cytoconference.org
isac-net.org
XXVIII Congress of the International
Society for Advancement of Cytometry
Innovation,
Discovery,
and Translation
May 19 – 22, 2013
San Diego Convention Center
San Diego, California, USA
Program
12810 CYTO 2013 Program cvr_V2.indd 1
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ISAC is grateful for the contributions of the following
sponsors for their generous support of CYTO 2013:
GOLD LEVEL___________________________________________________
SILVER LEVEL__________________________________________________
Sciences
BRONZE LEVEL__________________________________________________
MOBILE APP SPONSOR
KEY CARD SPONSOR
PRE-CONGRESS COURSE
CORE MANAGERS FORUMS
–– GOLD SPONSORS ––
––– SILVER SPONSOR –––
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DVS Sciences Mass Cytometry will be featured at:
Oral Presentation
Expanding the Capabilities of Mass Cytometry
Speaker: Scott Tanner
Sunday May 19, 2013 11:00 a.m.–12:30 p.m.
San Diego Convention Center
Commercial Tutorial
Mass Cytometry Reveals an Endogenous Immune
Response to Physiological Perturbation in Humans
Speakers: Gabi Fragiadakis and Dr. Brice Gaudilliere,
Stanford School of Medicine
Tuesday May 21st, 2013
Room 33AB – San Diego Convention Center
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Requirements:
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Standardization in Nano Cytometry Applications
Verifies instrument performance in Nano Cytometry fluorescent,
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Provides a size standard in Microbial and Microparticle Cytometry
Determines the low end size resolution of flow cytometers
· To be able run a CAP accredited
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Amerimmune is a clinical and laboratory diagnostics and research immunology program with a focus
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For more information, visit our website at
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BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2013 BD
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ISAC XXVIII International Congress
San Diego, California, USA
May 19 – 22, 2013
Dear Colleagues,
On behalf of the CYTO Organizing Committee and ISAC Council, it is our pleasure to welcome you to
CYTO 2013, the XXVIII Congress of the International Society for Advancement of Cytometry. ISAC’s mission
is to advance the science and practice of cytometry, the quantitative analysis of cells and cell systems.
Cytometry encompasses multiple disciplines from the physical sciences and engineering to basic research
and clinical practice, and CYTO is the signature venue for showcasing the state of the art and science of
cytometry. San Diego, our host city, is a center of biomedical research, and provides an excellent back drop
for the CYTO 2013 theme of Innovation, Discovery, and Translation.
The CYTO Program includes elements for scientists working in all areas of cytometry and at all career
stages. The CYTO 2013 Education Program starts on Saturday, May 18th, with Introductory Flow and Image
Cytometry Courses, the Intermediate Course on Advanced Flow Cytometry Data Analysis, and a program of
Scientific Tutorials providing focused updates on a range of research and clinical topics in flow and image
cytometry, as well as core facility management.
Also on Saturday, ISAC will host its 2nd CYTO-Innovation Forum, providing an opportunity to discuss the
challenges and opportunities of commercializing new cell analysis technologies and applications. Industry
experts, technology innovators, and others involved in the business of cytometry are welcome to participate
in this pre-CYTO event.
The CYTO 2013 Scientific Program opens Sunday, May 19th with the Wallace H. Coulter Centennial
Lecture, “Spatial Systems Biology”, presented by Joe Gray, commemorating the 100th birthday of this
pioneering inventor, entrepreneur, and philanthropist. The CYTO 2013 program continues with Frontiers
and Plenary Sessions featuring cutting edge cytometry technology and applications, Parallel Sessions with
examples of contemporary cytometry from the research lab to the clinic, and Poster Sessions that provide
an opportunity for detailed discussions between authors and delegates. A diverse program of interactive
workshops provides an opportunity for experts and novices alike to discuss and debate emerging or
controversial issues in cytometry.
The CYTO Commercial Exhibition features more than 70 companies displaying hardware, software, and
reagents for cytometry research. The popular lunchtime Commercial Tutorials provide opportunities to hear
in detail about new product offerings from leading companies. New this year is an Exhibitor Showcase, to
be held Monday during lunch in the Exhibit Hall, giving vendors a forum to highlight new products. We
appreciate the support of all of our exhibitors, and especially our Sponsors, for helping to make CYTO
possible.
We want to express our sincere thanks to the members of the CYTO Organizing Committee and to the
50+ members of the CYTO Program Committee, including the ISAC Scholars, who proposed themes and
speakers, and assisted with abstract review. Thanks go also to our Course and Tutorial faculty, Workshop
leaders, and Session Chairs for contributing their time and talents. Lastly, we want to thank the FASEB
Office of Scientific Meetings & Conferences team and Michelle Butler, ISAC Executive Director, for their
tireless work in ensuring that CYTO 2013 is a success.
We encourage all members to participate in ISAC’s efforts to advance cytometry. Please attend the Business
Meeting on Wednesday afternoon to hear about ISAC’s recent efforts and future plans, volunteer for service
on committees and task forces, and share your thoughts on the future of the Society and cytometry with the
ISAC Council and management at any time.
Finally, enjoy CYTO and San Diego!
John P. Nolan ISAC President-Elect
ISAC 2013 Program and Abstracts
Larry A. Sklar
CYTO 2013 Program Chair
1
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Table of Contents
Sponsors and Supporters.................................................................................................................... Inside Front Cover
Welcome Letter............................................................................................................................................................1
ISAC 2012 – 2014 Executive Committee and Councilors................................................................................................... 5
ISAC Leadership and Congress Organizers...................................................................................................................6
Isac Special Committees & Task Forces........................................................................................................................8
General Information
Registration .........................................................................................................................................................11
Badge/Access Control..........................................................................................................................................11
Business Services.................................................................................................................................................11
Cell Phones.........................................................................................................................................................11
Child Care...........................................................................................................................................................11
Closing Reception at the Stingaree.......................................................................................................................11
Commercial Exhibits – Exhibit Hall Hours...........................................................................................................12
Commercial Tutorials...........................................................................................................................................12
Companion/Guest Registration............................................................................................................................12
Concierge – San Diego Convention Center..........................................................................................................12
Cyber Café ..........................................................................................................................................................12
CMLE...................................................................................................................................................................12
CYTO Innovation.................................................................................................................................................12
Dining Options....................................................................................................................................................13
Disabilities and Special Needs.............................................................................................................................13
Exhibitor Showcase..............................................................................................................................................13
First Aid and Emergencies....................................................................................................................................13
ICCE....................................................................................................................................................................13
Internet/Wireless Access......................................................................................................................................13
ISAC Booth..........................................................................................................................................................13
Message Board....................................................................................................................................................13
Mobile APP.........................................................................................................................................................13
Poster and Multimedia Presentations....................................................................................................................13
Pre-Congress Courses..........................................................................................................................................14
Practical Information for San Diego.....................................................................................................................14
Recording............................................................................................................................................................15
Scientific Tutorials................................................................................................................................................15
Speaker Ready Room...........................................................................................................................................15
Transportation......................................................................................................................................................15
City Map....................................................................................................................................................................16
San Diego Convention Center Floor Plans..................................................................................................................17
Service Location & Telephone Numbers.....................................................................................................................19
Committee Meetings...................................................................................................................................................20
Congress Overview....................................................................................................................................................21
Special Lectures..........................................................................................................................................................24
Daily Program
Saturday, 18 May.................................................................................................................................................25
Sunday, 19 May...................................................................................................................................................27
Monday, 20 May..................................................................................................................................................31
Tuesday, 21 May..................................................................................................................................................36
Wednesday, 22 May............................................................................................................................................41
Multimedia and Poster Sessions..................................................................................................................................45
Commercial Tutorials & Exhibits
Commercial Tutorials...........................................................................................................................................66
Exhibitor Showcase..............................................................................................................................................75
Exhibitor Listing...................................................................................................................................................76
Exhibit Hall Floor Plan.........................................................................................................................................77
Exhibiting Companies..........................................................................................................................................78
Oral Session Abstracts................................................................................................................................................90
Poster Session Abstracts............................................................................................................................................135
Speaker/Author Index...............................................................................................................................................235
Program-at-a-Glance..........................................................................................................................Inside Back Cover
ISAC 2013 Program and Abstracts
3
Download the CYTO 2013 mobile app
via iPhone/iPad and Android native apps or
via Blackberry, Windows Phone,
and your desktop through the mobile web.
