2014   Program   Scientific Tutorials
New this year! Scientific Tutorials are open to all Full Congress Registrants; no additional fees apply.

SATURDAY, 17 MAY
1230 - 1400

TUTORIAL 1
Designing, Planning, and Performing Polychromatic Flow Cytometry Experiments
Pratip Chattopadhyay, ImmunoTechnology Section Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA

Polychromatic flow cytometry provides the power to analyze, in great depth, the complex relationships between cellular subsets and function.  We will discuss the latest hardware, reagent, and data analysis considerations for performing successful polychromatic experiments, and provide a practical roadmap for developing and improving this technology.  This year, there will be a new focus on executing large studies successfully, including unique aspects of planning and data analysis.

TUTORIAL 2
FIJI ImageJ: Open Source Image Informatics for Multidimensional Imaging
Kevin Eliceiri, University of Wisconsin - Madison, Madison, WI, USA

Modern biological imaging requires informatics tools for the acquisition, analysis, visualization and dissemination of complex multidimensional image datasets. Open Source software has become a powerful tool for image informatics due to its open access, easily customizable interfaces and flexibility. We present the ImageJ based toolkit FIJI (www.fiji.sc) as a platform for interoperability demonstrating how it can be used for basic and advanced analysis of complex image datasets and be interfaced directly with other software tools for visualization, dissemination and acquisition. FIJI can also be easily customized directly by the bench scientist through powerful scripting and plugin functionality. FIJI will be demonstrated and shown with functionality shown for specific advanced features including segmentation and tracking.

TUTORIAL 3
Cellular Proliferation Monitoring by Flow Cytometry: The Good, The Bad and the Ugly
Paul Wallace, Roswell Park Cancer Institute, Buffalo, NY, USA
Kathy Muirhead, SciGro, Inc., Middleton, WI, USA
Kylie Price, Malaghan Institute of Medical Research, Wellington, New Zealand


Course Objectives: Our objective in this tutorial is to stimulate problem- and solution- sharing for two flow cytometric methods commonly used to assess cell proliferation:
 
DNA cell cycle analysis:
• Cell division monitoring using dye dilution.
• To minimize didactic time and maximize discussion, we will use the
following format:
  1.    Brief overview of principles, terminology and data analysis methods (~15 min)
  2. Case studies illustrating typical problems, pitfalls and troubleshooting based on the organizers’ experience in their laboratories (~30 min)
  3. Group problem solving for participant-submitted examples of problematic issues and challenging applications encountered in their own work (~45 min)
All participants are strongly encouraged to submit their own examples of staining, data acquisition, and/or data analysis issues to the organizers before the meeting, by sending an email to proliferation@rpciflow.org. Data files and/or example histograms/dotplots may also be brought to the session for inclusion in the discussion if/as time allows. All data provided by participants will be discussed anonymously unless the provider wishes to identify themselves during the session.
 
Course Details or Outline: Relative time spent on different topics will be driven by participant interests, as expressed by the questions and data examples received by the organizers as of 12 May 2014.
 
Potential topics for discussion include:
  • Methods and reagents for identifying distribution among and progression through different cell cycle stages (e.g., including DNA binding dyes, thymidine analogues, and proliferation-related antigens
  • Principles and methods for analysis of DNA cell cycle data, including simple vs. complex mathematical techniques for deconvoluting single parameter DNA histograms and multiparameter methods for analysis of blocked or synchronized populations.
  • Methodological considerations for cell division monitoring by dye dilution, including advantages and limitations of different classes of proliferation tracking dyes (e.g., CFSE, PKH26 and newer analogs of each), critical controls and gating strategies.
  • Principles and methods for analysis of cell division based on dye dilution profiles, including proliferative fraction, proliferative index and precursor frequency.
Summary: This tutorial will focus on real world examples provided by the organizers and participants illustrating what can and does go wrong while performing DNA cell cycle analysis and dye dilution assays to monitor cellular proliferation. Troubleshooting techniques, practical solutions and consideration of how best to minimize typical problems will be shared in order to facilitate identification and use of ‘best practices’ as participants apply these methods in their biological systems of interest.

TUTORIAL 4
Education in a Shared Research Facility
Alfonso Blanco-Fernandez, University College Dublin, Dublin, Ireland
John C. Tigges, Harvard Stem Cell Institute, Cambridge, MA, USA

Flow cytometry and Imaging are well-established techniques although they are technologically in expansion. Core Technologies need to bring added value to their users and institutional setting. But how can core facility staff keep up with the latest developments, how can they receive appropriate continuing education and how can this be passed on to users of a facility? We will discuss different strategies for educating the core facility new and existing users and technical staff members. We will evaluate and deliberate the importance of educating on site, participation at relevant meetings, and how remote learning can benefit the facility.
 
At the end of the tutorial the delegate will be aware of the approaches that can be taken to bring added value to the core, its staff and its users.

