GLIIFCA 21 September 28th – September 30th, 2012 Buffalo, NY, USA www.gliifca.org 2011 Program Chairs: Site Organizer: David Hedley, President Michael Sramkoski Amanda (Nicole) White Alexander Nakeff Corporate Sponsors & Members: GLIIFCA 21 Program Table of Contents General Information…………………….….2 General Schedule……………………………5 Speaker Schedule – At a Glance…………...6 Industrial Science Symposium..……………7 Symposium I Schedule and Abstracts….... 14 Keynote Address Abstract ……………. 17 Luncheon Roundtable Topics ………….. 18 Symposium II Schedule and Abstracts….. 19 Symposium III Schedule and Abstracts….22 Poster Abstracts…………………………... 25 Steering Committee Info…………………. 36 List of Attendees………………………….. 37 Registered Vendor Representatives……... 39 Blank page for notes……...………………. 41 1 GLIIFCA 21 GENERAL INFORMATION (What You Always Wanted To Know About GLIIFCA 21 But Were Afraid To Ask!) CONFERENCE REGISTRATION DESK: Lobby Conference registration fee includes Friday reception, Sat/Sun breakfast, Sat. Lunch, evening reception, banquet and coffee breaks. Friday, Sept 28 Saturday, Sept 29 Sunday, Sept 30 6:00 to 11:00pm 8am to 11pm 9am to 12:30pm POSTERS: SET UP: Friday, September 28 after 12 noon in 106A&D Νumbers on posters correspond to poster abstract numbers in the program Poster board size= 3 ft wide and 4 ft high Please mount one poster on each side of a poster board using VELCRO only Viewing on Friday to Sunday 10:30am Presentation and Judging: Saturday 5:00 to 7:30pm EXHIBITS: Scheduled exhibitors will have booths in the Exhibit/Poster area (106A&D) Βooths will be open from 5:30pm Friday to the end of the meeting Αll activities other than the plenary sessions, roundtable luncheon, Steering Committee meeting and banquet will be located in the Exhibit/Poster area (106A&D) Please frequent the booths and show our appreciation for the generous financial support provided by the exhibitors who substantially help "pay the freight" for this meeting BREAKFASTS: Free continental breakfast provided for all registrants in the Exhibit/Poster area (106A&D) on: Saturday: 7 to 8am Sunday: 8 to 9am Steering Committee breakfast meeting, Sunday morning (7:30 to 9am) in 106C COFFEE BREAKS: Snacks and drinks available in the Exhibit/Poster area (106A&D) - no need to line up! 2 INDUSTRIAL SCIENCE SYMPOSIUM & FRIDAY RECEPTION: Industrial Science Symposium presentations: 7 to 9:30pm, 106B Reception: 5:30 to 8pm and 10 to 11pm, 106A&D Use 3 drink tickets for wine and beer SYMPOSIA LOCATION: 106B SATURDAY LUNCHEON ROUNDTABLES (12 to 1:30pm): Free lunch/pop; 2/3 ham/turkey and 1/3 veggie in 102 (Tables 1-3), 103 (Tab 4-6), 104 (Tab. 7-10), 105 & Boardroom for non-patricpants in workshops), Pick up box lunch (roast beef, turkey or veggie) and soft drink displayed in each room and move to roundtable of 10 labeled with the title of discussion topic - attendance at each table determined from sign up sheet at GLIIFCA registration desk. Non-participants can lunch in the Boardroom. SATURDAY WINE AND CHEESE HAPPY HOUR: 5:00 to 8pm in the Exhibit/Poster area (106A&D) with cheese and fresh fruit trays (use drink tickets). BANQUET: Free to registrants and paid guests Commences at 8pm, Ballroom. Numerous food stations (salad, entrée and dessert) provided to minimize waiting time Full service bar available for drinks (use drink tickets or pay cash) DJ with dance music until 11pm; requests encouraged (get up and have fun!) DRINKS: Full service bar located in the Exhibit/Poster area (106A&D) for Friday reception and Saturday afternoon for Wine & Cheese reception and Ballroom for banquet Saturday evening Three free drink tickets/registrant for beer and wine only – beer in bottles/cans Mixed drinks – cash bar (your cost). Αll pop in bottles/cans is free FACILITIES/SERVICES: Μessage Board: on easel next to the GLIIFCA Registration Desk, Foyer Xerox copying, faxing, etc: Ask at BNCC main desk 3 CMLE CREDITS: To receive CMLE credit, sign application form at the GLIIFCA registration desk NAME TAGS AND EVALUATION FORMS: Before leaving…. Fill out evaluation form and leave at Registration/Check Out Desk with your name tag ADDITIONAL ENQUIRIES: Contact Dr. Alexander Nakeff (Email: [email protected]; Cell: (313) 820-6227) or leave message for him at the GLIIFCA registration desk 4 GENERAL MEETING SCHEDULE: All symposia to be held in the Room 106B. Poster viewing, Vendor Exhibits, breakfasts and breaks will all be held in the Rooms 106A&D. Friday, September 28th 10a – 4p Resource Managers Workshop Room 106C Sponsored by: GLIIFCA & ISAC Sponsored by: Life Technologies, Stratedigm, Beckman Coulter, & Verity Software House 5p – 8p 5p – 10p Registration Lobby Poster Presentations and Judging 8p – 11p GLIIFCA Annual Banquet & Awards Ceremony Ballroom 6p – 11p Opening Reception Rooms 106A&D Sponsored by: iCyt 7p – 10p Industrial Science Symposium Room 106B Party Theme: “Casino Night” Costume Prize Sponsored by TreeStar Alex Nakeff Young Investigator Award Poster Awards Sponsored by: R&D Systems (2), Life Technologies (1), BD Biosciences (1) Saturday, September 29th 7a – 8a Continental Breakfast 8:15a – 11:00a Symposium I Monitoring Immune Function Travel Stipend Awards Sponsored by: R&D Systems (2), BD Biosceinces (1), Miltenyi Biotec (1), eBioscience (1) 9:30 – 10a Coffee Break Sponsored by: DeNovo Software The Carleton and Sigrid Stewart Keynote Lecture 11a –12p 12-1:30p Luncheon Roundtables Rooms 102-104, 105 & Boardroom for nonparticipants 1:30p – 4:15p Symposium II Emerging Applications of Translational Cytometry 3:00p – 3:30p Coffee Break Sponsored by: Spherotech, Inc. 5:00p – 8p Sunday, September 30th 8a – 9a Continental Breakfast 7:30a – 9a Steering Comm. Meeting Room 106C 9a – 11:45a Symposium III Rare Event Detection by Flow Cytometry 10:30a – 11a Coffee Break Sponsored by: Cytek Development Wine & Cheese Reception 11:45a–12p Closing Remarks BEFORE LEAVING: Please return evaluation form and name tag holder to Registration/Check out Desk. Thank you! 5 Speaker Schedule – At a Glance Resource Managers’ Workshop Friday, September 28th, 10:00a – 4:00p Convener: Sally Quataert 9:45-10:15 Coffee Break 10:15-11:00 Sharon Evans, RPCI, IL-6 – Friend or Foe? Targeting the Immune Microenvironment for Cancer Immunotherapy. Industrial Science Symposium Friday. September 28th, 7:00p – 10:00p Convener: Karen Domenico 7:00-7:20 Mark Munson, Verity Software House. Data Modeling: A Preferred Analysis Method for High-Dimensional Flow Cytometry Data. 7:20-7:40 Susan Reynolds, eBioscience, Inc. Using the Green Laser to Enhance Multi-Colored Flow Cytometry Experiments. 7:40-8:00 Darin Fogg, Amnis Corporation. Optimizing the Amnis ImageStreamX for CTC and other Rare Cell Analysis Applications. 8:00-8:20 Janet Horta, BD Biosciences. BD FACSJazzTM Cell Sorter Overview – A New Era in Cell Sorting. 8:20-8:40 Matthew Hsu, EMD Millipore. Cross Platform Solutions for Autophagy Detection using the Guava 8HT and Amnis ImageStream MarkII. 8:40-9:00 Lisa Nichols, Cytek Development. Back to the future: Updating your cytometer with affordable custom configurations. 9:00-9:20 Matthew Alexander, Sony iCyt. Microfluidics Cell Sorting Chip Platform. 9:20-9:40 Barbara Seredick, Life Technologies. Novel Apoptosis and ROS reagents from Molecular Probes®. 9:40-10:00 Shane Oram,Miltenyi Biotec, Inc. Search Deeper: Utilization of the MACSQuant® Analyzers for analysis of subcellular particles and microvesicles. 11a-12p The Carleton and Sigrid Stewart Keynote Lecture. Luncheon Roundtables 12:00p – 1:30p Conveners: Paul Champoux Symposium II – Emergin Applications of Translational Cytometry Saturday, September 29th, 1:30p – 5:00p Convener: Michael Sramkoski 1:30 – 2:15 Orla Macquire, Buffalo. Image Cytometry Cytometry Based Detection of Aneuploid by FISH-IS. 2:15 – 3:00 Robert Thacker, Cincinnati Children’s. Characterizing the crosspresentation of cell-associated antigens by merocytic dentric cells, a novel DC subset. 3:00-3:30 Coffee Break 3:30 – 4:15 Maria Watson, Universtiy of Toronto. Development of flow cytometry techniques for diagnosing and monitoring the epigenetic status of leukemia patients. 5:00p – 7:30p Poster Presentation and Judging Symposium III – Rare Event Detection Sunday, September 30th, 9:00a – 11:45a Convener: Amanda (Nicole) White 9:00 – 9:45 Alison Allan, London ON, From the bench to the bedside: image and flow cytometry analysis of circulating tumor cells. 9:45 – 10:30 Anna Porwit, Toronto General Hospital. Minimal residual disease in acute myeloid leukemia. Quantitation by Flow Cytometry: Why, How, and When? 10:30 – 11:00 Coffee Break 11:00 – 11:45 Vera Donnenberg, University of Pittsburgh. Cancer Stem Cells: a cell state or a cell type? Symposium I – Monitoring Immune Function Saturday, September 29th, 8:15a – 10:45a Convener: David Hedley (GLIIFCA 2012, President) 8:15-9:00 Hans Minderman, RPCI. Monitoring clinical pharmacodynamics response by image cytometry. 9:00-9:45 Sally Quataert, University of Rochester, Immune Monitoring: Development and Validation of Multichromatic Panels for Clinical Translational Studies. 6 Industrial Science Symposium Friday, September 28th 7:00pm – 10:00pm Room 106B Convener: Karen Domenico, Children’s National Medical Center 7:00-7:20 Data Modeling: A Preferred Analysis Method for High-Dimensional Flow Cytometry Data. Mark Munson, Verity Software House. 7:20-7:40 Using the Green Laser to Enhance Multi-Color Flow Cytometry Experiments. Susan Reynolds, eBioscience, Inc. 7:40-8:00 Optimizing the Amnis ImageStreamX for CTC and other Rare Cell Analysis Applications. Darin Fogg, Amnis Corporation. 8:00-8:20 BD FACSJazzTM Cell Sorter Overview – A New Era in Cell Sorting. Janet Horta, BD Biosciences. 8:20-8:40 Cross Platform Solutions for Autophagy Detection using the Guava 8HT and Amnis ImageStream MarkII. Matthew Hsu, EMD Millipore. 8:40-9:00 Back to the future: Updating your cytometer with affordable custom configurations Lisa Nichols, Cytek Development. 9:00-9:20 Microfluidic Cell Sorting Chip Platform. Matthew Alexander, Sony iCyt. 9:20-9:40 Novel Apoptosis and ROS reagents from Molecular Probes®. Barbara Seredick, Life Technologies. 9:40-10:00 Search Deeper: Utiliazation of the MACSQuant® Analyzers for analysis of subcellular particles and microvesicles. Shane Oram, Miltenyi Biotec, Inc. 7 Abstracts: Data Modeling: A Preferred Analysis Method for High-Dimensional Flow Cytometry Data Mark Munson Verity Software House The concept of data modeling is not new; you've actually been doing it in one dimension for years. What is new is the extension of modeling principles to multi-measurement space. We will begin with a familiar example, and then show you how to enter the realm of ndimensional modeling step-by-step, one measurement at a time. You will see how each additional measurement brings clarity to the analysis, not complexity. You may be surprised by how much the cells have to tell us - if we will only let them speak, and not muffle them with gating. Using the Green Laser to Enhance Multi-Color Flow Cytometry Experiments Susan Reynolds Field Application Scientist, eBioscience, Inc. Green (532 nm) and yellow-green (561 nm) lasers are increasingly common on today’s flow cytometers. In addition to being more powerful, these lasers also allow for optimal excitation of PE and PE tandem dyes and fluorescent reporter proteins. Learn how to successfully incorporate the use of these lasers in your experiments. Topics to be covered include: choices of dyes, optimization of filter sets, fluorochrome performance, and compensation issues. Optimizing the Amnis ImageStreamX for CTC and other Rare Cell Analysis Applications Darin Fogg, Ph.D. Sales Manager, Eastern Region, Amnis Corporation The ImageSreamX has been widely adopted for the analysy of CTCs and other rare cells. Such applications place stringent demands on instrument speed, sample handling efficiency, and real-time data analysis capabilities. This talk will describe a range of optimizations to the ImageStreamX that have greatly increased its utility for rare cell analysis and numerous other applications. 