To access the CYTO 2013 mobile app online,
visit http://cyto2013.crowdcompass.com/
ISAC 2012 – 2014 Executive Committee and Councilors
John P. Nolan
President
La Jolla Bioengineering
Institute
Andreas Radbruch
President-Elect
Deutsches RheumaForschungszentrum Berlin
Paul J. Smith
Past President
Cardiff University
Timothy Bushnell,
Secretary
University of Rochester
Paul K. Wallace,
Treasurer
RPCI Laboratory of Flow
Cytometry
Derek C. Davies
London Research
Institute, Cancer
Research UK
Grace Marie Chojnowski
Queensland Institute of
Medical Research
Andrea Cossarizza
University of Modena
and Reggio Emilia
Mario Roederer
Vaccine Research Center,
NIAID, NIH
Ryan Brinkman
British Columbia Cancer
Agency
Peter A. Lopez
New York University
ISAC 2013 Program and Abstracts
Rachel J. Errington
Cardiff University
Rui Gardner
Instituto Gulbenkian de
Ciência
Joanne Lannigan
University of Virginia
Alfonso Blanco-Fernandez
University College of
Dublin
5
ISAC Leadership and Congress Organizers
ISAC 2012 – 2014 Executive Committee
John P. Nolan, President
La Jolla Bioengineering Institute
Andreas Radbruch, President-Elect
Deutsches Rheuma-Forschungszentrum
Paul J. Smith, Past President
Cardiff University
Timothy Bushnell, Secretary
University of Rochester
Paul K. Wallace, Treasurer
Roswell Park Cancer Institute
6
Bruce Edwards
University of New Mexico
Virginia Litwin
Covance Central Laboratory Services
Zofia Maciorowski
Curie Institute
Robert Murphy
Carnegie Mellon University
John Nolan
La Jolla Bioengineering Institute
ISAC 2012 – 2014 Councilors
Jeff Price
Vala Sciences Inc. and Sanford Burnham Medical
Research Institute
Alfonso Blanco-Fernandez
University College of Dublin
Andreas Radbruch
Deutsches Rheuma - Forschungszentrum
Ryan Brinkman
British Columbia Cancer Agency
Mario Roederer
Vaccine Research Center, NIAID, NIH
Grace Marie Chojnowski
Queensland Institute of Medical Research
Paul Smith
Cardiff University
Andrea Cossarizza
University of Modena and RE
Paul Wallace
Roswell Park Cancer Institute
Derek C. Davies
London Research Institute, Cancer Research UK
CYTO 2013 Program Committee
Rachel J. Errington
Cardiff University
Mehrnoosh Abshari
National Institutes of Health
Rui Gardner
Instituto Gulbenkian de Ciência
Nima Aghaeepour
British Columbia Cancer Agency
Joanne Lannigan
University of Virginia
Donat Alpar
University of Pecs
Peter A. Lopez
New York University
Alireza Ardjmand
The University of Newcastle
Mario Roederer
Vaccine Research Center, NIAID, NIH
Kewal Asosingh
Cleveland Clinic
CYTO 2013 Organizing Committee
David Basiji
Amnis Corp
Larry Sklar, Chair
University of New Mexico
Anne Carpenter
Broad Institute of Harvard & MIT
Ryan Brinkman
British Columbia Cancer Agency
Pratip Chattopadhyay
Vaccine Research Center, NIAID, NIH
Tim Bushnell
University of Rochester
Estelle Glory-Afshar
Carnegie Mellon University
ISAC 2013 Program and Abstracts
Sung Hwan Cho
Nano Collect, Inc.
Ricardo Morilla
The Institute of Cancer Researach
Scott Cram
Los Alamos National Laboratory
Shazib Pervaize
National University of Singapore
Derek Davies
Cancer Research UK
Katarzyna Piwocka
Nencki Institute of Experiemental Biology
Albert Donnenberg
University of Pittsburgh Medical Center
Kylie Price
Malaghan Institute
Vera Donnenberg
University of Pittsburgh Medical Center
Bartek Rajwa
Purdue University
David Galbraith
University of Arizona
Paul Robinson
Purdue University
Steven Graves
University of New Mexico
Gustavo Rohde
Carnegie Mellon University
Philip Hexley
Shriners Hospitals for Children, Cincinnati
Gergely Toldi
Semmelweis University
Dayong Jin
Macquarie University
Rachael Walker
University of Cambridge
Tomas Kalina
Charles University in Prague
Robert Zucker
U.S. Environmental Protection Agency
Joanne Lannigan
University of Virginia
ISAC Executive Office
Anis Larbi
Singapore Immunology Network
Thomas Laurell
Lund University
James F. Leary
Purdue University
Silas Leavesley
University of South Alabama
Michael Lewis
University of Vermont
Er Liu
La Jolla Bioengineering Institute
Gerard Lizard
University of Bourgogne
Stephen Lockett
NCI - Frederick/SAIC
Michelle Butler, Executive Director
9650 Rockville Pike
Bethesda, MD 20814 USA
Tel. 301-634-7454, mbutler@isac-net.org
CYTO Meeting Management
Marcella Jackson, Director
Roya Jaseb, Meeting Manager
Taylor Shaw, Meeting Assistant
Janet Kearney, Exhibit Manager
Joni Friedman, Exhibit Coordinator
Josie Leftwich, Registrar
FASEB Office of Scientific Meetings &
Conferences
9650 Rockville Pike
Bethesda, MD 20814 USA
Tel. 301-634-7010; isac@faseb.org
Peter Lopez
NYU School of Medicine
Barry Moran
Trinity College Dublin
ISAC 2013 Program and Abstracts
7
Isac Special Committees & Task Forces
Task Forces & Committees 2012 - 2014 Members
Certification Advisory Committee
Robert Murphy, Chair
Mike Borowitz, Vice Chair
Derek Davies
Bruce Greig
Joanne Lannigan
Mara Neal
Paul Robinson
Ulrich Sack
Elizabeth Stone
Brent Wood
John Nolan (ex officio)
Andreas Radbruch (ex officio)
Core Managers Task Force
Derek Davies, Chair
Julie Auger
Kathy Brundage
Paula Campbell
Grace Chojnowski
Benjamin Daniel
Ian Dimmick
Elmar Endl
Marie Follow
Anna Fossum
Rui Gardner
Enid Keyser
Desiree Kunkel
Kathy Heel
Richard Konz
Zip Kruger-Gray
Charles A. Kuszynski
Joanne Lannigan
Peter Lopez
Lola Martinez
Simon Monard
Carol Oxford
Greg Perry
Joel Puchalski
Andy Riddell
Alan Saluk
Kathleen Schell
Adrian Smith
Greg Veltri
8
Rachael Walker
Maggie Wang
Kevin Weller
John Nolan (ex officio)
Andreas Radbruch (ex officio)
Council of ISAC Associated Societies
Grace Chojnowski, Chair
Gerald Lizard, Vice Chair
Mike Keeney
Oscar Segurado
Janos Szollosi
Ivan Vorobjev
Education
Zosia Maciorowski, Chair
Pratip Chattopadhyay
Awtar Krishan
Jonni S. Moore
Gustavo Rohde
John Nolan (ex officio)
Andreas Radbruch (ex officio)
Elearning Delivery Task Force
Pratip Chattopadhyay, Leader
Nima Aghaeepour
Kewal Asosingh
Alfonso Blanco
Ryan Brinkman
Lara Kreps
Mike Ormerod
Alan Saluk
Flow Cytometry Content Task Force
Jonni Moore, Leader
Grace Chojnowski
Derek Davies
Peter Lopez
Phil McCoy
Mario Roederer
Joe Trotter
Paul Wallace
Jennifer Wilshire
ISAC 2013 Program and Abstracts
Flow Cytometry Data Standards Task
Force
Ryan Brinkman, Chair
Kim RM Blenman
Chris Bray
James Cavenaugh
Francisco Chang
César Collino
Nicholas Crosbie
Chakradhar Dunna
Michael Goldberg
Mark Hubbard
Bill Hyun
Simon Lange
Ray Lefebvre
Robert Leif
Wayne Moore
David Novo
Leo Ostruszka
David Parks
Barclay Purcell
Josef Spidlen
Adam Treister
Jim Wood
Rober Zigon
Bob Zucker
John Nolan (ex officio)
Andreas Radbruch (ex officio)
FlowRepository Steering Committee
John Nolan, Interim Chair
Ryan Brinkman
Jeannine Holden
Wayne Moore
Mario Roederer
Finance
Paul Wallace, Chair
Rachel Errington
Tomas Kalina
Silas Leavesley
Peter Lopez
Bartek Rajwa
John Nolan (ex officio)
Andreas Radbruch (ex officio)
ISAC 2013 Program and Abstracts
Image Cytometry Content Task Force
Gustavo Rohde, Leader
Rachel Errington
Stephen Lockett
Anil Parwani
Bartek Rawja
Gyorgy Vereb
ISAC Scholars Review Committee
Alex Nakeff, Chair
Ryan Brinkman
Tim Bushnell
Zbigniew Darzynkiewicz
Rachel Errington
Stephen Lockett
Zosia Maciorowski
Paul Robinson
Gustavo Rohde
Mario Roederer
John Nolan (ex officio)
Andreas Radbruch (ex officio)
Live Education Delivery Task Force
Awtar Krishan, Leader
Umebreen Ahmad
Gulderen Yanikkaya Demirel
Paresh Jain
H. Krishnamurthy
Kovit Pattanapanyasat
Alan Saluk
Vivek Tanavde
Bill Telford
Qianjun Zhang
Membership Services
Tim Bushnell, Chair
Sung Hwan Cho
Grace Chojnowski
Peter Lopez
Bruno Paredes
Rachael Walker
Nicole White
John Nolan (ex officio)
Andreas Radbruch (ex officio)
9
Robert Hooke Distinguished Lecture &
Awards Committee
Paul Smith, Chair
Robert Murphy
Paul Robinson
Ian T. Young
Alan Waggoner
John Nolan (ex officio)
Andreas Radbruch (ex officio)
10
Scientific Communications
Andrea Cossarizza, Chair
Nima Aghaeepour
Zbigniew Darzynkiewicz
Elmar Endl
Enrico Lugli
Jose Enrique O’Connor
Mario Roederer
John Nolan (ex officio)
Andreas Radbruch (ex officio)
ISAC 2013 Program and Abstracts
General Information
All Congress activities will be held at the San
Diego Convention Center located at 111 W.
Harbor Drive, San Diego, CA 92101, unless
noted otherwise. CYTO Registration, Exhibits/
Posters, and the First Aid office are located on
the Ground Level of the Convention Center.
Sessions, Committee Meetings, Speaker Ready
Room and the Meeting Management Office are
located on the Upper Level of the Convention
Center.
Participation in CYTO 2013 is limited to
registered delegates. Full Congress registration
includes admission to all sessions, exhibits,
coffee breaks, happy hours, Opening Reception,
and exchange coupon for the Closing Reception
at the Stingaree. There is an additional fee for
the Pre-Congress Courses, CYTO Innovation, and
the Scientific Tutorials on Saturday, 18 May at the
San Diego Convention Center.
Congress Registration – Lobby
Registration for CYTO 2013 will be open during
the following days and hours:
Saturday, 18 May............................. 1100 – 1800
Sunday, 19 May.................................. 700 – 1900
Monday, 20 May................................ 730 – 1830
Tuesday, 21 May................................. 730 – 1830
Wednesday, 22 May........................... 730 – 1700
Refund Policy: Refunds will not be issued after
15 April, 2013
Exhibitor Registration – Lobby
Exhibitor registration is for booth personnel and
provides admittance into the Exhibit Hall only.