1415 - 1545

TUTORIAL 5
Algorithmic Analysis of Flow Cytometry Data (Part 1)
Tim Mosmann, University of Rochester Medical Center, Rochester, NY, USA
Richard Scheuermann, J. Craig Institute, La Jolla, CA, USA
Greg Finak, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
Ryan Brinkman, BC Cancer Agency, Vancouver, BC, Canada
Josef Spidlen, BC Cancer Agency, Vancouver, BD, Canada


This two-session tutorial will demonstrate the automated analysis of flow cytometry data using five different approaches.  Traditional manual gating analysis suffers from poor reproducibility and objectivity, and is very difficult for high-dimensional data.  Several algorithms have now been developed, using different approaches that may be more suited to different datasets and biological questions.  Five approaches will be highlighted in this tutorial, covering a range of approaches from just identification of targeted or unknown populations, to a full pipeline of data analysis.  The tutorials will consist of brief demonstrations of data analysis by each of the approaches.  After the tutorials, the students should have an appreciation of the power of automated analysis, the requirements for entering their data into an automated analysis system, and how to choose the most appropriate analysis method for their particular type of data/question.

TUTORIAL 6
Forensic Flow Cytometry
Jennifer Wilshire, Memorial Sloan-Kettering Cancer Center, New York, NY, USA

Do you sometimes look at flow cytometry results and wonder if something went wrong, but can’t put your finger on it?  In this tutorial we will take a “forensic” approach to study the most common issues that give bad flow cytometry data.  We will use case studies and real examples to illustrate the causes of bad data and how you can prevent it.  Many topics will be covered including staining issues, compensation, controls, and sorting.  The focus of this tutorial will be on understanding the potential pitfalls in flow cytometry experiments and learning practical methods to prevent these issues.  After participating in this tutorial, attendees will have practical suggestions of ways to ensure flow cytometry experiments will yield good data and purely sorted cells.

TUTORIAL 7
Ask the Experts: Biosafety
Kevin Holmes, NIH, Bethesda, MD, USA
Steve Perfetto, NIH, Bethesda, MD, USA
Hank Pletched, University of Pennsylvania, Philadelphia, PA, USA
Ingrid Schmid, University of California at Los Angeles, Los Angeles, CA, USA
Rich Konz, Univeristy of Massachusetts Medical School, Worchester, MA, USA

  1. This tutorial will provide a summary of biosafety principles as they apply to flow cytometry and cell sorting and an overview of applicable standards and risk assessment. It will also provide a forum in which to discuss with experts in the field, specific scenarios that operators or core facility managers encounter.
  2. After participation in this tutorial the attendee should have a clearer understanding of the principles and practices of biosafety as it pertains to flow cytometry, in particular cell sorting.  Additionally, the attendee will have a list of resources to aid in risk assessment and the development of Standard Operating procedures in their own lab.
TUTORIAL 8
Finances 101: Budgets, Service Fees & Equipment Service Costs
Julie Auger, University of California, San Francsico, CA, USA
Nicole White, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA


Every good core facility director recognizes that her or his facility operates as a small business.  However, most core directors are trained as scientists, not business specialists.  During this tutorial we will provide a basic interactive hands on tutorial reviewing the financial management of a core facility.  This will provide participants with the information they need manage equipment maintenance expenses, calculate service fees and understand their annual operating budget.  It will also prepare them for successful conversations with their financial manager.
 
After participating in this tutorial, the student will leave with various tools including an equipment maintenance workbook to make an informed decision regarding service contract versus self-insurance.  They will also have a workbook to calculate service fees and create an annual operating budget.

1600 - 1730

TUTORIAL 9
Algorithmic Analysis of Flow Cytometry Data(Part 2)
Tim Mosmann, University of Rochester Medical Center, Rochester, NY, USA
Richard Scheuermann, J. Craig Institute, La Jolla, CA, USA
Greg Finak, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
Ryan Brinkman, BC Cancer Agency, Vancouver, BC, Canada
Josef Spidlen, BC Cancer Agency, Vancouver, BD, Canada


This two-session tutorial will demonstrate the automated analysis of flow cytometry data using five different approaches.  Traditional manual gating analysis suffers from poor reproducibility and objectivity, and is very difficult for high-dimensional data.  Several algorithms have now been developed, using different approaches that may be more suited to different datasets and biological questions.  Five approaches will be highlighted in this tutorial, covering a range of approaches from just identification of targeted or unknown populations, to a full pipeline of data analysis.  The tutorials will consist of brief demonstrations of data analysis by each of the approaches.  After the tutorials, the students should have an appreciation of the power of automated analysis, the requirements for entering their data into an automated analysis system, and how to choose the most appropriate analysis method for their particular type of data/question.

TUTORIAL 10
Understanding Analytical Method Validation as Applied to Flow Cytometry
Virginia Litwin, Covance Inc., Chantilly, VA, USA

This tutorial will cover the Who, What, When, Why, and How of Analytical Method Validation. 

