Sunday, June 11, 2017
Small Core to Large Core: Recruiting, Retaining and Expanding Core Staff
The purpose of any cytometry core is to provide service, whether it be in the form of analysis, sorting or
training, to a variety of users. The essential components of how to do this are well-established. However
there are several issues that are less clear. For example: How to recruit and retain staff as the cores user
base and instrumentation grows? How the needs of staff members can be met while still providing
service? Many time the way these issues are addressed depends on how the core is staffed. It is obvious
that there are major differences between running a core that is staffed by one or two people compared to
one staffed by upwards of 10 people. However regardless of the size of your core it is import to
acknowledge strengths and areas for improvement of core staff, assess how work can be done efficiently
and spread fairly, and how the expectations of staff, users and management can be met. It is also
important to consider that as the core grows the goals of the facility may change. In this workshop, we
will present several scenarios highlighting how to deal with these issues and the major changes that need
to take place and be considered in moving from a single-staffed core, to four members to eight and
beyond. These issues will be addressed in the context of the Staffing section of the ISAC SRL Best
First Steps As An Innovator: Generate Your Value Proposition
Betsy Ohlsson-Wilhelm, SciGro, Inc.
Paul J Smith, OncoTherics Ltd.
Gary Durack, The TEKMILL, Univ. Illinois at Urbana-Champaign
Renold Capocasale, FlowMetric, Inc.
Problem Focus and Key Questions:
- What is a value proposition?
- Who will use your invention?
- What is the value to the user of that invention? What specific benefit will they obtain?
- Why should the user buy your product instead of that of a competitor?
- How will your value proposition affect your business plan?
- List of specific DIY tasks that can be carried out while still at your “day job”.
- List of information that attendees would like to see on the CytoInnovation page of the ISAC website to assist those interested in making their innovations available to the rest of the community.
- Feedback on the direction of the Innovation program within ISAC.
- List of mechanisms to enhance the general awareness on the part of the cytometry community of the potential for commercialization of innovations/inventions, whether the inventor is based in a research institution or a commercial site.
Photonic Calibration and Advanced Photon Detection in Flow Cytometry
Photo-sensors are key devices for flow and image cytometry. Understanding how the gain of a photosensor
influences the processed signal intensity and the compensation matrix in a flow cytometer saves
time and effort. A widely accepted method to characterize flow cytometer photo sensors and to compare
instruments uses the detection efficiency (Q value) and the signal background (B value). It will shown
how a high precision pulsed LED (e.g. quantiFlash™) is used to perform these measurements.
Additionally, the following can also be measured to facilitate reproducible instrument setup for interinstrument
comparisons: (i) the signal to noise ratio (SNR) and dynamic detection range (DNR), (ii) the
optimal photo-sensor gain to standardize the instrument scales for data presentation.
PMTs have been the mandatory and essential sensor for fluorescence detection and various biological
applications for many years. Recent advancements of PMT and solid-state photo-sensors provide new
opportunities to migrate to higher sensitivity and wider dynamic range devices including photocurrent
analog and digital photon detection. Some new flow cytometers using Si-APDs instead of conventional
As the field of flow and image cytometry moves forward, the use of standardized and high sensitivity
photo-sensors will be crucial to detecting and quantitating weak fluorescence and light scatter signals.
One trending application that will benefit from this attention to photo-sensor characterization is microparticle
Characterizing Instruments with Photon Statistics – Introduction
by Jim Wood (5 min)
Using Q & B to characterize, calibrate and optimize settings of photodetectors. (Steve Perfetto) (5 min)
Characterization of Flow cytometer dynamic range by using a LED light source. (Toralf Kaiser) (5 min)
Accuracy of Quadfit using Beads on Cytoflex. (Eric Chase) (5 min)
25 min discussion on this part of the workshop
APD and Si detectors - Introduction by JPR (5 min)
Fiber Optically Coupled Si APD Sensor - an Ideal Flow Cytometer Detector. (Yong Chen) (5 min)
Feasibility of photon sensing for cytometry. (M. Yamamoto) (5 min)
25 min discussion based on questions arising from the above or other interests of the audience.