Exhibitor registration will be open during the
following days and hours:
Saturday, 18 May............................. 1100 – 1800
Sunday, 19 May.................................. 700 – 1900
Monday, 20 May................................ 730 – 1830
Tuesday, 21 May............................... 1030 – 1830
Wednesday, 22 May......................... 1030 – 1700
Badges/Access Control
Participation in CYTO 2013 is limited to
registered attendees. The official badge is
required for admittance to all sessions, social
ISAC 2013 Program and Abstracts
activities and the Exhibit Hall. A fee may be
charged to reissue lost or misplaced badges.
Please do not place a business card into the
badge holder as identification. If there is an
error on a badge, please have it corrected at the
registration desk.
Business Services – Lobby
A FedEx Office as well as on-site full-service
business center providing fax, copying, express
mail, packaging, and printingis is located on the
ground level of the Convention Center.
Cell Phones
Cell phone use is prohibited. Please turn off all
cell phones and pagers prior to entering a session
room. If you must leave a session early, please
use the rear entrance and exit quietly.
Child Care
Please check with your hotel’s front desk or
concierge service for names of babysitters who
can provide care in your hotel room. Parents
and guardians are required to perform their
own reference checks and arrange child care
independently. ISAC is not responsible for child
care or the quality of care provided.
Closing Reception at the
Stingaree
The Closing Reception will take place at the
Stingaree, a night club located in the heart of
the Gaslamp District (only a few blocks from
the Convention Center). Enjoy a night of fun,
music, dancing, and refreshments with friends
and colleagues. the Stingaree also has a rooftop
lounge providing a relaxing atmosphere. The
event will take place on Wednesday, 22 May,
2013, from 1900 until 2300.
Full registration includes an exchange coupon
for the Closing Reception at the Stingaree. The
Closing Reception coupon must be exchanged
for an actual ticket on-site at the registration desk
beginning Saturday, 18 May, 2013 until Monday,
20 May, 2013. Coupons will be exchanged on
a first come first served basis until maximum
capacity is reached. A ticket is required for
admittance. Be sure to exchange your coupon
early so you don’t miss out on the fun!
11
Commercial Exhibits – Exhibit
Hall GH
Visit the commercial exhibits featuring displays
by leading suppliers and vendors. A complete
directory of exhibiting companies as well as
the Exhibit Hall floor plan is located under the
Exhibits tab of this program.
Exhibits will be open during the following days
and hours:
Monday, 20 May.............................. 1130 – 1900
Tuesday, 21 May............................... 1130 – 1900
Wednesday, 22 May......................... 1130 – 1630
Note: Children under the age of 16 are not
permitted in the Exhibit Hall without parent or
guardian supervision.
Commercial Tutorials
21 commercial tutorial sessions are offered from
1245 – 1345 on Sunday, 19 May, and 1215 –
1315 on Tuesday, 21 May, and Wednesday, 22
May. Please refer to the Commercial Tutorial tab
of this program for a complete list of offerings.
Companion/Guest Registration
Registered attendees of the CYTO 2013 Congress
may sign up a spouse/guest as a Companion
for $150 USD. Companion registration allows
entrance to the Opening Reception, Happy Hour
and the Closing Reception only. The Opening
Reception is scheduled to be held Sunday, 20
May, 2013, Happy Hours are on Monday, 20
May, 2013 and Tuesday, 21 May, 2013, and the
Closing Reception is on Wednesday, 22 May,
2013. Companion registrants are not permitted
in the session rooms or the Exhibit Hall at any
other time.
Concierge – Lobby
The Restaurant and Concierge Desk is located
in Lobby E of the Convention Center. It will
be open Saturday, 19 May, 2013, through
Wednesday, 22 May, 2013, from 900 – 1800.
A variety of services will be offered, including
restaurant reservations, city information, map,
brochures, directions, and the purchase of
attraction tickets. Visit the Congress website to
print coupons, or present your Congress badge at
select locations to receive discounts.
For baseball fans interested in seeing a Padres
game, Petco Park is a short walk from the
12
Convention Center. For more information visit
sandiego.padres.mlb.com.
Cyber Café – Exhibit Hall GH
For your convenience, ISAC has set up several
computers with free Internet access in the
Cyber Café. Attendees may use computers to
browse the Internet and/or to check email. In
consideration of others, please limit your use to
15 minutes. The Cyber Café will be open during
the Commercial Exhibit hours for attendee use
beginning Monday, 20 May, 2013.
CMLE
This Continuing Medical Laboratory Education
activity is recognized by the American Society for
Clinical Pathology as meeting the criteria for 23.5
hours of CMLE credit. ASCP CMLE credit hours
are acceptable to meet the continuing education
requirement for the ASCP Board of Registry
Certification Maintenance Program.
If you’re interested in earning CMLE credits,
please follow these steps:
1. Be sure to add your name to the sign in sheet
which will be located at the back of each
session room (sign in sheets will be present
in every session that is eligible to earn CMLE
credits);
2. Complete an evaluation form for each session
you attend and leave it in the box at the back
of the room, immediately following that
session;
3. Use the CMLE form available at registration
to track the sessions you attend. Please
follow the instructions on the form to finalize
the process. You will need to drop off your
completed form at the Meeting Management
Office (Room 28B) before leaving the
Congress.
CMLE certificates will be issues by the ISAC
Executive Office upon request via email to
isac@isac-net.org.
CYTO Innovation
ISAC presents CYTO Innovation 2013, a forum
for discussing the challenges and opportunities
for the development and commercialization of
cell analysis technologies (see page 25 for full
description). The registration fee for this session is
$75 USD.
ISAC 2013 Program and Abstracts
Dining Options
Internet/Wireless Access
Disabilities and Special Needs
Attendees may purchase “Instant Internet” for 24
hours at a rate of $12.95 per device. Purchasing
the “Instant Internet” will provide you with
access everywhere inside the Convention Center,
including session rooms, with the exception of
the Exhibit Hall.
Starbucks and Mrs. Fields will be open
throughout the week at the convention center.
A light lunch will be provided to congress
registrants in Exhibit Hall GH on Monday, 20
May at 1200. The concession stand in Exhibit
Hall GH will be open for the purchase of lunch
or snacks on Tuesday, 21 May and Wednesday
22, May from 1100 – 1400.
If you have a disability or special need that
may have an impact on your participation
in the meeting, please contact the Meeting
Management Office. ISAC cannot ensure the
availability of appropriate accommodations
without prior notification of need.
Exhibitor Showcase – Exhibit Hall GH
The exhibitor Showcase will take place on
Monday, 20 May, 2013, from 1200 – 1330. The
showcase will include presentations by several
exhibiting companies.
Complimentary WiFi Service is available in the
ground level lobby areas of the Convention
Center.
ISAC Booth – Exhibit Hall GH
Please visit the ISAC booth to learn more about
society activities and membership. The ISAC
Executive Director will be available to answer
your questions, as will the editor-in-chief of
Cytomerty Part A during the Exhibitor Showcase
on Monday, 20 May.
Message/Announcement Board
Messages can be posted on the board located in
the registration area. Participants should check
each day for messages.
First Aid and Emergencies –
Lobby
Mobile APP
Saturday, 18 May............................... 830 – 1800
Sunday, 19 May.................................. 830 – 1930
Monday, 20 May................................ 830 – 1930
Tuesday, 21 May................................. 830 – 1930
Wednesday, 22 May........................... 830 – 1800
Poster and Multimedia Sessions –
Exhibit Hall GH
The First Aid Office is located in Show Office
G on the ground level near the registration area
and will be open during the following days and
hours:
ICCE
ISAC is an approved provider of continuing
education for the ICCE certification. Any one
hour of ISAC educational programming is worth
one credit. Certified cytometrists will be asked to
report their credit hours earned when they renew
their certification. CYTO 2013 is worth a total of
23.5 credit hours.
For more information on the International
Cytometry Certification Examination and how to
become a certified cytometrist, visit
http://cytometrycertification.org.
ISAC 2013 Program and Abstracts
NEW!
Download the CYTO 2013 mobile app via
iPhone/iPad and Android native apps or via
Blackberry, Windows Phone, and your desktop
through the mobile web. To access the CYTO
2013 mobile app online, visit
http://cyto2013.crowdcompass.com/
Over 250 poster presentations will be on display
in the Exhibit Hall. Please refer to the Poster
Board Map in the Congress Addendum for the
assigned location of presentations. Please refer
to the schedule below for viewing hours.
Monday, 20 May
700 – 1100
Authors must set up posters
on assigned board
1130 – 1900
Poster Viewing
1715 – 1845
Poster Session 1
(Authors of odd numbered boards must be at
their poster to answer questions and discuss their
presentation.)
13
Tuesday, 21 May
700 – 1900
Poster Viewing
1715 – 1845
Poster Session 2
(Authors of even numbered boards must be at
their poster to answer questions and discuss their
presentation.)
Wednesday, 22 May
700 – 1600
Poster Viewing
1500 – 1600
Poster Session 3
(All authors present)
Posters must be removed by 1630 on Wednesday,
27 May
Outstanding Poster Awards
All poster presenters who are students or
postdoctoral researchers (who have received
their doctorate within the last five years) are
eligible. The posters will be judged at the time
they are scheduled to be presented by the author.
Those posters not attended at their scheduled
time will not be considered. The names of
authors selected for this award will be posted
on the Message/Announcement Board in the
Registration Area on Wednesday, 22 May. Poster
winners will be presented a prize and recognized
at the Awards Ceremony from 1645 – 1730 on
Wednesday, 22 May.
