cytometric assays in Clinical Trials
      • Applications of Flow Cytometry in early clinical trials
      • PD biomarker development
      • Flow cytometry as the method of choice for
        • Timely and robust immunomonitoring
        • Target Engagement (TE) / Receptor Occupancy (RO) assays
    • Case Studies: Novel application and approaches to avoid false-negative target assessment due to impact of Steric Hindrance in Multi-parameter Flow Cytometry
  •  

  • Lecture 13
    Advanced Applications in High Parametric Flow Cytometry: Characterization of the Activation Status of Innate Immune Cell Populations in Stabilized Human Whole Blood
    - Dr. Sandra Nuti, Head High Parametric Flow Cytometry & Immunomonitoring, GlaxoSmithKline
    • Use of High Parametric Flow Cytometry for the evaluation of immune responses of vaccine candidates
      • Method Development strategies for a whole blood assay
      • Sample Analysis from clinical trials
        • Evaluation of critical information regarding the immunogenicity of vaccine candidates
      • Assessment of Antigen-specific IgG functional titers and T-cell responses
      • Vaccines induction of early changes in circulating leukocytes that might be predictive of their safety and immunogenicity
      • Assay optimizationand evaluation as a proof-of-concept to the clinical trials
    • Challenges in High Parametric Flow Cytometry experiments
      • Importance of the right combination of fluorophores
      • Advances Flow Cytometry reagents
      • Selecting the right combination of fluorophores and antibody conjugates
    • Designing Panels for complex multicolor experiments
      • Performing single-cell interrogation with multiple markers
      • Increasing number of colors and antigens
      • Minimizing the need for compensation and spillover adjustments
    • Case Study: Robust High Parametric Flow Cytometry assay for evaluation of activation status of innate populations in stabilized human whole blood




Agenda at a Glance Agenda at a Glance