8 BD FACSJazzTM Cell Sorter Overview – A New Era in Cell Sorting Janet Horta Senior Product Manager, BD Biosciences The BD FACSJazz cell sorter incorporates design features that simplify operation of streamin-air cell sorters, to increase the operational efficiency of labs with high workloads, making the most commonly used sorting applications accessible to researchers with limited flow cytometry experience. The key features include factory-optimized settings, intuitive alignment, real-time video monitoring, and BD FACS™ Accudrop technology. Requiring less than 2 x 2 ft (20 x 20 in, 51 x 51 cm) of bench space, the BD FACSJazz is an easy fit in a core facility or an individual lab. The power supply, electronics, and fluid tanks are placed below the lab bench to allow the instrument to occupy a reduced footprint or be installed easily in a biological safety cabinet. The BD FACSJazz can be configured with up to three lasers and eight parameters to support application requirements for individuals and core labs. In a core lab, the BD FACSJazz can offload sorting demand and free high-end sorters by handling routine applications such as cloning. The system has also been designed to meet the needs of individuals for applications such as single-cell analysis, which are driving the accelerated pace of genomics and nextgeneration sequencing. To simplify setup and training, the BD FACSJazz runs with factory-optimized settings. It comes with BD FACS™ Sortware, an innovative software application specifically designed for comprehensive instrument control during acquisition, sorting, and analysis. With dependable BD performance, a benchtop fit and an affordable price, the BD FACSJazz™ signals a new era in cell sorting. Cross Platform Solutions for Autophagy Detection using the Guava 8HT and Amnis ImageStream MarkII Matthew Hsu R&D Director, EMD Millipore Autophagyis an intracellular catabolic pathway which causes cellular protein and organelle turnover, and is associated with diverse diseases such as Alzheimer’s disease, cancer, and Crohn's disease, in addition to aging. It is a tightly regulated process that plays a normal part in cell growth, development, and cellular homeostasis. Autophagy functions as a housekeeping mechanism through disposal of aging and dysfunctional proteins and organelles by sequestering and priming them for lysosomal degradation. Increasing evidence suggests that not only apoptosis, but also autophagy, can contribute to cell death and greatly influence general cell health. Malfunctions of autophagy can adversely impact longevity and the capability of cells to function at full capacity. Here, we describe two key platforms paired with a novel assay design which will allow for the study of autophagy by flow cytometry. Guava 8HT systems have the capabilities of higher throughput sampling utilizing a 96-well format and smaller sample sizes for acquisition. And when combined with the InCyte software module for data analysis and curve fitting algorithms for small molecule analysis, one can now screen autophagy inducers or inhibitors more efficiently. Amnis’s ImageStream system is a perfect complementary platform as it combines the capabilities of microscopy and flow cytometry in a single platform for quantitative image based cellular analysis of autophagy. 9 Back to the future: Updating your cytometer with affordable custom configurations Lisa Nichols Western Regional Director, Cytek Development Discover how our upgrade options can extend the useful life of your existing technology by adding capabilities and updating components. Cytek’s lines of cytometer upgrades are designed specifically for BD FACScanTM, FACSortTM and FACSCaliburTM flow cytometers. Extra Parameter (xP) and Digital Extra Parameter (DxP) customizable configurations and add-on options support a wide range of applications. By leveraging the use of existing proven fluidics, xP and DxP systems are a cost effective alternative to buying a new cytometer. In this workshop, Cytek will present various configuration options and demonstrate experimental data collected using Cytek’s acquisition software, FlowJo Collector’s Edition. Comparison QC and Biological data from multiple cytometer platforms will be presented. See for yourself how a Cytek upgrade compares to buying new in both quality and cost and how an affordable custom configuration allows you to purchase an instrument tailored to your applications. Microfluidic Cell Sorting Chip Platform Matthew Alexander Global Marketing Manager, Sony iCyt This session will describe a novel cell sorter platform which utilizes a microfluidic cell sorting chip. This innovative approach enables a unique cell sorter system that is capable of automated chip loading as well as automated optical alignment and a sorting parameters setup. The microfluidic cell sorting chip is manufactured using an industrial plastic material and a highly precise injection molding duplication. By using optical disc manufacturing process, the chip has low-cost and high analyzing and sorting performance. In this tutorial, we investigate a disposable microfluidic cell sorting chip for a sense in channel sorter. We will also discuss the sy3200 platform. This high end sorter is differentiated with true innovation in optics, electronics, and industrial design. Sony technology enables fast acquisition and sorting speeds while ensuring superior sensitivity and resolution. Additional topics will focus on the sy3200 unique capability to efficiently expand to a dual independent sorter system as well as the system’s innovative approach to ensuring the highest levels of biosafety. Search Deeper: Utilization of the MACSQuant® Analyzers for analysis of subcellular particels and microvesicles Shane Oram & Ashley Weant Flow Cytometry Specialist, Miltenyi Biotec, Inc. The use of flow cytometry as a technology for small particle analysis has increased in a growing number of fields, including microbiology and aquatic ecology. As most flow cytometers are generally designed for analysis of human blood samples and equally sized particles (~2 µm – 30 µm), some unique challenges arise when analyzing particles in the size range of 1 µm down to tens of nanometers. In this presentation, set up of the MACSQuant® Analyzer for analysis of such particles as mitochondria, viruses and microvescicles is discussed including the use of SSC or fluorescence as the trigger parameters. 10 Novel Apoptosis and ROS reagents from Molecular Probes® Barbara Seredick R&D Scientist, Flow Cytometry Systems, Life Technologies Multicolor flow cytometry provides answers to complex cell biology questions from population analysis to rare event detection of stem cells. Higher-plexed multicolor flow cytometry experiments reveal more information at the single cell or population level in less time. Life Technologies™ offers researchers many reagents and assays to accelerate the ability to analyze cell function far beyond immunophenotyping, including viability kits, dead cell stains, and reagents to study apoptosis, oxidative stress, membrane potential, cell cycle, metabolic activity, and phagocytosis. The CellEvent™ Caspase-3/7 Green Detection Reagent and the CellROX™ Reagents for oxidative stress detection are two new product lines that make it easier for researchers to study cell health. The CellEvent Caspase 3/7 Green Detection Reagent is a novel fluoregenic substrate that emits green fluorescence in the presence of activated caspase 3/7, a hallmark of programmed cell death. This reagent doesn’t rely on fixation and permeabilization, making it compatible with live cells and permits easy multiplexing with additional reagents for a more complex analysis of samples. The CellROX™ Deep Red reagent is a bright and stable ROS sensor that fluoresces in response to reactive oxygen species. Also compatible with live cells, CellROX™ offers significant advantage over existing ROS sensors because it can survive formaldehyde fixation and emits fluorescence in the far red region of the spectra. In this presentation we will cover use of these reagents with practical examples and details. Bringing cell sorting to your benchtop: reliable, affordable cell sorters for the core lab or your lab David Coder Bay bioscience In the past, high speed cell sorting has been difficult for non-specialist. That is, difficult to operate reliably, difficult to maintain, and certainly difficult to purchase and keep running. The new Bay bioscience sorters–the JSAN and the Jr/Swift–provide alternatives that are reliable, easy to use, easy to maintain, and are very cost effective. A wide range of lasers is available. The sense-in-quartz flow cell is easily removed for routine cleaning; flow cell replacement does not require extensive optical re-alignment. All components are easily accessible by the user. Systems are completely self-contained with an internal vacuum pump and compressor: Only a single wall socket is needed. Setup for sorting is rapid (less than 15’ from a cold start) and simple. While sorting, the drop delay is monitored and adjusted and the system can detect changes in stream dynamics to maintain optimal sort conditions. Sorting into tubes or microtiter plates is supported and you can switch between the two in 5 minutes. High speed sorting can be done effectively at 30psi, increasing the viability of fragile cell types. The entire systems are compact, and can fit inside smaller, less expensive Baker biosafety cabinets further decreasing the costs of sorting with Class 2a biosafety conditions. Overall, Bay bioscience enables, flexible high speed sorting for the non-specialist: Get more for your instrument budget! 11 Advanced Models of Spectral Unmixing David Novo President, DeNovo Software Proper software compensation is an integral component of successful polychromatic flow cytometry. It is also a continual source of confusion among novice (and experienced) users and requires considerable effort to perform and optimize. Even when applied properly compensation often results in highly non-intuitive results, such as negative fluorescence and excessive spreading of dim populations, that are very difficult to understand and interpret in light of the underlying biology. Much of these artifacts are a result of applying simplistic mathematical models to convert the observed fluorescence signal into a value proportional to dye abundance. This talk will discuss more advanced models of spectral unmixing and how they compares to standard compensation and apply to flow cytometry. Proper application of these techniques minimizes, and often eliminates, compensation artifacts and in addition can provide several new metrics for assessing of the quality of the unmixing. Multiple Flow Cytometric Approaches for Studying DNA Damage Pathways and Assessing the Level of the Cellular Responses to DNA Mark Santos, Wenying Zhang, Angelica Olcott, Jason Whalley, Patrick Schneider, and Matthew Hsu EMD Millipore Investigation of DNA damage pathways and assessment of the DNA damage levels in cells are extremely important for the study of cancer and developing anti-cancer drugs, as defects in DNA damage pathways can cause cancer and lead to genetic instability and ultimately uncontrolled cell growth. The activation of the ATM dependent DNA damage pathway by various agents is carefully monitored and confirmed using a group of directly conjugated and optimized phospho-specific antibodies against ATM, SMC1 and Histone H2A.X targets; then the level of DNA damage is quantitatively measured using a pair of antibodies against the total and phosphorylated H2A.X, in order to detect target-specific of phosphorylation especially in a mixed cell population. These and other related assays were successfully used in the evaluation of DNA damaging and anti-neoplastic agents, as well as their impact on cell cycle, which helped elucidate the mechanisms of cell proliferation, apoptosis, and DNA repair, ultimately advancing the study for cancer research. 