Pre-Congress Courses
The Introduction to Image-Based Cytometry,
Introduction to Flow-Based Cytometry, and the
Advanced Data Analysis Course will be held
on Saturday, May 18. For full description of the
courses, please visit http://cytoconference.org/
cyto/2013/Pre-Congress-Courses.aspx. There is a
separate registration fee of $150 USD for each of
the Intro Courses and a fee of $300 USD for the
Advanced Course.
Practical Information for San Diego
Language
The official language of the Congress is English.
Translation services will not be provided.
Banking and Foreign Exchange
The official currency in the United States is US
Dollars. The US Dollar ($) is divided into 100
cents; notes are in denominations of $100, 50,
20, 10, 5, and 1. Banking hours in the United
States are generally Monday through Friday,
900 – 1700. A few banks are open on Saturdays
14
as well. You can withdraw money from ATM
bank machines using your credit card and make
purchases in stores and restaurants. Signs display
accepted credit cards or you can ask the retailer.
Check with your local financial institution to
verify if you are able to use your home bank card
or credit card prior to travel.
ATM bank machine service is available in the
lobby of The San Diego Convention Center.
There are several banking institutions within
walking distance of The Convention Center as
well. Travelex offers a currency exchange kiosk in
Terminal 1 at the San Diego International Airport.
They also offer a mobile currency exchange
card in Terminal 2 East. Services include travel
insurance, traveler’s checks, money transfer,
notary, facsimile, copy, and phone cards.
International Currency Converters:
www.GoCurrency.com
www.Oanda.com
Climate
San Diego enjoys beautiful weather year round.
San Diego’s average high temperature is 70°F
and average low temperature is 59°F with 300
days of sunshine year round.
Time Zone
During CYTO 2013 San Diego will be on Pacific
Savings Time.
Electricity
Voltage in the United States is 120 voltage (60
HZ). Outlet sockets use either a Type A plug
which is a class II ungrounded plug with two
flat parallel prongs, or a Type B plug which is a
class I plug with two flat parallel prongs and a
grounding pin.
Dialing Codes
The International access code for the United
States is 1. The area code in San Diego is 619.
While in the United States, you can call the
directory information service number 411 for
assistance in finding a phone number. Calls to
area codes 800, 877, and 866 are toll-free in the
United States. To make an international call, dial
011 followed by the country code, city code, and
telephone number.
ISAC 2013 Program and Abstracts
Recording
Recording any presentation or session (oral or
poster) by any means (photographing, audio
taping, videotaping) is prohibited, except by an
ISAC authorized agent for official purposes or by
first authors who want to photograph their own
poster presentations. All other photography and/
or videotaping is prohibited in the Exhibit Hall.
Scientific Tutorials
Saturday,18 May, is dedicated to the scientific
tutorial program. Fifteen ninety-minute tutorials
will be offered. A registration fee of $80 USD
per tutorial (or $145 USD for two tutorials; $200
USD for three tutorials) is required to participate
in the program. You may purchase tutorial tickets
at the registration desk.
Speaker Ready Room – Room 28A
All speakers are requested to go to the
Speaker Ready Room to review and check the
compatibility of their presentation at least 4
hours prior to their session. Speakers must
arrive in the session room 30 minutes prior to
the scheduled start of their session to allow the
operator time to load their presentation onto
the computer. The operator will be seated at the
table next to the stage. ISAC is not responsible
for slides, laptops, or cables left in session rooms.
Speakers are not required to bring a laptop.
All session rooms will be equipped with a data
projector and PC. Please bring your presentation
on a Windows reusable USB flash drive or CDROM. We recommend that you bring a backup
presentation format.
Shuttles & Taxi Service
Shuttle services are a convenient way to navigate
the city and are ideal for airport pickup or
traveling to and from the Convention Center.
Some shuttle services offer site-seeing tours of
San Diego.
Taxicabs are located at the airport, most hotels,
attractions, and shopping centers. Base and fare
rates will be displayed on the meter and will
include a flag drop charge plus a per-mile and/or
a per-hour charge.
Approximate Taxi Cab Rate 2013:
••Getting in: $2.80
••Each mile: $3.00
••Per hour waiting time: $24.00
••From airport:
$1.50
Coronado Ferry
The Ferry runs regularly across San Diego Bay,
providing a gorgeous view of the downtown
skyline and Coronado Bridge. A Ferry Stop is
located right behind the Convention Center at
the San Diego Harbor Excursion Kiosk.
Water Taxi
San Diego Water Taxi offers on-call transportation
services in San Diego Bay. Travel serenely
from your hotel to local shopping centers and
restaurants.
For more information about transportation or
tour options, please visit the concierge desk in
the lobby at the San Diego Convention Center or
your hotel’s concierge service.
Transportation
San Diego Trolley
If you’re staying in the downtown San Diego
area, the iconic bright red trolley will take you
around the city. The San Diego Trolley provides
transportation to key downtown locations
including The Convention Center. Trolley fares
and passes are available at sdmts.com.
Pedicabs & Carriages
These are two popular forms of transportation
along downtown’s waterfront and in the Gaslamp
Quarter. There are also tours available, a unique
way to explore the city.
ISAC 2013 Program and Abstracts
15
City Map
CYTO 2013
May 19, 2013 to May 22, 2013
La
Airport
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rap
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5
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Trolley Line
Train
Depot
9th Ave.
8th Ave.
7th Ave.
6th Ave.
5th Ave.
4th Ave.
3rd Ave.
2nd
C St.
Broadway
Broadway
E St.
Pacific Hwy.
Harbor Drive
12th Ave.
Civic
Center
11th Ave.
B St.
3
Ferry/
Tour Boats
1st
A St.
10th Ave.
4
Front St.
Ash
Union St.
San Diego Bay
State St.
Kettner Blvd.
Beech
Columbia St.
Little Italy
Cedar
India St.
Embarcadero
Date St.
F St.
E St.
2
Horton
Plaza
F St.
G St.
G St.
Gaslamp
Quarter
Market Street
Island Ave.
Tro
ll
ey
Seaport
Village
Market Street
5
Lin
e
J St.
PETCO
Park
K St.
Marina
Embarcadero
Park South
vd
.
1
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VISITSANDIEGO.COM
619.525.5000
Downtown San Diego
Downtown San Diego
# ,FZ
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ive
0.12
2#Key
Ramada Inn - Gaslamp
3 W San Diego
1 Omni San Diego Hotel – 675 L St., San Diego, CA 92101
4 Wyndham San Diego Bayside
0.58 (in miles) from SDCC
Distance
0.80
(619-231-6664) 52 Stingaree
$:50$MPTJOH3FDFQUJPO
Ramada Inn
- Gaslamp – 830 6th Ave., San Diego, CA 92101
1.10
0.30
(619-231-8307) (619-398-3100) 5 Stingaree – the venue of CYTO’s Closing Reception – 454 6th Ave., San Diego, CA 92101
0.12
0.58
0.80
4 Wyndham San Diego Bayside (formerly Holiday Inn) – 1355 North Harbor Dr., San Diego, CA 92101
16
or
Distance (in miles) from SDCC
1 Omni San Diego Hotel
3 W San Diego – 421 W B St., San Diego, CA 92101
rb
(619-232-3861) (619-544-9500)
1.10
0.30
ISAC 2013 Program and Abstracts
ISAC 2013 Program and Abstracts
17
ESCALATOR TO
CYTO SESSIONS
UPPER LEVEL
ELEVATOR TO
UPPER LEVEL
WIFI
HOT SPOT
CYTO Registration
LOBBY
CYTO
Exhibits & Posters
Concessions
FIRST AID
VISIT SAN DIEGO CONCIERGE
BOX OFFICE E
DESK
San Diego Bay
Ground Level Exhibition Halls GH
San Diego Convention Center
GROUND LEVEL EXHIBIT HALLS GH
ESCALATOR TO
CYTO SESSIONS
UPPER LEVEL
ESCALATORS TO
CYTO EXHIBITS/POSTER
GROUND LEVEL
SPEAKER READY ROOM
MEETING MANAGEMENT
OFFICE
ELEVATOR TO
GROUND LEVEL
San Diego Bay
Upper Level Session Halls
San Diego Convention Center
UPPER LEVEL SESSION ROOMS
18
ISAC 2013 Program and Abstracts
ESCALATORS TO
CYTO EXHIBITS/POSTER
GROUND LEVEL
Service Location & Telephone Numbers
Meeting Management Office – Room 28B, Upper Level – (Tel. 619-525-6241)
Saturday, 18 May 1200 – 1700
Sunday, 19 May
730 – 1700
Monday, 20 May
730 – 1700
Tuesday, 21 May
730 – 1700
Wednesday, 22 May
730 – 1700
Congress Registration & Information – Ground Level, Lobby – (Tel. 619-525-6244)
Saturday, 18 May
1100 – 1800
Sunday, 19 May
700 – 1900
Monday, 20 May
730 – 1830
Tuesday, 21 May
730 – 1830
Wednesday, 22 May
730 – 1700
Exhibitor Registration & Information – Ground Level, Lobby – (Tel. 619-525-6245)
Saturday, 18 May
1100 – 1800
Sunday, 19 May
700 – 1900
Monday, 20 May
730 – 1830
Tuesday, 21 May
1030 – 1830
Wednesday, 22 May 1030 – 1700
Speaker Ready Room – Room 28A, Upper Level – (Tel. 619-525-6242)
Saturday, 18 May
1200 – 1700
Sunday, 19 May
Monday, 20 May
Tuesday, 21 May
Wednesday, 22 May
730 – 1700
730 – 1700
730 – 1700
730 – 1700
First Aid Room – Show Office G, Ground Level, Lobby – (Tel. 619-525-6243)
Saturday, 18 May
830 – 1800
Sunday, 19 May
830 – 1930
Monday, 20 May
830 – 1900
Tuesday, 21 May
830 – 1900
Wednesday, 22 May
830 – 1800
ISAC 2013 Program and Abstracts
19
Committee Meetings during CYTO 2013
All meetings are by invitation only unless specified otherwise.