Strategies, instrumentation, and best practices for NextGeneration characterization of single cells and nuclei flow sorted from complex tissues and organs
David Galbraith, Monica DeLay, and Byoung Koh.
Flow cytometry and cell sorting was originally devised for the analysis of natural single cell suspensions, prototypically cells of the mammalian erythropoietic system, and has ultimately developed to the point that cells can be analyzed for type and function at very high dimensionalities.
We now face two challenges. First, many eukaryotic cells do not naturally occur in the form of cell
suspensions, instead being complex three-dimensional assemblies of different cell types associated in the form of organs and tissues. Strategies for dissociation of tissues and organs into the individual cellular components therefore are needed. Second, the types of downstream analyses to be applied have developed considerably in sophistication, particularly those involving various ‘omics platforms and
methods applied at the level of single cells.
This workshop will outline strategies and provide examples that address both of these concerns when initiating and implementing NextGeneration analyses of cellular functions in organs and tissues, following flow cytometry and sorting to define and isolate single cells and nuclei. It will comprise short presentations to set the stage, followed by longer periods of open discussion to define concerns, reach
consensus, and establish best-practices.
: (90 mins)
David Galbraith and Monica Delay: Overview and identification of major concerns.
: Specific Examples
David Galbraith: Flow sorting of nuclei and characterization of transcripts within these nuclei.
Byoung Koh: Isolation of gastrointestinal cells by FACS and downstream applications.
Monica DeLay: Cell sorting to single cell RNASeq using commercial platforms
Presentations will be no more than 15 minutes each, including active audience participation, followed by a 30 minute question-and-answer session and general discussion.
Building a quantitative flow cytometry (FCM) measurement system
Lili Wang, NIST
Robert Hoffman, Consultant, CA
Gerald Marti, FDA
John Nolan, Scintillon Institute, CA
Focus and Key Questions:
Building a quantitative cytometry measurement system requires proper controls and standards, e.g. particles for instrument standardization and calibration, and biological cell reference materials. The purpose of this workshop is to foster the dialogue between ISAC standards committee/task force, NIST, FDA, NIH, CDC and user communities and address key questions: 1) how best to standardize flow cytometers using calibration beads with ERF value assigned traceable values according to NIST SRM 1934? 2) will any bead controls with ERF value assigned be of interest to assay manufacturers to be included in their assay kits? 3) is it useful to send out flow cytometry raw data files obtained from the reference cell characterization project to test users’ comparability of cytometry data analysis? 4) what types of evaluation study are of interest to general/specific user communities using reference bead and cell controls/standards?
The workshop will be formatted with three 10-15 minute presentations that are composed of current state of traceable calibration beads and characterized commercially produced cell reference materials, and applications needing these reference materials for ultimately building a quantitative flow cytometry measurement system. Presentations will be followed by discussion and conclusion.
Finding the Needle in the Haystack: Rare Event Detection and Validation of the Lower Limit of Quantification
Virginia Litwin, Hematology/Flow Cytometry, Covance Inc., firstname.lastname@example.org
MedImmune, One MedImmune Way, Gaithersburg, MD 20876, USA EckS@MedImmune.com
Alessandra Vitaliti, BioMarker Development, Novartis Pharma AG, Postfach CH-4002 Basel, Switzerland email@example.com
Jennifer Stewart, Flow Contract Site Laboratory, LLC, 18311 Bothell Everett Highway, Suite 180, Bothell, WA 98012, USA firstname.lastname@example.org
This workshop is intended for translational and clinical researchers wishing to tap into current thoughts around trends in ensuring that the performance of rare event analyses assays are well characterized and fit-for-purpose, particularly in regulated environments such as for lab developed clinical tests, toxicology studies and clinical trials. This workshop is aimed at producing a recommendation paper on the validation of rare event and establishing a LLOQ.