12 Detection of Mouse CD4+ Th Cell Differentiation by Flow Cytometry and Multiplex Mark Santos, Yuko Williams, Don Weldon, Wen-Rong Lie, Angelica Olcott, Jason Whalley, Patrick Schneider, and Matthew Hsu EMD Millipore Naïve CD4+ T cells can give rise to a variety of subsets of Th cells depending on the nature of the immune response, and subsequently release a distinct subset of cytokines. Conventionally these cytokines can be measured in a secreted format (e.g. Luminex or ELISAs), but here we describe a novel method to reliably differentiate mouse CD4+ T cells, and further characterize the differentiated lineages by measuring intracellular cytokines using flow cytometry. In addition, we also describe the use of a proprietary fixable viability dye in order to eliminate the false positives associated with non-specific staining. Based on an optimized protocol for differentiating CD4+ T cells, we have developed differentiation tools designed to obtain the desired Th cell lineages. Following naïve T cell differentiation toward specific Th lineages, we further developed a flow cytometry assay to measure intracellular cytokine production by blocking the secretion of the cytokine with Brefeldin A. We further eliminate nonspecific staining due to dead or dying cells using a proprietary fixable viability dye and accurately determine the percentage of Th lineages using guava bench top flow cytometer and InCyte analysis software. 13 Symposium I Saturday, September 29th 8:15am-11:00am Monitoring Immune Function Room 106B Convener: David Hedley (GLIIFCA 2012, President) 8:15 – 9:00 Clinical Applications of Imagestream Cytometry Hans Minderman, Roswell Park Cancer Institure 9:00 – 9:45 Immune Monitoring: Development and Validation of Multichromatic Panels for Clinical Translational Studies. Sally A. Quataert, University of Rochester 9:45 – 10:15 Coffee Break (Sponsored by DeNovo Software) – Posters and Exhibits 10:15 –11:00 IL-6 - Friend or Foe? Targeting the Immune Microenvironment for Cancer Immunotherapy Sharon S. Evans, Roswell Park Cancer Institute Abstracts: Clinical Applications of Imagestream Cytometry Hans Minderman Roswell Park Cancer Institute, Buffalo, NY The ImageStream is a flow cytometry-based image analysis platform that acquires up to 12 spatially correlated, spectrally-separated images of cells in suspension at rates of up to 1000 cells/sec. By combining the high throughput and multiparameter capability of flow cytometry with the high image content information of microscopy it enables quantitative image analysis in immunophenotypically defined cell populations in statistically robust cell numbers. One area of its clinical application is in the study of cellular signal transduction pathways for which the intracellular localization of signaling intermediaries correlate with activity. Examples of this will be presented with regards to the cytoplasmic to nuclear translocation of NFAT1 and p65 upon activation of the NFAT and NFkB transcription factor pathways, respectively. Examples will be presented how these assays can provide determinants of response to immunosuppressive and anti-leukemic therapies. Correspondence: [email protected] Immune Monitoring: Development and Validation of Multichromatic Panels for Clinical Translational Studies. 14 Sally A. Quataert, Ph.D., RQAP-GLP Rochester Human Immunology Center Core Laboratory, University of Rochester, Rochester, NY Multichromatic flow cytometry technology provides the opportunity for increasing depth of analysis and discovery in clinical translational research. However, development of validated panels capable of giving scientifically sound results for a study across different individuals, time points, reagent lots and instruments is challenging. Often clinical studies require following the immune status of subjects over a period of years rather than weeks as in nonclinical animal studies. The cost can also be prohibitive in terms of both reagents and instrument time. The Rochester Human Immunology Center has worked with investigators in multiple studies to develop and validate immune monitoring panels that can be readily adapted to other projects to lower the cost of initial set up. The panels include T, B, natural killer (NK), dendritic cell (DC), T regulatory phenotyping and intracellular cytokine staining using 12 to 16-colors in a micro-method minimizing cost while maintaining precision and optimal staining. Activation markers are accessed in the development phase in order to insure that direct ex-vivo as well as in vitro stimulation is detectable in rare populations. Strategies for validation and quality control of results over the study include standardization of sample handling, use of fluorescent beads, instrument calibration, cryopreserved assay controls, use of FMO and isotype controls, and careful titration and qualification of new lots of reagents. When control methods are designed into the flow cytometry panels employed in a clinical study, it is possible to achieve consistent and comparable results over the entire course of the study. Examples from a pediatric study for stability of populations during processing of cord blood specimens, a lymphoma study and an infectious disease study will be used to illustrate the development process. Correspondence: [email protected] IL-6 - Friend or Foe? Targeting the Immune Microenvironment for Cancer Immunotherapy Sharon S. Evans, Ph.D. Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY Efforts to harness the exquisite specificity of cytolytic CD8 T lymphocytes to destroy tumors have had minimal clinical success, in part because the mechanisms impeding cancer immunity are incompletely understood. We have recently identified restricted homeostatic trafficking of CD8 T cells across tumor-vascular gateways as a rate-limiting step during the effector phase of tumor immunity. In contrast to the protumorigenic activity commonly ascribed to IL-6, our findings revealed a novel IL-6 regulatory program triggered by systemic thermal therapy that drives a switch to an adhesive vasculature that supports trafficking of CD8 effector T cells, thereby improving immune-mediated cytolysis of tumor targets. A concomitant decrease in infiltration of immunosuppressive CD4+ CD25+ FoxP3+ regulatory T cells (TReg) was further detected following acute systemic thermal therapy that was 15 dependent on IL-6 bioactivity. As a result, we detected a substantial increase in the intratumoral CD8 TEM:TReg ratio which is considered a strong prognostic indicator of overall survival in cancer patients. This work suggests that IL-6 derived from non-hematopoietic stromal cells within the tumor microenvironment can be exploited to create a therapeutic window that boosts T cell-mediated antitumor immunity. Supported by grants from the National Institutes of Health (CA79765, CA094045, and AI082039) and the Joanna M. Nicolay Melanoma Foundation. Correspondence: [email protected] 16 The Carleton and Sigrid Stewart Keynote Lecture Saturday, September 29, 2012 11:00 am – 12:00 pm Room 106B Flow Cytometry: Let’s Keep this Party Goin’ Jonni Moore, Ph.D. Professor, Pathology and Laboratory Medicine Director, Clinical and Research Flow Cytometry Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA Technologies for single cell analysis have become central to emerging life sciences markets. Genomics, proteomics and metabolomics are all making daily headlines, but cytomics, or high dimensional single-cell analyses utilizing systems biology approaches, is rapidly becoming the technology of choice for the elucidation of relevant phenotypic patterns that can be directly utilized in the clinical arena. Thus cytometric profiling is poised to play a major role in research, discovery, diagnosis, prognosis and even therapy. New areas of application are appearing such as cardiology that will extend the power of flow cytometry to new, previously untapped areas. While the tools, technology and applications are rapidly advancing, a significant gap is the lack of experts in the technology. Science careers are not enticing the new generation so it is up to today’s scientists to encourage the next generation so that we can “Keep the Party Goin”! Correspondence: [email protected] 17 Luncheon Roundtables Rooms 102-105 Saturday, September 29th 12:00-1:30PM Convener: Paul Champoux, University of Minnesota Free box lunch available at each table – drinks available at side tables. The primary aim of these discussions is to provide a forum for participants to address their interests in a variety of cytometry and image topics. R1—  Future  of  Flow  Cytometry: Moderator: Phil Hexley, Cincinnati Shriner’s Hospital R2— 10-Parameter Social Media Marketing for Core Management: Moderator: Ryan Duggan, University of Chicago R3— Do-it-Yourself Toolkit for Flow Cytometry Repair and Troubleshooting: Moderator: Lisa Nichols, Cytek Development Inc. R4— Analysis of Microparticles by Flow Cytoemtry: Moderator: Nancy Fisher, University North Carolina, Chapel Hill R5— Personal Cytometers: Bane or Boon to Cores: Moderator: Charles Kucznski, University of Nebraska R6— Cell Sorting: Moderator: Justin Meyers, Purdue University R7— Fluorescent Protein Chemistry: Some New and Some Old but Always Exciting: Moderator: David LeClerc, University of Chicago R8— Choosing and Optimizing Funding Opportunities: Moderator: Paul Champoux, University of Minnesota R9— Imaging flow cytometry: The Dark Field Rises: Moderator: Hans Minderman, Roswell Park Cancer Institute R10— Data Analysis: Moderator: John Quinn, TreeStar, Inc, Room 102: Tables 1-3 for R1-3 Room 103: Tables 4-6 for R4-6 Room 104: Tables 7-10 for R7-10 Room 105 and Boardroom: for non-participants 18 Symposium II Saturday, September 29th 1:30 pm – 4:15 pm Emerging Applications of Translational Cytometry Room 106B Convener: Michael Sramkoski All Speakers in this Session are Nakeff Young Investigator Candidates 1:30 – 2:15 Image Cytoemtry-Based Detection of Aneuploidy by FISH-IS. Orla Maguire, Roswell Park Cancer Institute. 2:15 – 3:00 Characterizing the cross-presentation of cell-associated antigens by merocytic dentritic cells, a novel DC subset. Robert I. Thacker, Cincinnati Children’s Hospital. 