Sunday, 19 May, 2013
ISAC Scholars/Student Travel Awards Breakfast
730 – 830
Room 28D
Cytometry Part A Editorial Board Meeting
1230 – 1400
Room 28E
Associated Societies Luncheon
1245 – 1345
Room 28D
Monday, 20 May, 2013
Finance Committee
730 – 830
Room 28C
Certification Advisory Committee
730 – 830
Room 28E
Membership Services Committee
730 – 830
Room 28D
Tuesday, 21 May, 2013
Current Protocols Board Meeting
1200 – 1330
Room 28E
Wednesday, 22 May, 2013
Flow Cytometry Data Standards Task Force
730 – 830
Room 28C
CYTO 2014 Planning Meeting
730 – 830
Room 28E
ISAC Scholar Review Committee
730 – 830
Room 28D
ISAC Scholars Luncheon
1200 – 1300
20
Room 28D
ISAC 2013 Program and Abstracts
Congress
Overview
Congress Overview
Special
Lectures
All Congress activities (with the exception of the Closing Reception at the Stingaree) will be held at the
San Diego Convention Center. Exhibits/Posters, Cyto Registration and First Aid offices are located on
the Ground Level. All other rooms are located on the Upper Level of the Convention Center.
Saturday, 18 May 2013
Introduction to Image-Based Cytometry Course
Room 29C
900 – 1215
Introduction to Flow-Based Cytometry Course
Room 29D
930 – 1600
Advanced Data Analysis Course
1100 – 1800
Scientific Registration OpenLobby
1100 – 1800
Exhibitor Registration OpenLobby
1230 – 1400
Scientific Tutorials 1 – 5
1300 – 1700
CYTO Innovation
1415 – 1545
Scientific Tutorials 6 – 10
Rooms 30 – 33
1600 – 1730
Scientific Tutorials 11 – 15
Rooms 30 – 33
Saturday,
18 May
900 – 1215
Room 29AB
Sunday,
19 May
Rooms 30 – 33
Room 28E
Monday,
20 May
700 – 1900
Scientific Registration OpenLobby
700 – 1900
Exhibitor Registration OpenLobby
900 – 1030
Opening Remarks:
Wallace H. Coulter Centennial Lecture
1030 – 1100
Coffee Break
1100 – 1230
Parallel Sessions
1245 – 1345
Exceptional Student Award Presentations
1245 – 1345
Commercial Tutorials
1400 – 1530
State-of-the-Art Lectures
1530 – 1545
Coffee Break
1545 – 1715
Workshops
1730 – 1830
Robert Hooke Lecture
1830 – 1930
Opening Reception
Tuesday,
21 May
Sunday, 19 May 2013
Wednesday,
22 May
Ballroom 20D
Foyer/Terrace, Upper Level
Rooms 30 – 33
Poster
Session
Room 28C
Rooms 29 – 33
Ballroom 20D
Commercial
Tutorials &
Exhibits
Foyer/Terrace, Upper Level
Rooms 30 – 33
Ballroom 20D
Foyer/Terrace, Upper Level
Oral Session
Abstracts
Monday, 20 May 2013
Scientific Registration OpenLobby
730 – 1830
Exhibitor Registration OpenLobby
830 – 1000
Frontiers Session 1
1000 – 1015
Coffee Break
1015 – 1145
Parallel Sessions
1130 – 1900
Commercial Exhibits & Poster Viewing
Exhibit Hall GH
1200 – 1330
Exhibitor Showcase
Exhibit Hall GH
Ballroom 20D
Foyer/Terrace, Upper Level
Rooms 30 – 33
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Poster Session
Abstracts
730 – 1830
21
Congress
Overview
Special
Lectures
Saturday,
18 May
1200– 1330
Lunch for CYTO Attendees Exhibit Hall GH
1330 – 1500
Plenary Session 1
1500 – 1545
Coffee Break
1545 – 1715
Workshops
1715 – 1845
Poster Session 1
Exhibit Hall GH
1800 – 1900
Happy Hour
Exhibit Hall GH
1900 – 2200
Core Managers Forum
Ballroom 20D
Exhibit Hall GH
Rooms 30 – 33
Room 29
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Tuesday, 21 May 2013
700 – 1900
Poster Viewing
Exhibit Hall GH
730 – 1830
Scientific Registration OpenLobby
800 – 1000
Frontiers Session 2
1000 – 1015
Coffee Break
1015 – 1145
Parallel Sessions
1030 – 1830
Exhibitor Registration OpenLobby
1215 – 1315
Presidential Award for Excellence Presentations
1130 – 1900
Commercial Exhibits
Exhibit Hall GH
1215 – 1315
Commercial Tutorials
Rooms 29 – 33
1330 – 1500
Plenary Session 2
1500 – 1545
Coffee Break
1545 – 1715
Workshops
1715 – 1845
Poster Session 2
Exhibit Hall GH
1800 – 1900
Happy Hour
Exhibit Hall GH
Ballroom 20D
Foyer/Terrace, Upper Level
Rooms 30 – 33
Room 28C
Ballroom 20D
Exhibit Hall GH
Rooms 30 – 33
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday, 22 May 2013
700 – 1600
Poster Viewing
Exhibit Hall GH
730 – 1700
Scientific Registration OpenLobby
830 – 1000
Frontiers Session 3
1000 – 1015
Coffee Break
1015 – 1145
Parallel Sessions
1030 – 1700
Exhibitor Registration OpenLobby
1130 – 1630
Commercial Exhibits
Exhibit Hall GH
1215 – 1315
Commercial Tutorials
Rooms 29 – 33
1330 – 1500
Plenary Session 3
1500 – 1545
Coffee Break
Exhibit Hall GH
1500 – 1600
Poster Session 3
Exhibit Hall GH
1615 – 1645
ISAC Business Meeting Ballroom 20D
1645 – 1730
Awards Ceremony Ballroom 20D
Ballroom 20D
Foyer/Terrace, Upper Level
Rooms 30 – 33
Ballroom 20D
1900 – 2300
Closing Reception Stingaree
(see page 11 for details)
22
ISAC 2013 Program and Abstracts
Congress
Overview
Special
Lectures
Visit the Exhibits & Posters
Monday, 20 May
1130 – 1900 Poster Viewing and Commercial Exhibits
1200 – 1330 Exhibitor Showcase
1200 – 1330
Lunch Provided
1500 – 1545 Coffee Break
1715 – 1845 Poster Session 1 (Authors of odd numbered posters present)
1800 – 1900 Happy Hour
Sunday,
19 May
Authors Must Place Posters on Boards
Saturday,
18 May
700 – 1100 Poster Viewing
1100 – 1400
Concession open for purchase of lunch/snacks
1130 – 1900 Commercial Exhibits
1500 – 1545 Coffee Break
1715 – 1845 Poster Session 2 (Authors of even numbered posters present)
1800 – 1900 Happy Hour
Tuesday,
21 May
700 – 1900 Monday,
20 May
Tuesday, 21 May
Wednesday,
22 May
Wednesday, 22 May
Poster Viewing
1100 – 1400
Concession open for purchase of lunch/snacks
1130 – 1630 Commercial Exhibits
1500 – 1545 Coffee Break
1500 – 1600 Poster Session 3 (all authors present)
Poster
Session
700 – 1600
Commercial
Tutorials &
Exhibits
Posters must be removed by 1630 on Wednesday, 22 May.
Oral Session
Abstracts
Don’t forget to exchange your coupon for a ticket to the
Closing Reception at the Stingaree!
Poster Session
Abstracts
(ticket required for admittance; see page 11 for details)
Speaker/Author
Index
ISAC 2013 Program and Abstracts
23
Congress
Overview
Special Lectures
Special
Lectures
Wallace H. Coulter Centennial Lecture
Sunday, 19 May, 900 – 1030
Ballroom 20D
Sunday,
19 May
Saturday,
18 May
Wallace Coulter was a pioneering figure in the field of cytometry. His invention of the
Coulter principle for electronic particle sizing revolutionized the counting and sizing of
blood cells, and stimulated the design of the first flow cytometers and cell sorters. The
Coulter Corporation sold some of the first flow cytometers, and employed many pioneers
in the field of cytometry. Wallace Coulter’s legacy lives on through the Wallace H. Coulter
Foundation, which actively supports the development and translational application of new
biomedical technologies. The Foundation is also a valued partner of ISAC and supporter
on issues of mutual interest, including education, data sharing, and certification. ISAC
is pleased to recognize this pioneering inventor, entrepreneur, and philanthropist on the
100th anniversary of his birth with the Wallace H. Coulter Centennial Lecture to open CYTO 2013. For more
information on Wallace H. Coulter and the Wallace H. Coulter Foundation, visit www.whcf.org.
Monday,
20 May
Spatial Systems Biology, Joe Gray
Wednesday,
22 May
Tuesday,
21 May
The Wallace H. Coulter Centennial Lecture will be presented by Professor Joe Gray, of the
Knight Cancer Center and the Oregon Health and Sciences University. Dr. Gray, a pastpresident of ISAC and Fulwyler Award winner, has been a pioneer in the development and
translational application of cytometry technologies. He currently leads the OHSU Center
for Spatial Systems Biomedicine, which is dedicated to the use of advanced measurement
technologies, especially omic and multiscale imaging, to improve medical diagnosis and
treatment.
Marylou Ingram Lecture
Wednesday, 22 May, 1430 – 1500
Ballroom 20D
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Marylou Ingram’s contributions to the field of cytometry span nearly 70 years. Dr. Ingram’s
distinguished career in academic medicine and research, includes faculty service at the
University of Rochester, Caltech and the University of Miami, at Los Alamos National
Laboratory, as a consultant to the National Cancer Institute, the FDA, Brookhaven National
Laboratory, NASA and other organizations. She was a pioneer in automated cell analysis,
played a key role in developing automated cell analysis systems, and was founding director
of the Institute for Cell Analysis at the University of Miami. Dr. Ingram spent two of her
sabbaticals working directly with Wallace Coulter in the very early days of automated
cell analysis, and she regularly interacted with Wallace Coulter as he developed new
technologies for analysis. Since 1982, she has been at the Huntington Medical Research Institute in Pasadena,
California, where she is a Senior Research Scientist and heads the Tissue Engineering & In Vitro Systems program,
and leads a research program on tumor spheroids with the aim of creating better models for studying tumor
growth and drug responsiveness.