Both the American Association of Pharmaceutical Scientists (AAPS) and the International Clinical Cytometry Society (ICCS) have published white papers on the analytical validation of flow cytometric methods (1, 2). But neither of these publications has adequately addressed best practices for the validation of rare event and establishing a lower limit of quantification (LLOQ).
- How do the concepts of LLOQ, limit of blank (LOB), and limit of detection (LOD) apply to flow cytometry?
- What defines a ‘blank’ sample?
- Experimental design for LLOQ validation and creation of validation samples
- LLOQ vs total number of events collected.
- What is the minimal acceptable number of events?
- Application of quantitative validation principles to rare event analyses
- Data review
- Acceptance criteria
- Application of cut-point approaches and presenting ranges of numbers.
- Introduction—Overview and Definitions (15 min)
- Topic 1. How do the concepts of LLOQ, limit of blank (LOB), and limit of detection (LOD) apply to flow cytometry? What defines a ‘blank’ sample
- Introduction (5 min)
- Open discussion (20 min)
- Topic 2. Experimental design for LLOQ validation and creation of validation samples. LLOQ vs total number of events collected
- Introduction (5 min)
- Open discussion (15 min)
- Topic 3. Application of quantitative validation principles to rare event analyses
- Introduction (5 min)
- Open discussion (15 min)
- Summary and Next Steps (10 min)
Lifting the ‘curse of dimensionality’ from single-cell data
Nikolay Samusik, Stanford School of Medicine, USA
Zinaida Good, Stanford School of Medicine, USA
Modern cytometry devices provide an ever-expanding number of detection channels, which on one hand enables comprehensive survey of complex biological systems, but on the other hand presents significant challenges for data analysis and visualization. One of the common problems is the ‘curse of dimensionality’, which manifests in that some cell populations that can be easily separated by biaxial gating become inseparable by clustering methods and invisible in dimensionality reduction techniques, such as single-cell force-directed layouts or tSNE. Here we will recast the ‘curse of dimensionality’ as a signal-to-noise problem and discuss potential methods to overcome it: noise filtering, nonlinear data compression and specially designed distance measures.
Introductory talk and a review code examples (30 minutes), facilitated group discussion (30 minutes), brainstorming session (15 minutes).
Monday, June 12, 2017
Self-Service Cell Sorting in an SRL: User Training, Implementation and Other Concerns
Michael Gregory, DART Cytometry, NYULMC
Benjamin Daniel, Microbiology and Immunology, UTHealth San Antonio
This workshop aims to discuss the placement of self-serve sorters in SRLs, focusing on the training and support that needs to be provided by SRLs for their proper use.
Multilaser Cytometry 2017 - Optimising Wavelength, Power and Optics for High Parameter Flow Cytometry
The ongoing development of high-dimensional flow cytometry has required advances in both the palette
of reagents and in highly-optimised instrumentation. While much of the focus of this development has
been aimed at maximising the number of parameters (the “emission” side of the technology), there has
also been concurrent development on the “excitation” side. In this workshop we will concentrate on
excitation, in particular, the lasers that drive the emission from fluorophores, and consider best practice in
developing high-performance and optimised multilaser cytometry platforms.
Laser technology has advanced significantly in the past ten to fifteen years and it is now possible to
purchase lasers of almost any wavelength and outfit (and potentially retro-fit) commercial flow
cytometers with ten or more different high-powered laser lines. This great flexibility significantly
enhances our cytometric capabilities by allowing us to optimise our excitation characteristics to maximise
the signal from individual reagents and/or facilitate the combining of reagents into multiparametric
panels. However, this flexibility can come with substantial financial cost and can significantly increase
instrument complexity in ways that can hinder our ability to obtain maximum benefit from the
A similar workshop was held at CYTO2015 and this year we plan to revisit this topic and survey what
progress has been made in both the instrumentation and in our ability to optimise laser configurations for
particular applications. Through a combination of short presentations and panel discussion, we aim to:
1. review current laser technologies and options to consider when purchasing new (or upgrading
2. discuss the advantages and disadvantages of different wavelengths in the context of different
3. examine issues around laser power optimisation - how much power is enough and at what cost?