3:00 – 3:30 Coffee Break (Sponsored by Spherotech, Inc.) – Posters and Exhibits 3:45 – 4:15 Development of flow cytometry techniques for diagnosing and monitoring the epigenetic status of leukemia patients. Maria Watson, Prince Margaret Hospital. Abstracts: Image Cytometry-Based Detection of Aneuploidy by FISH-IS Orla Maguire, Ph.D. Roswell Park Cancer Institute, Buffalo, NY Fluorescent In Situ Hybridization, (FISH), is a slide-based molecular cytogenetic technique designed to detect and locate specific nucleic acid sequences. FISH has many applications, and is one of the conventional methods by which cytogenetic abnormalities are detected in hematologic malignancies. Sensitivity of this microscopy-based method is limited by the relatively low numbers of cell events analyzed (usually 200-1000 cells) and the subjective nature of operator based analysis. We have developed a flow cytometry-based imaging approach to detect chromosomal abnormalities following FISH in suspension (FISH-IS) which enables the automated analysis of several log-magnitude higher number of cells compared to the microscopy-based approach. The present study demonstrates the applicability of FISH-IS for detecting numerical chromosome aberrations, establishes accuracy and sensitivity of detection compared to conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R2=0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples 19 procured from 10 AML patients with trisomy 8. Additionally, FISH-IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual trisomy 8 cells indicating that this approach may be used to detect minimal residual disease and associated chromosomal defects. The current FISH-IS protocol can be modified for use in other FISH applications, including diagnosis of syndromes associated with aneuploidy, and detection of bacteria/pathogens in infection, and for use in other FISH techniques, such as RNA-FISH. Correspondence: [email protected] Characterizing the cross-presentation of cell-associated antigens by merocytic dendritic cells, a novel DC subset Robert I. Thacker, Rachel A. Reboulet, Cassandra M. Hennies, Edith M. Janssen Division of Cellular and Molecular Immunology Cincinnati Children’s Hospital Research Foundation University of Cincinnati College of Medicine, Cincinnati, OH Cross-presentation of cell-associated antigens (Ag) plays an important role in the induction of anti-tumor responses, autoimmune diseases, and transplant rejection. Recently, the novel DC subset designated merocytic DCs (mcDCs), has been shown to potently prime both CD8+ and CD4+ T cells to cell-associated Ag. This priming leads to increased primary expansion, enhanced effector function, and increased memory formation. The superior priming of the mcDC is critically dependent on type I IFN production upon uptake of apoptotic material. Because this breach of tolerance facilitated by mcDCs may prove to be essential for successful cancer vaccines and interference with autoimmune disease development, studies to discover distinguishing features among these novel DCs have become essential. As the uptake, processing and presentation of cell-associated Ag is highly dynamic we have utilized multiple tools in our research including ImageStream, flow cytometry, confocal microscopy and electron microscopy. This multifaceted approach has facilitated the mechanical distinction of mcDC from other subsets. Comparatively, mcDC take up smaller particles from apoptotic cells utilizing a Pi3Kinase independent pathway. These small phagosomes showed a reduced lysosomal acidification rate. In addition, phagocytosed particles were stored in non-acidic compartments resulting in sustained Ag presentation. The mcDC are further characterized by type I IFN production, most likely induced by the slow release of nucleotide structures from early phagosomes wherein apoptotic material is sequestered. Taken together, these data allow us to correlate functional differences in T-cell activation to mechanical differences in antigen processing among the DC subsets. Correspondence: [email protected] 20 Development of flow cytometry techniques for diagnosing and monitoring the epigenetic status of leukemia patients. Maria Watson Prince Margaret Hospital, University of Toronto Epigenetics is defined as heritable and acquired changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. Epigenetic regulation of genes involved in cell growth, survival, or differentiation through histone modifications have been consistently shown to be an important determinant of cancer development. There are many post-translational histone modifications but the best investigated are methylation and acetylation of lysine residues on the histone tails. These modifications have been shown to influence the structure of chromatin and to affect gene regulation. For example, H3K9Ac and H3K79me2/3 are associated with transcriptionally active euchromatin whereas H3K9me2/3 and H3K27me3 are associated with transcriptionally inactive heterochromatin. In the current project we are building a multiparametric flow cytometry method for studying epigenetic regulation via histone modifications. These techniques have been applied to leukemia patient samples with the goal of developing a robust platform able to link epigenetic changes to biology and clinical outcome. Once fully optimised we will be able to use this assay to monitor the effects of drug-like compounds, which target histone-modifying enzymes, in leukemia cancer patients. We have so far generated novel preliminary data that establishes the feasibility of building complex single cell assays to monitor histonemodifying enzymes and their specific lysine targets. There have been several studies into global histone modifications in myeloid malignancies. Some studies report that depletion of EZH2 along with inhibition of HDACs leads to apoptosis in AML cells (Fiskus et al, 2009). Others have found inactivating mutations of EZH2 in various myeloid disorders and the concomitant decrease in H3K27me3, suggesting EZH2 acts as a tumor suppressor (Ernst et al, 2010). In addition to this, increased DOT1L methyltransferase activity (a specific methyltransferase of the H3K79 mark) has been linked to MLL-rearranged leukemia (Bernt et al 2011). There have also been some studies reporting the occurrence of inhibited demethylases in various myeloid malignancies (Klose et al, 2006, Xu et al, 2011). These studies highlight the importance of monitoring the epigenetic status in myeloid disorders and the need for an assay to screen for changes. Although our clinical focus will be on acute leukemia, where we have access to large numbers of patient samples, the flow cytometry methods being developed in this project will be broadly applicable to other types of cancer. References Bernt et al, Cancer Cell. 2011 Vol 20(1), 66–78. Ernst et al, Nature genetics. 2010, Vol 42, 722-727. Fiskus et al, Blood. 2009, Vol 114(13), 2733–2743. Klose et al, Nature review, genetics. 2006, Vol 7, 715-727. Xu et al, Cancer cell. 2011, Vol 19, 17-30. Correspondence: [email protected] 21 Symposium III Sunday, September 30th 9:00 am – 11:45am Rare Event Detection Room 106B Convener: Amanda (Nicole) White 9:00 - 9:45 From the bench to the bedside: image and flow cytometry analysis of circulating tumor cells. Alison L. Allan, London Regional Cnacer Program & Western University 9:45 – 10:30 Minimal Residual Disease in Acute Myeloid Leukemia. Quatitation by Flow Cytometry: Why, How, and When? Anna Porwit, University of Toronto, Universtiy Health Network, Toronto, ON 10:30 – 11:00 Coffee Break (Sponsored by Cytek Development) 11:00 – 11:45 Cancer Stem Cells: a cell state or a cell type? Vera Donnenberg, University of Pittsburgh Abstracts: From the bench to the bedside: Image and flow cytometry analysis of circulating tumor cells Alison L. Allan, Ph.D. London Regional Cancer Program and Western University, London, ON Given the multi-step nature of cancer development, there should be several opportunities for therapeutic targeting of tumor cells and/or the tumor microenvironment. The ideal way to identify and monitor disease progression is through surrogate marker approaches that are minimally invasive and allow for longitudinal testing, such as blood tests. Our current research focuses on the development of such approaches, in particular rare event detection of circulating tumor cells (CTCs) by image and flow cytometric methods. Identifying rare populations requires a different approach than standard cytometry techniques, which rely mainly on positive and negative decisions made in either one, or at most, two dimensional space. This presentation will discuss the issues that must be addressed when designing an assay to accurately detect rare populations of CTCs in the blood of patients with solid tumors and in experimental mouse models of cancer. Cytometry-based methods provide the opportunity to elucidate the mechanistic details of early steps in cancer progression and determine how these steps relate to the development, monitoring, and treatment of metastatic disease. Correspondence: [email protected] 22 Minimal Residual Disease in Acute Myeloid Leukemia. Quantitation by Flow Cytometry: Why, How, and When? Anna Porwit, Ph.D. Department of Laboratory Medicine and Pathobiology University of Toronto, University Health Network Toronto General Hospital, Ontario, Canada WHY: Minimal residual disease (MRD) monitoring in acute myeloid leukemia (AML) allows detection of subclinical levels of disease when bone marrow (BM) is morphologically normal. Both PCR based and multiparameter flow cytometry (FCM) based MRD studies showed that patients with MRD levels >0.1% of BM cells post induction therapy have higher risk of relapse. Therefore, MRD levels can be considered as the major independent post-treatment prognostic factor. First preliminary studies have been published proposing individual pre-transplant assessment, which combined MRD levels with the well-established pre-treatment prognostic factors such as cytogenetic/molecular characteristics. HOW: Sensitivity of MRD detection varies based on the test involved but both PCR and FCM reach sensitivity 0.01%. For molecular methods multiple probes are required since no single genetic abnormality is present in more than 50% of patients. FCM detection of MRD is based on abnormal immunophenotypes displayed by leukemia cells in comparison to normal BM cells. Using modern FCM, aberrant phenotypes (also called Leukemia Associated Immunophenotypes, LAIP) can be detected in >90% of patients with AML. For correct interpretation of follow-up samples and detection of MRD, the immunophenotypic pattern of diagnostic sample and thorough knowledge of immunophenotype of various cell populations in normal and regenerating BM is necessary. Most reported studies using 3-5 color FCM included construction of patient specific panels, dependent on immunophenotype at diagnosis. New 8-10 color approaches (sc. polychromatic FCM) rely on common comprehensive panels applied at both diagnosis and follow-up, allowing detection of aberrant cells in most patients using sequential gating strategy. WHEN: Early time points (day 15 and 30 post-induction) identify as soon as possible high-risk patients in need of for very intensive treatment. Testing may also help to differentiate leukemic blasts from regenerating BM in patients with borderline blast numbers in the smears (4-6%). However, there may be patients with slower blast clearance, which carries some risk for overtreatment. Later time points (post-consolidation) identify patients with persistent low-level disease, who may be candidates for SCT. However, the prognostic significance of pre-transplantation