Speaker/Author
Index
Poster Session
Abstracts
The Marylou Ingram Lecture will be presented by Dr. Vera Donnenberg of the McGowan
Institute of Regenerative Medicine at the University of Pittsburg. Dr. Donnenberg is an
Associate Professor of Cardiothoracic Surgery in the School of Medicine at the University of
Pittburgh, where her lab focuses on the application of flow cytometry to cancer biology and
regenerative medicine. Dr. Donnenberg serves on the Editorial Board of Cytometry Part A,
and is a member of the International Society for Advancement of Cytometry, the American
College of Clinical Pharmacology, and American Association for Cancer Research.
24
ISAC 2013 Program and Abstracts
Congress
Overview
Daily Program
SATURDAY, 18 MAY 2013
Special
Lectures
Pre-Congress Courses
(separate registration required)
Saturday,
18 May
900 – 1215
Room 29C
Introduction Image-Based Cytometry
900 – 1215
Room 29D
Introduction to Flow-Based Cytometry
Sunday,
19 May
930 – 1600
Room 29AB
Advanced Data Analysis Course
Monday,
20 May
Scientific Tutorials
(separate registration required)
1230 – 1400
Tutorials 1 – 5
1230
1
Tuesday,
21 May
Room 33AB
Apoptosis, Autophagy and DNA Damage W. Telford and Z. Darzynkiewicz.
NCI, NIH and New York Med. Col.
1230
2
Wednesday,
22 May
Room 32AB
Cytometer Performance Characterization and Standardization R. Hoffman.
BD Biosciences, San Jose, CA.
Room 31ABC
1230
3
Poster
Session
Approaches in Image-Based High Content Screening S. Heynen-Genel and
J.H. Price. Sanford-Burnham Med. Res. Inst., La Jolla and Vala Sciences Inc., San
Diego.
Room 30CDE
4
Biosafety: Risk Assessment and SOP Development K. Holmes. NIAID, NIH.
Commercial
Tutorials &
Exhibits
1230
Room 30AB
1230
5
High Throughput and High Content Screening for Flow Cytometry J.P. Robinson,
P. Narayanan and R. Jepras. Purdue Univ., Amgen, Seattle and GSK, Middlesex, U.K.
Oral Session
Abstracts
CYTO Innovation
(separate registration required)
1300 – 1700
Poster Session
Abstracts
Room 28E
ISAC 2013 Program and Abstracts
Speaker/Author
Index
ISAC presents CYTO lnnovation 2013, a forum for discussing the challenges and opportunities for
the development and commercialization of cell analysis technologies. The CYTO Innovation Program
will include short talks offering perspectives on current trends in the industry, a panel discussion,
and a showcase of short presentations by innovators and entrepreneurs on new technologies and
applications for cell analysis. If you are involved in the development, evaluation, or commercialization
of new cell analysis technologies, plan to join us for this stimulating program.
25
Congress
Overview
Scientific Tutorials
(separate registration required)
1415 – 1545
Special
Lectures
Tutorials 6 – 10
Room 33AB
6
Saturday,
18 May
1415
Proliferation Tutorial: Cell Cycle Progression and Division – Unraveled by Flow
Cytometry K. Price, K.A. Muirhead and P.K. Wallace. Malaghan Inst. of Med. Res.,
Wellington, NZ, SciGro Inc./MidWest Ofc., Madison, WI and Roswell Park Cancer
Inst., Buffalo.
Room 32AB
Sunday,
19 May
1415
7
Cell Sorting: Fundamentals, Applications and Troubleshooting G. Osborne. Univ. of
Queensland, Australia.
Room 31ABC
Monday,
20 May
1415
8
Image Quantification and Analysis Using CellProfiler D. Logan. Broad Inst. of
Harvard and MIT.
Room 30CDE
1415
9
Analysis of Receptor Dynamics Using Flow Cytometry A. Chigaev and Y. Wu. Univ.
of New Mexico.
Tuesday,
21 May
Room 30AB
Wednesday,
22 May
1415
10
Evaluation and Purchase of an Analytical Flow Cytometer: Some of the Numerous
Factors to Consider R. Zucker and N. Fisher. U.S. EPA, Durham, NC and Univ. of
North Carolina at Chapel Hill.
Scientific Tutorials
(separate registration required)
1600 – 1730
Poster
Session
Tutorials 11 – 15
Room 33AB
Commercial
Tutorials &
Exhibits
1600
11
Seeing a More Colorful World: A Guide to Polychromatic Flow Cytometry
P. Chattopadhyay. NIAID, NIH.
Room 32AB
Oral Session
Abstracts
1600
12
Analysis and Sorting of Rare Cell Populations J.P. McCoy. NHLBI and Ctr. for Human
Immunol., NIH.
Room 31ABC
1600
13
Quantitative FRET Microscopy G. Vereb and J. Szollosi. Univ. of Debrecen, Hungary.
Room 30CDE
Poster Session
Abstracts
1600
14
Growing a Cytometry Core Facility: Adding Value with Hardware and Education
D. Davies and A. Blanco. Cancer Res. UK, London and University Col. Dublin.
Room 30AB
Speaker/Author
Index
1600
26
15
Cell Organizer: Building Models of Cell Structure from Microscope Images and
Using Them for High-Content Screening and Cell Simulations R. Murphy, G. Johnson
and D. Sullivan. Carnegie Mellon Univ.
ISAC 2013 Program and Abstracts
Congress
Overview
Sunday, 19 May 2013
Wallace H. Coulter Centennial Lecture
Special
Lectures
900 – 1030
Ballroom 20D
Chair: John Nolan
Saturday,
18 May
Spatial Systems Biology Joe Gray. Knight Cancer Center and Oregon Health & Sciences University.
Coffee Break
1030 – 1100
Foyer/Terrace, Upper Level
Sunday,
19 May
Concurrent Parallel Sessions
1100 – 1230
Monday,
20 May
Parallel 1: Flow Cytometry Instrumentation: Spectroscopy
Room 33AB
Chair: Diether Rechtenwald
Cochair: Michael Zorden
1120
21
Simultaneous Analysis of Multiple Fluorescent Proteins and Fluorochromes by a
Novel Spectral Flow Cytometer M. Tomura, K. Futamura, N. Nitta M. Kakuta and
M. Furuki. Kyoto Univ. Grad. Sch. of Med. and SONY Corp., Tokyo.
1140
22
Quantitative Real Time Single Cell Spectroscopy in Flow J. Nolan, D. Condello and
E. Duggan. La Jolla Bioengineering Inst.
1200
23
Fluorescence Lifetime-Dependent Flow Cytometry in the Time-Domain W. Li,
G. Vacca, M. Naivar and J. Houston. New Mexico State Univ., Kinetic River Corp.,
San Jose, CA and DarklingX LLC, Los Alamos.
Poster
Session
Expanding the Capabilities of Mass Cytometry S. Tanner, A. Loboda, D. Bandura,
V. Baranov and O. Ornatsky. DVS Sciences Inc., Markham, ON.
Wednesday,
22 May
20
Tuesday,
21 May
1100
Parallel 2: Personalized Medicine 1
Commercial
Tutorials &
Exhibits
Room 32AB
Chair: Virginia Litwin
Cochair: Kewal Asosingh
1120
25
Functional Characterization of Lymphoid Subsets in Chronic Myelogenous Leukemia
by Mass Cytometry Phospho-Flow Analysis J. De, R. Fernandez, N. Shah and H.
Maecker. Stanford Univ. and UCSF Helen Diller Family Comprehen. Cancer Ctr.
1140
26
Maternal BMI Affects Expression Pattern of Cord Blood T- and NK-Cell-Subtypes A.
Tárnok , J. Bocsi, C. Blatt, A. Szabó, S. Melzer and I. Dähnert. Univ. of Leipzig and
LIFE Leipziger Res. Ctr.
ISAC 2013 Program and Abstracts
Speaker/Author
Index
Remarkable Durability of Ag85a-Specific CD4 T Cell Memory Responses Up to 6
Years after Mva85a Vaccination E. Smit, M. Tameris, E.J. Hughes, A. Veldsman, L. van
der Merwe, H. Geldenhuys, M. Hatherill, H. McShane, W. Hanekom, H. Mahomed
and T. Scriba. Univ. of Cape Town and Univ. of Oxford.
Poster Session
Abstracts
24
Oral Session
Abstracts
1100
27
Congress
Overview
Special
Lectures
1200
27
Compromised Innate and Adaptive Immune Responses during Yellow Fever
Vaccination in Elderly — A Multiparametric Longitudinal FACS Study R.A. Schulz,
R. Stark, J.N. Maelzer, C. Domingo-Carrasco, T. Jelinek, K. Juerchott, N. Babel, A.U.
Neumann, M. Niedrig and A. Thiel. Charité, Berlin, Robert Koch Inst., Berlin, Berlin
Ctr. for Travel and Trop. Med. and Humboldt Univ. of Berlin.
Parallel 3: Flow Cytometry Software
Room 31ABC
1100
28
Reproducing Manual Gating of Flow Cytometry Data by Automating Cell Population
Identification J. Taghiyar, R. Droumeva, M. Malekesmaeili, G. Finak R. Gottardo and R.
Brinkman. British Columbia Cancer Agcy. and Fred Hutchinson Cancer Res. Ctr., Seattle.