4. discuss some of the pitfalls and cautions in multi-laser cytometry,
5. provide an update on new and forthcoming technologies, including the latest trends in single
wavelength lasers and the emergence of tunable laser systems, and
6. identify areas where the further investigation/experimentation is required.
Attendees are encouraged to submit questions and issues to the facilitators prior to the workshop for
inclusion in the discussion.
Single Cell Genomics
Single cell genomics brings a 3 order of magnitude increase in our interrogation of single cells and the
promise to transform our understanding of disease and deliver on the promise of precision medicine. But
with operational challenges, collaborating with the genomics core, and new data types, where do you
start? Participants in our workshop will roll up their sleeves, learn from experts, and work in groups to
create 2 take-home frameworks for successful adoption of new single cell sequencing
technologies. Herein, we will introduce single cell sequencing technology and application, discuss the
advantages and disadvantages (e.g. cost per cell, cost per gene, throughput), and technical hurdles (e.g.
library preparation) of various approaches. In addition, we will discuss experimental workflows that
combine single cell genomics and flow cytometry / FACS and educate on the analysis approaches used
for sequencing data, with a focus on single cell sequencing and its unique challenges and considerations.
Finally, we will bring it all together with small group case study discussion of best practices for
collaborating with a genomics SRL, and strategies for bringing single cell genomics into the flow SRL.
Automating High Volume Cytometry Data Analysis for Central Facilities
More than 60 approaches for automated flow cytometry data analysis are now available to address many
of the bottlenecks in manual analysis, including quality checking, cell population identification (gating)
and high dimensional data analysis. With careful standardization, bioinformatics approaches can provide
superior sensitivity and specificity and save time. While there is anecdotal evidence that bioinformatics
tools have been adopted in some centers, there are few published reports that highlight their use. As such
there is the assumption that hurdles remain in pushing big data sets through existing tools including ease
of use, and this workshop is intended to explore these with end users in order to map a path forward in
concert with input from tool developers.
Tool developers and even more importantly users - people who run and analyze data for central service
facilities and are interested in pushing large volumes of data through automated analysis pipelines.
Brief (2 x 10 minute) presentations on the state of the art and challenges to adoption of automated
methods based on experience of the facilitators addressing the points below, with substantial group
Set expectations: define the distinction between discovery and routine reproducible analysis of large data
Import and preprocessing.
Data annotation, gating and export.
Modeling and data analysis.
Discuss the challenges to pushing big data sets through existing tools
Usability of existing tools (interface, documentation, scalability)
Standardization of data and assays. (SOPs, gating, marker, channel, file names)
Challenges of data sharing (tool interoperability, size of data, other concerns)
What can we improve?
Priorities for the future (user needs, developer improvements)
Moving beyond automated gating, thinking about analysis in terms of end goals.
Identify gaps in current automated analysis tools that limit adoption and uptake.
Connect users with resources for training, analysis, and collaboration.
Foster discussion and communication between tool developers and users.
Suggested background reading
Kvistborg et al., Thinking outside the gate: single-cell assessments in multiple dimensions. Immunity.
2015 Apr 21;42(4):591-2. doi: 10.1016/j.immuni.2015.04.006. PMID: 25902473 PMCID:PMC4824634
Finak et al., OpenCyto: an open source infrastructure for scalable, robust, reproducible, and automated,
end-to-end flow cytometry data analysis. PLoS Comput Biol. 2014 Aug 28;10(8):e1003806. doi:
10.1371/journal.pcbi.1003806. eCollection 2014.
Detecting and Characterizing Heterogeneity in Single Cell Data-or-How Not to Leave Cytometric
Money on the Table- or- What is Behind the Heat map?
Intended audience: Primarily Image and Flow Cytometrists involved in drug screening
What are the sources and implications of population heterogeneity as revealed by single cell analysis?
How canonical / typical data reporting ignores underlying biology
For example: how two very different distributions can have the same mean.