MRD levels is still unclear. CONCLUSIONS: Use of polychromatic MRD assay may increase sensitivity, improve qualitative information and allow standardization of MRD measurements. Measurement of MRD levels might refine the prognostic relevance of defined risk-groups based on pre-treatment characteristics. Correspondence: [email protected] 23 Cancer Stem Cells: a cell state or a cell type? Vera S. Donnenberg, Albert D. Donnenberg, James B. Hicks, Michael Wigler University of Pittsburgh, Pittsburgh, PA Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity recapitulates the normal multilineage differentiation processes remains unknown. The classical differentiation scheme, as described in the hematopoietic system, is a unidirectional differentiation tree with self-replicating stem cells giving rise to progenitor cells which in turn generate the differentiated progeny. However not all tissues follow this unidirectional differentiation paradigm. Experiments from nuclear-somatic cell transfer, from the creation of induced pluripotent stem cells as well as study of regenerating liver underscore that stemness may be viewed as a cell state and not a cell type. A change of state resulting in dedifferentiation of more prevalent “mature” tumor cells into a stem-like tumor phenotype is compatible with the cancer stem cell paradigm and can only be definitively distinguished from clonal selection at the single-cell level. Viewing stemness as a state that can be conditionally reexpressed when differentiation signaling pathways are blocked by environment, mutation or epigenetic reprogramming may help us appreciate the important analogy between tumorigenicity and normal tissue renewal, without locking us into a one-way differentiation paradigm that views cancer stem cells as a unique cell type. As the most developed single cell technology, cytometry and particularly multidimensional cell sorting, provides critical tools for molecular and functional analysis of cancer cell states. Therefore in this presentation I will review the current state of the art in the identification and isolation of non-malignant and malignant stem and progenitor cells. I will address several questions such as: What markers have been used to detect stem cells in normal human bone marrow, epithelial and adipose tissues? Do markers define populations with biologically unique properties? Are the same phenotypes found in cancer tissue? Several markers have been proposed to identify stem/progenitor cells in normal lung and other epithelial tissues. I will describe a systematic multiparameter investigation of the expression of stem/progenitor markers on primary clinical tumor isolates in cancer and normal non-malignant tissue. Further I will demonstrate how the use of immunofluorescence on paraffin sections and multiparameter flow cytometry determine the histologic location of tumor stem cells. Lastly, I will provide a guidance concerning avoidance of pitfalls associated with flow cytometry on disaggregated tissues. Correspondence: [email protected] 24 POSTER ABSTRACTS Abstract number corresponds to poster board number. All posters displayed in Rooms 106A&D 1. Quantitative assessment of instrument sensitivity and performance. Ryan Duggan UCFlow, The University of Chicago, Chicago, IL When consumers are looking to purchase a new flow cytometer and are gathering the specification sheets for instruments from different manufacturers, they typically see a few common specs; Number and type of lasers, number and type of detectors, max event rate, and some form of sensitivity measurement - undoubtedly represented by the Detection Threshold (DT) for FITC and PE. The DT is the number of Molecules of Equivalent Soluble Fluorophore (MESF) their instrument can detect above electronic noise. Today’s best instruments typically report somewhere around 100 MESF for FITC and <100 MESF for PE. With all instruments meeting this ungoverned specification, how is a consumer supposed to differentiate these instruments? Does having a DT of 50 for FITC make an instrument better across the board? What about the other 10, or 15 channels on this instrument? Will they follow suit and perform as well as the FITC and PE channel? Considering that unstained lymphocytes have MESF values in the FITC channel of approximately 500 units or more, is the DT even useful as a predictor for instrument sensitivity and resolution? What is proposed here is a method for instrument sensitivity that can be used by consumers to yield a composite picture of an instrument’s ability to detect low levels of antigen expression above autofluorescence background in a quantitative fashion. NORM (Normalized Overlap Ratio Metric), can be calculated for any fluorochrome that can be coupled to an antibody, and as such, can allow for the direct comparison of all channels of an instrument, the same channel between instruments, or the same channel within an instrument whilst changing filters or laser power, or laser wavelength. The unit of NORM is the minimum number of antibodies bound to a cell that can be detected above lymphocyte autofluorescence. 2. Fluorochrome Choice Influences Escapee Formation Andrew Bantly, Richard Schretzenmair, Lifeng Zhang, Amy Steinmetz, Jonni Moore Flow Cytometry & Cell Sorting Resource Laboratory Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA Introduction: The apparent loss of mononuclear populations during staining with fluorescent antibodies, the so-called lymphocyte escapee phenomenon, was noted as early as 1994. The effect was most evident with FITC in ammonium chloride lysed whole blood. Although there has been a large expansion of fluorescent probes, it has not been determined if the escapee phenomenon occurs with the new fluorochromes and if it is restricted to certain subsets. This information is critical to panel design and to accurate measures of individual leukocyte subsets. Materials and Methods: Pre-lysed whole blood was stained with CD3 antibodies conjugated to more than 20 different fluorochromes and analyzed on a BD FACSCanto with 11-color upgrade or a BC Gallios. Events were analyzed using a pan-Leukocyte gate, excluding only debris and CD3 vs. SSC plots were generated. Escapees were classified as 25 events with CD3 intensity similar to T cells (CD3+ low SSC) which had SSC properties more appropriate to that of Granulocytes or Monocytes. We also evaluated the use of several commercial blocking reagents, plasmas and immunoglobulins. Results: Escapee formation is evident with multiple probes and is dependent upon the fluorochrome tag and not on the antibody clone. Large difference between donors was noted, some having virtually no escapees while others with substantial numbers. The use of several commercial blocking reagents or the addition of murine immunoglobulins had no effect. The addition of autologous plasma, human AB serum or rabbit sera substantially reduced escapee formation. 3. Different Sorts for Different Folks: The Importance of Technological Diversity in a Cell Sorting Facility. Monica L. DeLay1, A. Nicole White1, Edith Janssen2, George Babcock3, Christopher A. Worth4, Sherry Thornton1. 1 Research Flow Cytometry Core, Division of Rheumatology and 2Division of Molecular Immunology, Cincinnati Children’s Hospital, 3333 Burnet Ave., Cincinnati, Ohio 45229, 3 Flow Cytometry Lab, Shriner’s Hospitals for Children-Cincinnati, 3229 Burnet Ave, Cincinnati, Ohio 45229, 4Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street Louisville, KY 40202 Flow cytometry core facilities have the challenge of offering appropriate technologies to diverse client needs. Therefore, there is no one-size-fits-all technology that can handle any and all cell types or applications. For cell sorting, this becomes even more of a challenge when a variety of cells need to be purified for an even wider variety of downstream applications. There are two types of technologies currently available for droplet based cell sorting; cuvette and jet-in-air. While there are advantages and disadvantages to both technologies, no cell sorter design can fit all needs, especially when the cell type to be sorted is sensitive to manipulations. There have been reports (published and anecdotal) of phenotypic and functional changes to dendritic cells after isolation using different techniques. In our core facility, DC populations that were sorted on the FACSAria II showed an increase in cell death and were found to be nonfunctional in an assay testing their ability to process cell associated antigen and stimulate proliferation of T cells. In contrast, cells sorted on a FACSVantage, MoFlo Legacy or MoFlo XDP were able to function in the same in vitro assay. We attribute this difference to the differences in fluid dynamics through the sample path that may damage the cells and decrease their ability to function. Here we present data comparing the functionality of DCs with each system. We conclude that it is best to have a diversity of technologies in a cell sorting facility to better meet the needs of all clients. 26 4. TRAVEL AWARD WINNER - Cell cycle reprogramming blocks apoptosis in Drosophila follicle cells Christiane A. Hassel, Brian R. Calvi Department of Biology, Indiana University, Bloomington, IN Correct programming of and progression through the canonical cell cycle is necessary to sustain eukaryotic life. A variation of this cell cycle, the endocycle, is conserved across species and results in duplication of genomic DNA content without cell division. I have used the invertebrate Drosophila to explore cell cycle remodeling that results in a mitotic to endocycle switch (M/E switch) and abrogation of the DNA damage response. When subjected to irradiation and genotoxic stress, endocycling cells do not apoptose (Mehrotra et al., 2008). Here, I examine whether experimental induction of an M/E switch blocks apoptosis. To induce reprogramming of the mitotic cell cycle, I blocked mitosis by knocking down or overexpressing mitotic regulators. Specifically, I knocked down cyclin A (cycA) or overexpressed fizzy-related (fzr/cdh1). cycA RNAi or fzr/cdh1 overexpression resulted in an M/E switch in ovary follicle cells. When subjected to gamma-irradiation, apoptosis was repressed in these cells. This shows that an endocycle can be induced by alteration of a single mitotic regulator and this is sufficient to repress apoptosis. These data suggest that cell cycle reprogramming in some cells is sufficient to repress apoptosis. Some tumors may have chemotherapy-resistant endocycling cells. Further investigating how cell cycle reprogramming is linked to apoptosis-resistance may lead to the discovery of new cancer treatments. 5. Modification of a Canto A for Simultaneous 10 Fluorescent Color Detection. Edward Podniesinski, Alexis Conway, Terry Donahue, Aileen Cinquino, Paul Wallace Laboratory of Flow & Image Cytometry, Roswell Park Cancer Institute, Buffalo N.Y. 14263 The number of fluorochromes conjugated to monoclonal antibodies commercially available for routine flow cytometry continues to grow. With the addition of PE Texas Red/PE-CF594 and Alexa 700 flours routine 10 color flow cytometry is now a reality. Our original 6 color Canto-A’s instrument architecture (4/2) was first upgraded to 3 lasers within the recent past through a purchase of a SORP (Special Order Research Option). This produced a fluorescence emission detection count on par with the CANTO-II (4/2/2). We found the opportunity existed to easily add two additional spectral detection channels to the existing 3 laser interrogation/ intercepts. To increase the current 3 laser CANTO-A detection capability to 10 color (5/3/2), required 2 additional PMTs (Photo Multiplier Tubes), 2 integrated PMT/ HV control sockets, 2 remote Pre-Amplifier circuit boards, Red/PE-CF594 filter/ mirror set, Alexa 700 filter/ mirror set and some additional cabling. The CANTO-A software configuration needed change necessary to map or associate the 2 additional detectors for acquisition. The performance characteristics of the Canto A was compared to itself before and after upgrading. We will show our findings as a result of the upgrade from 8 to 10 color. 