1120
29
FlowCAP: Comparison of Automated and Manual Gating of Standardized Lyoplate
Flow Cytometry Data G. Finak, J. Ramey, J. Taghiyar, R. Stanton, A. Brandes, P. Qui,
J.P. McCoy, D. Hafler, H. Maecker, T. Mossman, R. Scheuermann, R. Brinkman and
R. Gottardo. Fred Hutchinson Cancer Res. Ctr., Seattle, British Columbia Cancer
Agcy., J Craig Ventner Inst., San Diego, Broad Inst., Cambridge, MA, Univ. of Texas
MD Anderson Cancer Ctr., NHLBI, NIH, Yale Univ. Sch. of Med., Stanford Univ. and
Univ. of Rochester Med
1140
30
A Computational Method for Creating and Using Pre-defined Gating Sequence
Templates to Automatically Gate High-Dimensional Flow Data S. Meehan, C.
Meehan, W.A. Moore, D.R. Parks and L.A. Herzenberg. Stanford Univ., Burnaby,
BC and Stanford, CA and Univ. of Toronto.
1200
31
Simplicial Analysis in Flow Cytometry Data Processing: Enabling SVM and HDP Cell
Classification via Standardization of Cell-Signal Simplices B. Rajwa, F. Akova, A. Pothen
and M. Murat Dundar. Purdue Univ. and Indiana Univ.-Purdue Univ. Indianapolis.
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
Chair: Nima Aghaeepour
Cochair: Enrico Lugli
Parallel 4: Image Cytometry Technology
Poster
Session
Room 30CDE
Chair: Gustavo Rohde
Cochair: Bartek Rajwa
32
Next-Generation FLIM: Modulated All Solid-State Camera System I. Young, Q. Zhao,
B. Schelen, B. Schelen, R. Schouten, J. Bosiers, R. Leenen, I. Peters and K. Jalink, Delft
Univ. of Technol., Teledyne DALSA, Eindhoven, NKI - Dutch Cancer Inst., Amsterdam
and Lambert Instruments, Roden, Netherlands.
1120
33
Intravital Marker-Free NAD(P)H Fluorescence Lifetime Imaging – In Vivo Selective
Enzyme Detection R. Niesner, A. Mossakowski, J. Pohlan, M. Radbruch, J. BayatSarmadi, F. Paul, A.E. Hauser and H. Radbruch. Charité - Univ. Hosp. and DRFZ, Berlin.
1140
34
Ultra High Throughput Image Cytometry via Continuous Scanning J. Price, G.
Gemmen, B. Azimi, M. Guigli and J. Price. Vala Sciences Inc., San Diego and
Sanford-Burnham Med. Res. Inst., La Jolla.
1200
35
Automated Intracellular FRET Measurements Using Hyperspectral Microscopy and
Feature Extraction S. Leavesley, A. Britain and T. Rich. Univ. of South Alabama.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
1100
28
ISAC 2013 Program and Abstracts
Congress
Overview
Parallel 5: Multiplexed and Microparticle-based Assays
Room 30AB
Amplifying DNA Beads Assay in Flow Cytometry: A Platform Technology Based on
Exonuclease III-Aided Target Recycling J. Lu and D. Jin. Macquarie Univ., Australia.
1120
37
Development of a Glycoprofiling Method Using Multiplex Microspheres L. Yang,
C. Leoff and R. Woods. Glycosensors and Diagnostics, San Diego, Univ. of Georgia
and Glycosensors and Diagnostics, Athens, GA.
1140
38
Multiplexed Microsphere Protease Assays and High Throughput Flow Cytometry for
Drug Discovery J. Zhu, L. Sklar, B. Edwards and S.W. Graves. Univ. of New Mexico.
1200
39
Significantly Improve Bead Recovery of Flow Cytometry Assay by Utilising a New
Technology Platform for Sample Incubation M. Tenje, N. LeBlanc, M. Evander,
B. Hammarström, H. Xia, A. Tojo, S. Belák and T. Laurell. Lund Univ., Natl. Vet. Inst.,
Uppsala, Swedish Univ. of Agr. Sci. and Dongguk Univ., South Korea.
Sunday,
19 May
36
Saturday,
18 May
1100
Special
Lectures
Chair: Marie Iannone
Cochair: Yang Wu
Monday,
20 May
Exceptional Student Award Presentations
12:45 – 13:45
Room 28C
Tuesday,
21 May
Award Finalists
Ali Vaziri Gohar
Determining Intracellular Protein Localization with Fluorescence Lifetime-Based Flow Cytometry
Wednesday,
22 May
Irina Polshchitcina
Kinetic Study of Morphological Changes in Human Lymphocytes during Early Stages of Apoptosis using Scanning
Flow Cytometry
Charles Shields IV
Acoustofluidic Cell Sorting via Negative Acoustic Contrast Capture Colloids
Poster
Session
Jiangbo Zhao
SUPER Dots: The Next-Generation Bio-Labels
Commercial Tutorials
Commercial
Tutorials &
Exhibits
1245 – 1345
Featured Companies
Oral Session
Abstracts
Cytek Development – Room 33AB
Beckman Coulter Life Sciences – Room 32AB
Sony Biotechnology, Inc. – Room 31ABC
EMD Millipore – Room 30CDE
eBioscience, an Affymetrix company – Room 30AB
Beckman Coulter Life Sciences – Room 29CD
Fluidigm – Room 29AB
Poster Session
Abstracts
See pages 66– 68 for full details.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
29
Congress
Overview
State-of-the-Art Lectures
1400 – 1530
Ballroom 20D
Sunday,
19 May
Saturday,
18 May
Special
Lectures
Chair: J. Paul Robinson
Cochair: Andrea Cossarizza
1400
17
Translating Acoustophoretic Cell Handling to Clinical Applications T. Laurell. Lund
Univ., Sweden.
1430
18
Signaling Networks Regulating Cellular Growth and Form C. Bakal. Inst. of Cancer
Res., London.
1500
19
Discovery of Small Molecules That Control Cell Differentiation P. Bartunek. Inst. of
Molec. Genet., Prague.
Coffee Break
Monday,
20 May
1530 – 1545
Foyer/Terrace, Upper Level
Concurrent Workshop Sessions
1545 – 1715
Tuesday,
21 May
Workshop 1
Room 33AB
Wednesday,
22 May
1545
40
Navigating the Labyrinth of Regulated Flow Cytometry in Drug Development
V. Litwin, J.J. Stewart, J. Olsen, J. Puchalski, C. Green and C. Wiwi. Covance Inc.,
Flow Contract Site Lab., Amgen Inc. and Celgene Corp.
Workshop 2
Room 32AB
41
Poster
Session
1545
The Delivery of Image-Based Cytometry Education J. Nolan, B. Rajwa, R. Errington,
G. Vereb, S.J. Lockett, A. Parwani and G.K. Rohde. La Jolla Bioengineering Inst.,
Purdue Univ., Cardiff Univ., U.K., Univ. of Debrecen, Hungary, SAIC- Frederick Natl.
Lab. and Carnegie Mellon Univ.
Commercial
Tutorials &
Exhibits
Workshop 3
Room 31ABC
Oral Session
Abstracts
1545
42
Biosafety: Biosafety Policy Meets Real Life Scenarios K. Holmes and S.P. Perfetto.
NIAID, NIH.
Workshop 4
Room 30CDE
Poster Session
Abstracts
1545
43
Quantitative Cytometry — Calibration and Standardization L. Wang and
R. Hoffman. NIST, Gaithersburg, MD and BD Biosciences, San Jose, CA.
Workshop 5
Speaker/Author
Index
Room 30AB
1545
30
44
Functional Analysis of Mitochondria and Transporters G. Babcock and P Narayanan.
Univ. of Cincinnati and Amgem
ISAC 2013 Program and Abstracts
Congress
Overview
Robert Hooke Lecture
1730 – 1830
Ballroom 20D
Special
Lectures
Chair: Larry Sklar
TBA
Saturday,
18 May
Opening Reception
1830 – 1930
Foyer/Terrace, Upper Level
Sunday,
19 May
Monday, 20 May 2013
Monday,
20 May
Frontiers Session 1: Innovation
830 – 1000
Ballroom 20D
46
Use of Kinetic Imaging Cytometry to Develop Pharmacological Approaches to
Cardiac Regeneration and Preservation of Contractile Function M. Mercola. SanfordBurnham Med. Res. Inst., and UCSD, La Jolla.
915
47
Non-genetic Cell Population Heterogeneity: Implications for Cell Differentiation
and Cancer Progression S. Huang. Inst. for Systs. Biol., Seattle.
Wednesday,
22 May
830
Tuesday,
21 May
Chair: Jeff Price
Cochair: William Telford
Coffee Break
Poster
Session
1000 – 1015
Foyer/Terrace, Upper Level
Concurrent Parallel Sessions
Commercial
Tutorials &
Exhibits
1015 – 1145
Parallel 6: Cell Proliferation and Death
Room 33AB
Live Cell Analysis of NCAM Polysialylation in Micro-Communities Using the Novel
Combination of an Antibody-Mimetic EGFP-Endosialidase and the Viability Dye
DRAQ7 P. Smith, M. Wiltshire, S. Chappell, L. Patterson, S. Shnyder, R. Falconer and
R. Errington. Sch. of Med., Cardiff Univ. and Univ. of Bradford, U.K.
1035
49
Cytometric Assessment of DNA Damage- and mTOR-Signaling, the Factors
Contributing to Aging and Senescence. Z. Darzynkiewicz, H. Zhao and D. Halicka.
New York Med. Col.
ISAC 2013 Program and Abstracts
Speaker/Author
Index
48
Poster Session
Abstracts
1015
Oral Session
Abstracts
Chair: Rachel Errington
Cochair: Katarzyna Piwocka
31
Congress
Overview
Special
Lectures
1055
50
Dissecting the Intricate Network of the Cell Death Machinery: Simultaneous
Detection of Apoptosis and Autophagy Using Flow Cytometry R. Pal, Y. OheneAbuakwa, T. Rutherford and F. Gibson. St George’s Univ. of London.