How distribution implies biology
How to detect and characterize heterogeneity
What are the tools, how do they work, what do they mean?
Introduction to heterogeneity and sources
Why do this for drug discovery
What is compound screening? To distinguish compounds from each other based on biology,
how distribution implies biology
How canonical reporting e.g. EC50 is “blinded” to distribution
Different distributions can have the same EC50
Introduction to tools to detect and characterize heterogeneity-
Pros/ Cons- Interpretation
What are implications of heterogeneity analysis in the Age of High Content?
Gough, AH et.al, (2014) Identifying and Quantifying Heterogeneity in High Content Analysis:
Application of Heterogeneity Indices to Drug Discovery. PLOS ONE, Vol 9, e102678
Huang, S, (2009) Non-Genetic Heterogeneity of Cells in Development: More Than Just Noise.
Development 136, 3853-3862
Launching and Supporting Basic and Translational Studies Incorporating Mass Cytometry
Jonathan Irish, Vanderbilt University
Adeeb Rahman, Mount Sinai
Bill O’Gorman, Genentech
This interactive workshop will focus on successfully moving your group, collaborators, and core users to higher dimensional panels for basic and translational research (>15 features per cell). We especially encourage core facility staff and managers, graduate students, and postdocs – the people immersed in the details of designing and validating cytometry panels – to attend this workshop. Attendees will learn how organizers and others at the workshop maximize the information revealed in single cell experiments by balancing measurement of project-specific “must have” cellular features, well-studied “backbone” cell identity markers, and opportunities to make new discoveries. The workshop will address the following questions: What are the important considerations in contemplating mass cytometry experiments encompassing antibody panels with 15 or more markers? How do you tailor a panel and controls to answer a particular question? What type of experiments work well to generate pilot data? How do the design and analysis strategies change for projects of different sizes or with different goals (discovery vs. validation)? What are the important factors to consider in experimental design and setup in order to maximize the results obtained on precious translational clinical research samples? What do reviewers expect in mass cytometry manuscripts and what are current "best practices"?
Tuesday, June 13, 2017
Developing an ISAC Shared Resource Laboratory Recognition Program
Adrian Smith, Rui Gardner, Joanne Lannigan, Michael Gregory
Overview: In November 2016 a group of SRL staff serving on the ISAC Shared Resource Lab Taskforce published ‘International Society for Advancement of Cytometry (ISAC) Flow Cytometry Shared Resource Laboratory (SRL) Best Practices’ in Cytometry A. The purpose of this publication was to ‘define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas.’ This was also the first step in the development of a program to recognize SRL’s that adhere to these best practices and provide an example of a level of excellence to which other facilities can aspire. This workshop will provide a forum for discussion and to gain feedback and ideas that will help inform the development, implementation and benefits of this new ISAC program.
Panel members will introduce the idea of the ISAC SRL recognition program, give a brief history of its inception and share the progress made thus far. Topics of discussion will be introduced including:
· Application and evaluation process,
· Selection criteria
· Potential benefits for participating SRLs
· Renewal process
Feedback will be gathered by audience members via either online tools or in app tools using the CYTO app. Depending on attendance levels, the workshop may be broken out into smaller discussion groups that focus on particular topics before reporting back to the whole group.
Cytometry Via High Throughput Single Cell RNA Seq, Why All the Fuss About Microfluidics?
David Weitz, Experimental Soft Condensed Matter Group, Harvard, Boston, MA, United States
Rob Salomon, Garvan Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Sydney, Australia
This workshop will focus on the use of microfluidics to perform high throughput single cell transcriptomic characterisation of cells. Whilst there are various ways to achieve high-throughput Single cell RNAseq, this tutorial will allow attendees to physically see many of the components of a microfluidics system and will focus on the setup, operations and expected results from the inDrop system (as published by Klein et. Al., Cell 2015).