27 6. Performance Evaluation of A Flow Cytometer’s Optical Filters From New to 8 years Later Edward Podniesinski, Paul Wallace Roswell Park Cancer Institute, Buffalo, NY The detection performance of aged optical filters can get overlooked from Flow Cytometers of long service. An 8 year old flow cytometer’s detection optics were spectrally swept by a slightly modified spectrophotometer and compared to when the filters were new and to new identical replacements. Data from the spectrometer and multi-peak broad spectrum calibration particles, used to critique detector channel performance, will also be presented. 7. Assessing Containment and De-Contamination of Cell Sorters Matthew Cochran University of Rochester, Rochester, NY Contamination of aseptic sorts can cause frustration on the part of the investigator, not to mention the lost time and effort caused by this problem. Many sorting facilities take special care to prepare and maintain their instruments in a �clean’ state using different decontamination procedures to minimize the chance that the system is the source of contamination. In multi-user facilities where samples range from mammalian primary cells and cell lines to yeast and bacteria, cleaning protocols between sorts are especially important. Coupled to this is the fact that many testing protocols require 24-48 hours after a sort to determine if the system was clean, which may be too late for the investigators who are possibly impacted by the contamination. Our current protocols use a permissive plate to detect aerobic bacteria at concentrations as low as 25/ml, but take 48 hours to complete. Recently BD Biosciences released a flow-based assay for detecting bacterial contamination, the FACSMicro count. The system is composed of a flow cytometer and bacterial testing reagents, offering results in as little as 5 minutes, with sensitivities between 30-50 bacteria/ml. In this report, we discuss the optimization of these reagents for use with standard flow cytometers and compare it to current plate based testing protocols used in our facility. 8. Nuclear translocation of NFAT and NF-κB in T-cells as an early quantitative response parameter of immune-activity pre- and post-solid organ transplantation. Orla Maguire, Kieran O’Loughlin, Oleh Pankewycz, Mark R. Laftavi, Hans Minderman Roswell Park Cancer Institute, Buffalo NY With the development of more effective immunosuppressive therapies as well as better combination strategies for their use, the rates of acute rejection after kidney transplantation have plummeted to single digit levels and one year graft survival rates of 90%. Paradoxically, this early success has not led to improved long-term allograft survival. Durable graft survival over the course of years depends on finding the right balance between overimmunosuppression leading to drug toxicity or infectious complications and underimmunosuppression resulting in the formation of donor specific antibodies and chronic rejection both of which cause premature graft failure. Effective post-transplant patient management lacks an adequate method for determining the optimal level of immunosuppression required for a given individual to prevent toxicity or rejection. We aim 28 to develop an effective early-response assay which can gauge the overall immune status in the post-transplant recipient, thus, allowing for personalized therapy. The most commonly used immunosuppressants function by inhibiting the activation of the transcription factors NFAT and NF-κB, thereby reducing the transcription of immune response genes. The function of these transcription factors depend on their translocation from the cytoplasm to the nucleus which is quantifiable by imaging cytometry. Preliminary studies in our lab show that we can measure transcription factor activation in T-cells using HLA (Human Leukocyte Antigen) coated beads which corresponds to a proliferative response. A peak in signaling activity is demonstrated at 16hrs post-stimulation. Proliferation is measurable 7 days following stimulation. This time frame is longer than e.g., CD3/CD28 stimulation, where signaling occurs within a few hours, and proliferation can be measured in four days. We believe this difference is due to the increased specificity of the anti-HLA response. Using the immunosuppressant tacrolimus, we can inhibit CD3/CD28-induced NFAT and NF-κB nuclear localization in healthy donor PBMCs ex vivo at concentrations equivalent to those achieved in post-transplant patients. These early results indicate that this assay could be useful in measuring potential reactivity towards the donor graft before/immediately following transplant and monitoring immunosuppressant activity in patients in the months following transplant. 9. Sorting Renin Positive Cells from Mouse Bone Marrow Craig A Jones, Joseph Tario, Paul Wallace and Kenneth W. Gross Roswell Park Cancer Institute, Buffalo, NY The existence of a functional Renin-Angiotensin system in mammalian bone marrow has been postulated for many years. In fact, expression of many of the genes comprising the RAS has been documented. However, evidence of renin expression, which catalyzes the first step of this enzymatic cascade, in normal bone marrow has been sketchy. We use PCR and RTPCR to confirm expression of the renin gene in mouse bone marrow cells. Furthermore, Affymetrix microarrays were used to characterize renin-expressing cells sorted from the bone marrow of transgenic mice carrying a renin-GFP reporter gene. In addition, immunephenotyping was used to show these cells were multipotent and had characteristics of early B-cell lineage. 10. Statistical classification of multivariate flow cytometry data analyzed by manual gating: Stem, progenitor and epithelial marker expression in non-small cell lung cancer and normal lung Bratislav Janjic, Albert D. Donnenberg, Vera S. Donnenberg, Daniel P. Normolle UPMC, University of Pittsburgh, Pittsburgh, PA The use of unsupervised classification to extract markers from primary flow cytometry data is an emerging field which has made significant progress, spurred by the growing complexity of multidimensional flow cytometry. Whether markers are extracted without supervision or by conventional gate and region methods, the number of candidate variables identified is typically larger than the number of specimens (p > n) and many are highly inter-correlated. Thus comparison across groups or treatments to determine which markers are significant is challenging. Here we utilize a data set in which 86 variables are created by conventional manual analysis of individual listmode data files, and compare the application of five multivariate classification methods to discern subtle differences between the stem/progenitor 29 content of 35 non-small cell lung cancer and adjacent normal lung specimens. The methods compared included elastic-net, lasso, random forest, diagonal linear discriminant analysis and best single variable (best-1). We describe a broadly applicable methodology consisting of: 1) Variable transformation and standardization; 2) Visualization and assessment of correlation between variables; 3) Selection of significant variables and modeling; and 4) Characterization of the quality and stability of the model. The analysis yields both validating results (tumors are aneuploid and have higher light scatter properties than normal lung), as well as clues that require followup: Cytokeratin+ CD133+ progenitors are present in normal lung but reduced in lung cancer; diploid (or pseudo-diploid) CD117+CD44+ cells are more prevalent in tumor. We anticipate that the methods described here will be broadly applicable to a variety of multidimensional cytometry problems. 11. Perforin Deficienc Presenting in an Adolescent as IBD Trivikram Dasu1, Jerome Sigua2, Cindy Bauer2, James Casper3, John Routes2 and James Verbsky1,4 1 Clinical Immunodiagnostic & Research Laboratory, Medical College of Wisconsin 2 Division of Pediatric Allergy, Asthma & Clinical Immunology 3 Divisions of Pediatric Hematology-Oncology, Blood & Marrow Transplantation 4 Division of Pediatric Rheumatology, Children’s Hospital of Wisconsin, Milwaukee, WI A 14 year-old Caucasian male with a 1-year history of ulcerative colitis presented with increased ostomy output, vomiting, abdominal pain, anorexia, and fatigue 1-month postcolectomy. Endoscopy showed new onset pan-enteritis. Tests for humoral deficiency, Chronic Granulomatous Disease, Toll-like receptor 4, Immune Dysregulation Polyendocrinopathy Enteropathy X-linked, X-linked Lymphoproliferative Disease, and NFkB Essential Modulator Deficiency were normal. Natural Killer (NK) cell cytotoxicity was severely reduced with normal absolute NK cell numbers. Immunophenotyping for expression of perforin and granzymes revealed perforin deficiency in cytotoxic NK and CD8 T cells. Genetic testing demonstrated a homozygous PRF1 272 C>T (A91V) mutation, which has been reported to be a disease causing variant in familial hemophagocytic lymphohistiocytosis (HLH). Furthermore, the patient’s lymphokine activated NK cells did not upregulate expression of perforin in presence of IL-2 compared to the control cells. The patient’s fraternal twin brother demonstrated detectable, but reduced perforin expression in his lymphocytes, indicating haploinsufficiency. The subject’s sister and mother, however, displayed normal expression levels and profiles of perforin and granzymes in the cytotoxic lymphocyte subsets. Rituximab and immunoglobulin replacement were initiated while awaiting evaluation for allogenic bone marrow transplant. This report demonstrates that perforin deficiency, one of the familial causes of HLH, can also present as inflammatory bowel disease in adolescence. 