WHO: Everyone—Regulated and non-regulated labs including basic research labs.

WHAT: The tutorial will begin with an overview of method validation. We will define each of the validation parameters, and how those parameters can be addressed in a flow cytometry method validation.  The ICSH/ICCS validation recommendations will be presented (Cytometry B: 85:315, 2013).

WHEN: We will discuss when the various types of validation (Fit-for-Purpose; Technology Transfer; GLP; GMP; CLIA LDT and IVD; Companion Diagnostics) should be applied, and the corresponding regulatory agency. 

WHY: The value of method validation in data interpretation, and in transitioning assays to other laboratories will be reviewed.

HOW: Most of the tutorial will focus on how to complete a method validation from start to finish. We will discuss the specifics: number of samples; number of analytical runs; acceptance criteria; data analysis; and troubleshooting.  The tutorial will also include recommendations for writing a user-friendly validation report.
 
Participants will gain:
  1. An understanding of basic validation terminology
  2. An awareness of regulatory agency oversight
  3. The ability to design a meaningful analytical method validation
  4. Tips on writing a validation report
TUTORIAL 11
Establishing & Growing a Shared Research Facility
Andy Riddell, Univeresity of Cambridge, Cambridge, United Kingdom
James Marvin, University of Utah, Salt Lake City, UT, USA
Mehrnoosh Abshari, National Institute of Health/NIDCR, Bethesda, MD, USA
Barry Moran, Trinity College of Dublin, Dublin, Ireland
Marie Follo, University of Freiburg Medical Center, Freiburg, Germany
Patricia Simms, Loyola University Medical Center, Chigaco, IL, USA


Many established scientific institutes are moving toward a centralized model of shared resource facilities. This has a number of advantages to the institute, such as concentration of experts and access to state of the art instrumentation. Also, it can prove economical to the institute as there is no duplication of expensive and fragile instrumentation or dilution of the skill base. Good quality staff is the limiting resource
 
Creating such a valuable resource requires detailed knowledge of areas not generally associated with scientists running the facility. For example, use of a water-cooled gas laser requires knowledge of the dew point in the lab area where the system is housed. Other non-related skills such as the development of a web site to enable communication with the user base, scheduling, billing, reporting and tracking and calculating instrument power consumption to evaluate heat load in the lab are also needed. Furthermore, tracking usage, evaluating impact and charging may be required to be reported, as well as the “impact” of the lab, and how you evaluate this.
 
In general, when setting up a new resource, a number of considerations have to be evaluated and decisions made about their utility. Considerations include geographical issues: are users housed in a 3 building medical campus or  miles area?. Do you consolidate the equipment in one area or spread it to where it’s getting used the most? Making a decision will have further implications, such as staffing levels, access for the users and may even be adding some level of confusion as to the location of instruments!
 
This course will teach the skill set needed for Establishing and Growing a Shared Resource Facility.

TUTORIAL 12
Ask the Experts: Apoptosis
William Telford, NIH, Bethesda, MD, USA
Zbigniew Darzynkiewicz, New York Medical College, Valhalla, NY, USA
Karen Tamul, EMD Milipore, Washington D.C., USA
Jolene Bradford, Molecular Probes, Eugene, OR, USA

  1. The course faculty will be reviewing methods for the detection of apoptosis, particularly more advanced applications like fluorogenic caspase substrates and FRET based biosensors.  DNA damage detection will also be covered, including both the underlying basic science and the practical assays for detecting this phenomenon by flow cytometry.  The recent area of autophagy detection will also be reviewed, with data demonstrations of both imaging and flow cytometric assays.  Sample data will be analyzed in real-time to illustrate the key issues for these assays. The format will be very informal, with the emphasis on practical aspects of these assays, and troubleshooting to avoid technique pitfalls and data analysis artifacts. 
  1. Upon completion of this tutorial, the attendee should be able to both perform a
    variety of assays for apoptosis and autophagy, and should be able to correctly analyze and critically interpret flow cytometric data.  They should be sensitive to both technique-related and data analysis and interpretation problems with these assays.
TUTORIAL 13
Image Cytometer Performance Characterization and Calibration
Michael Halter, NIST, Gaithersburg, MD, USA
Silas Leavesley, University of South Alabama, Mobile, AL, USA
Stephen Lockett, NIH, Bethesda, MD, USA


Fluorescence microscopes are an important tool for measuring cellular properties. The use of standard instrument performance criteria, such as limit of detection, linear dynamic range, and resolution (point spread function), and reference materials can allow minimal performance criteria to be established for an assay. By applying these minimal performance criteria, data generated by image cytometry instrumentation can assured to be comparable from day to day and from laboratory to laboratory. This tutorial will instruct students on methods available for establishing minimal instrument performance criteria for an image cytometry assay. Students will also be provided with a checklist that can be used to assure that fluorescence microscopy image intensity data is appropriate for quantitative analysis.
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