Optimizing Biomarkers for Immunotherapy and Adoptive Cell Therapy
- Virginia Litwin, Hematology/Flow Cytometry, Covance Inc., Indianapolis, IN
- Cherie Green, Flow Cytometry Biomarker Development Sciences, Genentech, Inc., a Member of the Roche Group, South San Francisco, CA, USA
- Ulrike Sommer, BioMarker Development, Novartis Pharma AG, Basel, Switzerland
This workshop will address the challenges associated with analyzing cellular biomarkers for Immunotherapy and Adoptive Cell Therapy. The workshop is applicable to non-regulated as well as regulated laboratories. This workshop is aimed at producing recommendation papers on the optimal processes and controls for cellular biomarkers and monitoring cellular therapies.
- Standardization. We will discuss approaches to standardizing assays and instruments between laboratories. The focus will be on establishing standards that will facilitate successful assay and data exchange.
- Beads and Controls for Believable Biomarkers. The use –and usefulness- of various beads, assay controls, quality controls, etc. will be discussed. The focus will be on ensuring robust data sets from multicenter, longitudinal studies.
- Biomarkers Supporting Adoptive Cellular Therapies. Strategies and challenges associated with successfully measuring biomarkers for adoptive cellular therapies and rare event detection will be tackled! CAR-T therapeutic monitoring will be emphasized.
- Introduction—Overview of biomarkers for Immunotherapy and Adoptive Cell Therapy (5 min)
- Topic 1. Standardization
- Introduction (5 min)
- Open discussion (20 min)
- Topic 2. Beads and Controls for Believable Biomarkers
- Introduction (5 min)
- Open discussion (20 min)
- Topic 3. Unique Requirements for Adoptive Cell Therapy and Rare Event Detection
- Introduction (5 min)
- Open discussion (20 min)
- Summary and Next Steps (10 min)
3-Dimensional Image Analysis and Image Cytometry
Organizers: Silas Leavesley, Stephen Lockett, Gustavo Rohde, Anne Carpenter
3-dimensional (3D) analysis of cells, organoids, and tissues is an area of growing interest to the image cytometry field. Of particular importance are approaches that allow quantitative measurements to be made from 3D image cytometry data. The goals of this workshop are to discuss the need and potential uses of 3D image cytometry techniques, currently available tools and approaches for analyzing 3D image data, potential areas and needs for further software development, and future directions for the 3D image cytometry and image analysis fields.
Flow Cytometry for Cohort, Longitudinal and Multi-Center Studies
As increasing numbers of clinical studies are including Flow Cytometry as a technology platforms, mainly to perform immune phenotyping, there is a need for standardization and consensus on the minimal requirements. This Workshop is a follow-up to CYTO1016 where we discussed such studies and some of the attemps to minimize site to site variations and the limitations clinicians and scientists are facing. We will discuss the most recent updates for Flow Cytometry standardization in such studies to ensure reliability of the data. CYTO attendees will hear experienced scientists in large-scale and multi-center studies and we will provide enough space for interaction and interventions from the participants.
Participants are invited to send their comments, questions and items to be raised during the workshop to email@example.com.
We aim at 1) raise interest in this topic 2) disseminate the concept of standardization 3) enable attendees to identify the gaps of their own studies 4) Identify feasible strategies developed by cytometrist 5) create a network of cytometrist involved in this effort. The overall goal is provide the opportunity to the participants to raise their issues and/or how they implemented procedures to improve the outcome of Flow Cytometry measures in their studies.
Moving Towards Reproducible Flow Cytometry-based Extracellular Vesicles Measurements
John Nolan, The Scintillon Institute
Joanne Lannigan, University of Virginia
The Flow Cytometry Working Group of the Tri-Society (ISEV-ISAC-ISTH) collaboration charged with developing protocols to standardize the calibration and reporting of flow cytometry measurements of extracellular vesicles (EVs), will review the results and experiences of a recent multi-lab study aimed at standardizing EV fluorescence measurements. Following a few short presentations, we will open the floor for discussion of these results and collect input from the audience on other ways this group can support those in the cytometry community who wish to measure EVs. Live polling of the audience via the CYTO app will be used to collect input.