30 12. Hyperbaric oxygen stimulates adipose- derived mesenchymal stem cells growth and differentiation in streptozocin-induced diabetes mellitus type 1 mice in vivo Tatyana N. Milovanova1, Veena M. Bhopale1, Sorokina Elena M1 ,Gunalp Uzun1, Ming Yang1, Jonni S. Moore3 and Stephen R. Thom 1,2 1 Institute for Environmental Medicine, 2Departments of Pathology and Laboratory Medicine and Emergency Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania; 3Department of Flow Cytometry, University of Pennsylvania Medical Center, Philadelphia. Hyperbaric oxygen modified function of stem/progenitor cell (SPCs) in new blood vessels formation which is a required step in wound healing in diabetic patients. In wounds healing VEGF secreted from adipose-derived mesenchymal stem cells (ASCs) induces migration and proliferation of endothelial cells, increasing the vascularity of wound bed. We hypothesized that oxidative stress from hyperbaric oxygen (HBO2, 2.8 ATA for 90 min daily) exerts a trophic effect on adipose- derived mesenchymal stem cells in streptozocin-induced diabetes mellitus type 1 mouse model via reactive oxygen /reactive nitrogen species (ROS/RNS)dependent mechanism. Adipose-derived stem/progenitor cells (ASCs:Sca-1+/CD31-/DAPI-), were sorted from omentum of additional WT mouse, labeled with the fluorescent dye 5,6carboxyfluorescein diacetate succinimidyl ester (CFSE), added to one of the Matrigel plugs subcutaneously injected one on either side of the thoracic vertebrae and stimulated by HBO2. In combination, HBO2 and ASCs in vivo demonstrated cumulative effects. Vascular channels lined by CD34+ SPCs were identified and they were significantly reduced in diabetic animals. In the Matrigel with seeded ASCs after HBO2 simulation accelerated channel development, cell differentiation based on surface marker expression and cell cycle entry was identified. CD34+ SPCs of diabetic animals in blood and bone marrow defined down regulated thioredoxin-1 (Trx1), Trx reductase, hypoxia-inducible factors (HIF)-1, -2, and -3, phosphorylated mitogen-activated protein kinases, vascular endothelial growth factor, and stromal cell-derived factor-1 in. Cell recruitment to Matrigel and protein synthesis responses was abrogated in STZ-mice. Thioredoxin system activation leads to elevations in HIF-1 and 2, followed by synthesis of HIF-dependent growth factors. HIF-3 has a negative impact on SPCs. By causing an oxidative stress, HBO2 activates a physiological redox-active autocrine loop in recruited SPCs and increased paracrine secretion in seeded ASCs, resulted in the stimulated neovasculogenesis. 13. TRAVEL AWARD WINNER - Looking at Microvesicles (MVs) by Flow Cytometry Phil Hexley Shriners Hospitals for Children, Cincinnati, OH Microvesicles (MVs) are small blebs released from mammalian cells following apoptosis or activation. With mounting evidence suggesting MVs act as distal effectors of pathology, they are increasingly relevant in clinical settings. However, due to their size range (0.1 – 1 µm diameter) and relative novelty, they are notoriously difficult to measure: even with known caveats of flow cytometry in this size range it is still a popular method of analysis. In addition, many analytical and patient variables have been shown to artificially increase circulating plasma MV numbers yet protocol standardization is still lacking. We acknowledge there are still limitations, but by controlling and/or minimizing known 31 confounding variables, we obtained data indicating detectable circulating plasma MV number correlates strongly with total body weight (R2=0.91). The relationship we have observed should be an important point to consider in future research into circulating plasma MVs, furthermore the consistency in intra-donor sampling over time may be encouraging for using this number as a clinical parameter. 14. Cross Platform Solutions for Autophagy Detection using the guava 8HT and Amnis ImageStream mkII Mark Santos, Kevin Su, Haley R. Pugsley, Angelica Olcott, Jason Whalley, David Basiji, Patrick Schneider, and Matthew Hsu EMD Millipore, Hayward, CA, United States Autophagy is an intracellular catabolic pathway which causes cellular protein and organelle turnover, and is associated with diverse diseases such as Alzheimer’s disease, cancer, and Crohn's disease, in addition to aging. It is a tightly regulated process that plays a normal part in cell growth, development, and cellular homeostasis. Autophagy functions as a housekeeping mechanism through disposal of aging and dysfunctional proteins and organelles by sequestering and priming them for lysosomal degradation Increasing evidence suggests that not only apoptosis, but also autophagy, can contribute to cell death and greatly influence general cell health. Malfunctions of autophagy can adversely impact longevity and the capability of cells to function at full capacity. Here, we describe two key platforms paired with a novel assay design which will allow for the study of autophagy by flow cytometry. Guava 8HT systems have the capabilities of higher throughput sampling utilizing a 96-well format and smaller sample sizes for acquisition. And when combined with the InCyte software module for data analysis and curve fitting algorithms for small molecule analysis, one can now screen autophagy inducers or inhibitors more efficiently. Amnis’s ImageStream system is a perfect complementary platform as it combines the capabilities of microscopy and flow cytometry in a single platform for quantitative image based cellular analysis of autophagy. 15. Enhanced Dynamic Range Capabilities for Microcapillary Cytometry Katherine Gillis, Asima Khan, Julie Clor, Kamala Tyagarajan, Ray Lefebvre, Bruce Goldman, Rick Pittaro EMD Millipore, Hayward, CA, United States Sensitive and multi-parametric cell analysis by flow cytometry typically requires substantial expertise, a confusing array of protocols, and cumbersome software analysis, thereby limiting its utility for on-demand use. Flow cytometric set-up can often be cumbersome and time consuming as it requires multiple steps for instrument configuration and data analysis. Samples often require gain adjustment to view positive and negative samples on screen, or else both populations cannot be visualized together. Conversely, small shifts within experiments may be overlooked if display scaling is not utilized to its fullest. In this work, we demonstrate how features of InCyteTM software allow the user to not only display samples comparing large fluorescence shifts and minimal fluorescence shifts, such as GFP expressing cells and CFSE stained cells, but additionally we highlight how small fluorescent shifts in 32 Cytochrome c loss from the mitochondria can be detected with no loss in data integrity. Additionally, utilizing features such as six-parameter heat-mapping and IC50/EC50 curve generation offer sophisticated data analysis in just a few steps, with updates in real time and easy export of data. As a result, users now have complete flexibility regarding their choice of assay and analysis methods. Experiments can utilize a set of samples run in a single day, across multiple days, or even between multiple assays or experimental questions. Taken as a whole, these novel and innovative advancements of InCyteTM Software enable analysis of multiparametric cellular data in a format which is even faster, simpler and more accurate. 16. Simplified Cell Cycle Analysis on the MuseTM Cell Analyzer Katherine Gillis, Asima Khan, Kimvan Tran, Arni Barican, Anh Nguyen, Ray Lefebvre, Kamala Tyagarajan EMD Millipore, Hayward, CA, United States Cell Cycle analysis and the assessment of cell cycle impacts has traditionally been restricted to more expensive and elaborate instrumentation platforms that required greater user expertise. In this study we present highly simplified cell cycle analysis solutions on a novel, cytometry platform, the Muse Cell Analyzer. The Muse Cell Analyzer is a small footprint instrument that utilizes innovative optics and electronics along with a touch-screen interface to guide users to obtain quantitative cellular data and obtain comprehensive cell health analysis. In this study, we present results using the Muse Cell Cycle Assay and dedicated software module, for the identification of cells in different phases of the cell cycle. Using suspension (Jurkat) and adherent (PC3) cell line changes in the phases of the cell cycle could be clearly distinguished using compounds that are know to arrest cell cycle phases, nocodazole and aphidicolin as well as compounds that are known apoptotic inducers, staurosporine and etoposide. Impacts on both cell count and apoptotic percentages were evaluated in parallel using other count and viability and Annexin-V based assays on the platform. Comparative studies with predicate methods established that comparable and accurate results for determination of % of cells in different phases of cell cycle could be obtained on the novel system. The simplicity of the assay and data interface and its output further allowed for highly precise results to be obtained on the platform with %CV of replicates being <10%. The Muse Cell Cycle Assay solution thus greatly simplifies a traditionally complex assay and makes it accessible to users of multiple expertise level while still offering rapid, accurate and precise cell cycle results. 17. Approaches to Evaluate Immune Cell Health and Apoptosis Using MicroCapillary Cytometry Julie Clor, Kimvan Tran, Katherine Gillis and Kamala Tyagarajan EMD Millipore Corporation, Hayward, CA The study of apoptosis and cell death mechanisms in immune cell subpopulations has become increasingly important to understand the pathways that lead to elimination of cells during normal development, disease, drug treatment or autoimmune responses. These studies are complicated and cumbersome to perform since they require parallel identification of immune cells and identification of impact of the treatment condition on immune cell health 33 and often have to be performed on limited sample sizes. We present here highly simplified approaches using microcapillary cytometry and small cell sample sizes to investigate changes in annexin V–based apoptosis detection, mitochondrial membrane potential changes, FAS expression levels and caspase activity in in CD4 and CD8 T cell populations simultaneously using a series of FlowCellect Immune Cell Health kits. The approaches thus provide information in change in immune cell population and a snapshot of immune cell health. Our results demonstrate that the approaches can be applied to plate-based screening studies to screen PBMC’s with ~80 cytotoxic compounds from blood donors. The studies allowed for the rapid identification of compounds such as thimerosal, phenylmercuric acetate, 2,6 dimethoxyquinone and sanguinarine sulfate that caused high levels of apoptosis in immune sub-populations and provide mechanistic information on their mode of action. The facile identification and dissection of immune cell apoptosis pathways can greatly enhance our understanding of how compounds interact with the immune system. In a screening study with 80 cytotoxic compounds, PBMC’s from multiple donors were treated with cytotoxic compounds and then evaluated for their impacts on CD4 and CD8 T cells and their immune cell health using immune cell health assays followed by flow cytometry on the guava easyCyte cytometry platform. The studies identified several compounds such as gambogic acid, thimerosal, phenylmercuric acetate, 2,6 dimethoxyquinone and sanguinarine sulfate which caused high levels of apoptosis in both CD4 and CD8 T cells with overnight treatment with some of them having differential effects on individual sub-populations. 18. A Networking Guide for A Multi-parametric Community White, AN; Delay, ML; Thornton, S Cincinnati Children’s Hospital Medical Center Networking is the framework essential to building relationships, forming collaborations, and the exchanging ideas. The field of cytometry is a multi-faceted community of researchers, suppliers, and technical specialists from varying areas of disciplinary interests and expertise. These indviduals come from both academic and industrial backgrounds and are trained in different disciplinary fields of study in engineering, biology and chemistry. The diversification, along with geographical location and access to funding for travel to meetings can also inhibit the exchange of ideas between heterogenous groups. The complex networking structure in a field like cytometry introduces several potential impediments that work against a network’s competency to achieve productive mechanisms for the exchange of ideas. Utilizing established organizations and affiliations within the cytometry community can (1)enhance its networking competency between local, regional, and international groups; (2) create a stronger interconnectedness among diverse cytometrists; and (3) increase the degree of impact cytometrists have on each other. 34 19. Analysis and Sorting of Fragile Cells (Adipocytes, Protoplasts etc.), Cell Clusters and Model Organisms Using BIOSORTER® Instrument Mikalai Malinouski, PhD, Union Biometrica, Inc. Many objects are too large or fragile for conventional flow cytometry. Union Biometrica instruments are engineered to automate the analysis and sorting of fragile cells and “large” objects such as hepatocytes, adipocytes, cardiomyocytes, kidney tubular cells, plant protoplasts, embryoid bodies, pancreatic islets. Other suitable samples are small model organisms such as all stages of C.elegans, Drosophila eggs, embryos and larvae, and Zebrafish eggs, embryos and the early larval stages. The BioSorter instrument uses interchangeable fluidic modules and is capable of analyzing and sorting a wide range (11,500 micron) of object sizes at rates of 10-500 objects/second. Using object size, optical density and fluorescence intensity as sorting criteria, selected objects can be dispensed in multi-well plates or bulk receptacles. A gentle pneumatic sorting mechanism avoids harming sensitive objects thereby making the instrument suitable for fragile cells and live biological materials. Several applications (e.g. model organisms) require extensive analysis of object shape and fluorescence localization before sorting. A unique feature available with these instruments is Profiler II™ which allows setting complex sorting parameters. Each object passing through the flow cell is digitized into a 2D profile. These axial profiles are analyzed and can be used as sort criteria based on the localization of optical density and fluorescence within each object, including peak height, peak width, peak counting and relative peak position. This approach is much faster than imaging and proves to be useful in the analysis and sorting of cells, cell clusters and model organisms. 35 GLIIFCA STEERING COMMITTEE - 2012 Contact info Canada David Hedley California Maurice R. G. O’Gorman Illinois Ryan Duggan Indiana Lisa Green Bartek Rajwa Iowa Bruce Pesch Massachusetts Betsy Ohlsson-Wilhelm Michigan Alexander Nakeff Louis King Minnesota Paul Champoux New York Matt Cochran Sally Quataert Paul Wallace Ohio R. Michael Sramkoski A. Nicole White Pennsylvania Vera Donnenberg Mike Meyer Andrew Bantly Tennessee Keith Shults Washington DC Karen Domenico Wisconsin Katharine Muirhead Brian DuChateau Kathy Schell Emeritus Carleton & Sigrid Stewart Charles Goolsby [email protected] 2012 President [email protected] [email protected] Co-Executive Secretary [email protected] [email protected] Education & CMLE Chair [email protected] [email protected] [email protected] [email protected] Site Committee Chair [email protected] Roundtable Chair [email protected] [email protected] [email protected] Core Manager Meeting Chair Treasurer [email protected] [email protected] 2012 Program Committee 2012 Program Committee [email protected] [email protected] [email protected] Co-Executive Secretary [email protected] [email protected] Vendor Liaison [email protected] [email protected] [email protected] Treasurer [email protected] [email protected] Emeritus Members Emeritus Member 36 GLIIFCA 21 Registered Attendees First Name Last Name Email address Institution David Adams [email protected] University of Michigan Alison Allan [email protected] London Regional Cancer Program David Allman [email protected] University of Pennsylvania George Babcock [email protected] Shriners Hospital for Children Matthew Balys [email protected] University of Rochester Andrew Bantly [email protected] University of Pennsylvania Suzanne Buck [email protected] Life Technologies Tim Bushnell [email protected] University of Rochester Paul Champoux [email protected] University of Minnesota Juan Chen [email protected] University of Illinois at Chicago Carolyn Cheney [email protected] Ohio State University Sue Chow [email protected] Princess Margaret Hospital Aileen Cinquino [email protected] Roswell Park Cancer Institute Matthew Cochran [email protected] University of Rochester Bunny Anne Cotleur [email protected] Cleveland Clinic Kamila Czechowska-Kusio [email protected] Yale Medical School Trivikram Dasu [email protected] Medical College of Wisconsin Monica DeLay [email protected] Cincinnati Children's Hospital Priscilla Do [email protected] The Ohio State University Ree Dolnick [email protected] Roswell Park Cancer Institute Karen Domenico [email protected] Children’s National Medical Center Terry Donahue [email protected] Roswell Park Cancer Institute Vera Donnenberg [email protected] University of Pittsburgh Albert Donnenberg [email protected] University of Pittsburgh Ryan Duggan [email protected] University of Chicago Ann Marie Eckel [email protected] Roswell Park Cancer Institute Masilamani Elangovan [email protected] Idea Elan Karen Ersland [email protected] UWCCC Flow Cytometry Laboratory Sharon Evans [email protected] Roswell Park Cancer Institute Nancy Fisher [email protected] University of North Carolina Rosemary Furlage [email protected] Roswell Park Cancer Institute Balaji Ganesh [email protected] University of Illinois at Chicago Jeannie Gaylor [email protected] DVS Sciences Jewell Graves [email protected] University of Illinois at Chicago Christiane Hassel [email protected] Indiana University David Hedley [email protected] Princess Margaret Hospital Erika Heninger [email protected] UWCCC Flow Cytometry Laboratory Phil Hexley [email protected] Shriners Hospital for Children Jan Hoffman [email protected] Roswell Park Cancer Institute Chris Holley [email protected] Washington University Siteman CC Matthem Hsu [email protected] EMD Millipore Jackie Hughes [email protected] Washington University Siteman CC Bratislav Janic [email protected] Pittsburgh Cancer Institute, U. of Pitt. James Jacobberger [email protected] Case Western Reserve University Craig Jones [email protected] Roswell Park Cancer Institute Louis King [email protected] Michigan State University Geoff Kraker [email protected] Northwestern University Charles Kuszynski [email protected] University of NE Medical Center Joanne Lannigan [email protected] University of Virginia 37 David Leclerc [email protected] University of Chicago Deborah Lee [email protected] Bristol-Myers Squibb Orla Maguire [email protected] Roswell Park Cancer Institute Roland Marcus [email protected] FluoroFinder Bryan Mc Elwain [email protected] The Ohio State University Mike Meyer [email protected] University of Pittsburgh Justin Meyers [email protected] Purdue University Tatyana Milovanova [email protected] University of Pennsylvania, IFEM Hans Minderman [email protected] Roswell Park Cancer Institute Jonni Moore [email protected] University of Pennsylvania Alex Nakeff [email protected] Carole Anne Nakeff [email protected] Jeffrey Nelson [email protected] Northwestern University Mario Nunez [email protected] Beckman Coulter Kieran O’loughlin kieran.o’[email protected] Roswell Park Cancer Institute Betsy Ohlsson-Wilhelm [email protected] SciGro, Inc./NorthEast Office Mike Olson [email protected] University of Chicago Andrew Osterburg [email protected] University of Cincinnati Jennifer Piraino [email protected] Roswell Park Cancer Institute Hank Pletcher [email protected] University of Pennsylvania Edward Podniesinski [email protected] Roswell Park Cancer Institute Anna Porwit [email protected] Toronto General Hospital Sally Quataert [email protected] University of Rochester Aaron Robida [email protected] University of Michigan Wade Rogers [email protected] University of Pennsylvania Kathleen Schell [email protected] UW Retired Heather Schumacher [email protected] Van Andel Research Institute Dan Schweppe [email protected] Washington University Siteman CC Dagna Sheerar [email protected] UWCCC Flow Cytometry Laboratory Keith Shults [email protected] IncellDX,Inc. Sukhwinder Singh [email protected] UMDNJ – New Jersey Medical Sharleen Slaunwhite [email protected] University of Rochester Michael Sramkoski [email protected] Case Western Reserve Univ Kim Stojka [email protected] Joseph Tario, Jr. [email protected] Roswell Park Cancer Institute Robert Thacker [email protected] Cincinnati Childrens Hospital Sherry Thorton [email protected] Cincinnati Children’s Research Rachel VanWinkle [email protected] Beckman Coulter Paul Wallace [email protected] Roswell Park Cancer Institute Maria Watson [email protected] Prince Margaret Hospital Nicole White [email protected] Cincinnati Children’s Hospital Wojciech Wojciechowski [email protected] University of Rochester Philip Woost [email protected] Case Western Reserve University Daniel Wu [email protected] Princess Margaret Hospital 38 Registered Vendor Representatives Alexion Pharmaceuticals, Cheshire, CT http://www.alxn.com/ Dan Hayden Kim Coleman Lohr DVS Sciences, Inc., Sunnydale, CA http://www.dvssciences.com/ Jeannia Gaylor Clare Rogers Amnis Corporation (see EMD Millipore) eBioscience, San Diego, CA www.ebioscience.com Elizabeth Dobbs Hether Ide Keith Olszens Susan Reynolds Bay Bioscience, San Francisco, CA www.baybio.co.jp David Coder Beckman Coulter, Miami, FL www.beckmancoulter.com Cheryl Guyre Peter Racanelli Patricia Sardina John Stojka EMD Millipore, http://www.millipore.com/ Andy Arena Darin Fogg Sabrina Hawthorne Dave Kennedy Cassandra Singer Christine Valway BD Biosciences, San Jose, CA www.bdbiosciences.com Eva Chan Darci Gorgone Bill Gunderman Janet Horta Thomas Lendrihas Susan Merill Nicole Ovadia iCyt Mission Technology, Champaign, IL www.i-cyt.com Matt Alexander Joanie Capps Jeff Clapper Mike Kanavos Sharlene Wright BioLegend, San Diego, CA www.biolegend.com Rebecca Bultema Thomas Williams Immudex, Copenhagen, Denmark http://www.immudex.com/ Stephen Haley Kivin Jakobsen Cedarlane Labs, http://www.cedarlanelabs.com/ Joel Koolstra Leinco Technologies, St. Louis, MO http://www.leinco.com/ Patrick Leinert Cell Signaling Technology, Beverly, MA www.cellsignal.com Gordon Miller Life Technologies, Eugene, OR http://www.lifetechnologies.com/home.html Jolene Bradford Neil Bristol Suzanne Buck Dolores Ciufo Pat Jacobs Barb Sereick Cytek Development, Gordonsville, VA www.cytek.com Kim Belenzom Raymond Lannigan Lisa Nichols Nick O’Gorman Miltenyi Biotec, Inc., Auburn, CA www.miltenyibiotec.com Dan Graziano Shane Oram Ashley Weant De Novo Software, Thornhill, Ontario www.denovosoftware.com David Novo 39 Partec North America, Inc., Swedesboro, NJ www.partec.com Roy Overton Allan Schell Stratedigm, Inc., San Jose, CA http://stratedigm.com/ Thermo Fisher (Dako), Waltham, MA http://www.thermofisher.com/global/en/home.asp Brian DuChateau Mike Stinson Propel Labs, Fort Collins, CO http://www.propel-labs.com/ George McDonough TreeStar, Inc., Ashland, OR www.treestar.com John Quinn Nick Ostrout R&D Systems, Minneapolis, MN http://www.rndsystems.com Chris Larson Union Biometrica, Holliston MA http://www.unionbio.com/ Michael Fazzio Mikalai Malinouski Spherotech, Inc, Libertyville, IL www.spherotech.com Andy Wang Brian Shah Ayesha Hamid Verity Software House, Topsham, ME www.vsh.com Mark Munson 40 NOTES: 41
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