1115
51
Development and Qualification of a Whole Blood Assay to Monitor pHH3 and
Cell Cycle in Patients with Acute Myeloid Leukemia Treated with Aurora Kinase
Inhibitors C. Green, C. Ma, J. Ferbas and G. Juan. Amgen, Ventura and Thousand
Oaks, CA.
Saturday,
18 May
Parallel 7: Image Cytometry Applications
Room 32AB
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Chair: Stephen Lockett
Cochair: Anis Larbi
1015
52
Understanding Subcellular Filament Networks from Fluorescence Microscopy
Images G. Rohde, S. Basu, J. Li, K. Dahl and R. Murphy. Carnegie Mellon Univ.
1035
53
New Approaches to Study Neuronal Connectivity in a High-Content Format
N. Prigozhina, J. Seldeen, F. Cerignoli, R. Basa, J. Price and P. McDonough. Vala
Sciences Inc., San Diego.
1055
54
Addressing Uncertainty in the Automated Evaluation of Stem Cell Colony Quality
M. Halter, Y-S. Li-Baboud, A. Peskin, P. Bajcsy, D. Hoeppner and A.L. Plant. NIST,
Gaithersburg, MD and Leber Inst. for Brain Develop., Baltimore.
1115
55
A Non-parametric Method for the Automated Discovery of Perturbagen Responses
in High-Content Analysis G. Johnson, J. Kangas, A. Dovzhenko, K. Voigt, S. Bhavani,
K. Palme and R. Murphy. Carnegie Mellon Univ. and Albert Ludwigs Univ. Freiburg,
Germany.
Parallel 8: Flow Cytometry Instrumentation
Room 31ABC
1015
56
Accelerated Cell Sorting Using In-Line Sample Pre-enrichment B. Warner, L. Yu,
J. Trotter, M. Jaimes, M. Blom, W. Buesink, A. Lenshof, T. Laurell and T. Lau BD
Biosciences, San Jose, CA, Micronit Microfluidics, Netherlands and Lund Univ.,
Sweden.
1035
57
Microflow1, a Portable Flow Cytometer for Space Travel: In Preparation for the
International Space Station Demonstration C. Riviere, O. Mermut, G. DubeauLaramee and L. Cohen. INO, Quebec City and Canadian Space Agcy., Saint-Hubert,
QC.
1055
58
An Extremely Parallel Flow Cytometer for Rapid Cellular Analysis S. Graves,
P.P. Austin Suthanthiraraj, M.E. Piyeasena and A. Shreve. Univ. of New Mexico.
1115
59
Acoustofluidic Cell Sorting via Negative Acoustic Contrast Capture Colloids
C. Shields IV, L. Johnson, L. Gao and G. Lopez. Duke Univ. and Triangle MRSEC,
Durham, NC.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Chair: Geoffrey Osborne
Cochair: Sung Hwan Cho
32
ISAC 2013 Program and Abstracts
Congress
Overview
Parallel 9: Personalized Medicine 2
Room 30CDE
1035
61
Immune Monitoring of Human Kidney Allografts with Biopsy Multicolor Flow
Cytometry and Cytokines K. Muczynski, N. Leca and S. Anderson. Univ. of
Washington.
1055
62
Metastatic Breast Cancer Stem/Progenitor Populations Survive Consolidation
Chemotherapy, Circulate and Disseminate to Bone Marrow A. Donnenberg,
V.S. Donnenberg, R. Landreneau and A. Brufsky. Univ. of Pittsburgh Sch. of Med.
1115
63
Relapsing-Remitting MS Patients Have Significant Differences in Circulating B
Cells Subset and Phenotype Compared with Healthy Controls, and IFN&[beta]-1b
Treatment Can Alter the Composition and Phenotype of These Subsets D. Mielcarz,
J. DeLong, A. Bergeron, K. Smith, A. Heyn, L. Kasper and J. Channon. Geisel Sch. of
Med. at Dartmouth.
Monday,
20 May
Complex Changes in Invariant Natural Killer T Cells in Patients with Different
Clinical Forms and Treatments of Multiple Sclerosis S. De Biasi, M. Nasi,
A.M. Simone, D. Ferraro, F.F. Vitetta, L. Gibellini, M. Pinti, A. Del Giovane, P. Sola
and A. Cossarizza. Univ. of Modena and Reggio Emilia and Nuovo Ospedale Civile
Sant’Agostino Estense (NOCSAE), Italy.
Sunday,
19 May
60 Saturday,
18 May
1015
Special
Lectures
Chair: Cherie Green
Cochair: Padma Narayanan
Tuesday,
21 May
Parallel 10: Cellular Assay Systems
Room 30AB
1035
65
Analysis and Sorting of Antigen-Specific Antibody Secreting Cells in Mixed Cultures
Using the Affinity Matrix Technology A. Taddeo, H-D. Chang, V. Gerl, B. Hoyer,
A. Radbruch and F. Hiepe. DRFZ and Charité - Med. Univ. Berlin, Campus Mitte.
1055
66
Developing Measures of Cellular Potency for Therapeutic Transplantation D. Kaplan,
H.M. Lazarus and N.M. Kaye. Pathfinder Biotech, Cleveland and Case Western
Reserve Univ.
1115
67
Sorting of Specific Lymphocyte Populations from Peripheral Blood Progenitor Cell
Products Using a Novel Micro-Chip Based Acoustophoretic Platform A. Urbansky,
A. Lenshof, A. Jamal, J. Dykes, I. Åstrand-Grundström, T. Laurell and S. Scheding.
Lund Univ., Sweden.
Oral Session
Abstracts
A High-Throughput Method for Creating Uniform 3D Tissue Models J. De Lora,
D. Kalb, A. Martinic, A. Trujillo, T. Woods, A. Shreve and J. Freyer. Univ. of New
Mexico.
Commercial
Tutorials &
Exhibits
64
Poster
Session
1015
Wednesday,
22 May
Chair: Tim Bushnell
Cochair: Alfonso Blanco-Fernandez
Commercial Exhibits
Poster Session
Abstracts
1130 – 1900
Exhibit Hall GH
See pages 76– 89 for full details.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
33
Congress
Overview
Poster Viewing
Special
Lectures
Exhibitor Showcase
1130 – 1900
Exhibit Hall GH
1200 – 1330
Exhibit Hall GH
Saturday,
18 May
1215
BD Biosciences
1235handyem
1255
NewSouth Innovations
1315BioStatus
Sunday,
19 May
Lunch for CYTO Attendees
1200 – 1300
Exhibit Hall GH
Monday,
20 May
Plenary Session 1: Immunology
1330 – 1500
Ballroom 20D
Wednesday,
22 May
Tuesday,
21 May
Chair: Andreas Radbruch
Cochair: Pratip Chattopadhyay
1330
68
Image Cytometry of Cell Adhesion K. Ley. La Jolla Inst. for Allergy & Immunol.
1400
69
Single-Cell Approaches to Investigate the Immune Response M. Cahalan. Univ. of
California, Irvine.
1430
70
Immune Profiles in the Central Nervous System: What We Know, What We Need to
Know, and What It Means N. Monson. Univ. of Texas Southwestern Med. Ctr.
Poster
Session
Coffee Break
1500 – 1545
Exhibit Hall GH
Commercial
Tutorials &
Exhibits
Concurrent Workshop Sessions
1545 – 1715
Workshop 6
Oral Session
Abstracts
Room 33AB
Poster Session
Abstracts
1545
71
Integrating Standards at the Interface of Flow and Image Cytometry H. Minderman,
A. Filby, A. Plant, M. Halter, P. Narayanan and A. White. Roswell Park Cancer Inst.,
Buffalo, Cancer Res. UK, London, NIST, Gaithersburg, MD, Amgen, Seattle and
Cincinnati Children’s Hosp.
Workshop 7
Room 32AB
Speaker/Author
Index
1545
34
72
Writing, Publishing and Reviewing: Advice and Tips from Cytometry A A. Tarnok,
R. Brinkman, V. Donnenberg, H. Ulrich and S. Vice. Univ. of Leipzig , British
Columbia Cancer Agcy., Univ of Pittsburgh Hillman Cancer Ctr., Univ of São Paulo
and Wiley.
ISAC 2013 Program and Abstracts
Congress
Overview
Workshop 8
Room 31ABC
1545
73
Special
Lectures
High Throughput Flow Cytometry in Pharma B. Edwards and R. Jepras. Univ. of New
Mexico and GlaxoSmithKline, Stevenage, U.K.
Workshop 9
Room 30CDE
74
Mesenchymal Stem Cell Identification and Characterization V. Donnenberg,
K. Wonnacott, A.D. Donnenberg, H. Ulrich and A. Plant. Univ of Pittsburgh Hillman
Cancer Ctr., OD, NIH, Univ. of São Paulo and NIST, Gaithersburg, MD.
Saturday,
18 May
1545
Workshop 10
1545
75
Sunday,
19 May
Room 30AB
Malaria Cytometry H. Shapiro and G. Chojnowski. Howard M. Shapiro, MD, PC and
Queensland Inst. of Med. Res., Australia.
Monday,
20 May
Poster Session 1
1715 – 1845
Exhibit Hall GH
Authors of odd numbered boards will present.
Tuesday,
21 May
Happy Hour
Wednesday,
22 May
1800 – 1900
Exhibit Hall GH
Core Managers Forum
1900 – 2200
Room 29
Poster
Session
This year the Core Managers Forum will have the theme “How can ISAC best serve the Shared
Resource Laboratory and its staff”. As facility staff, we know there can be issues with being able to
attend CYTO meetings, accessing ISAC member benefits, developing a career track and developing
the skill set needed to run or work in a successful cytometry core. How can ISAC help? What are the
programs or added value benefits that would aid life in the core? How should they be implemented?
There will be two short presentations - Derek Davies (Cancer Research UK, London) “The ISAC
SRL Task Force” and Peter Lopez (School of Medicine, New York) “Synergy between ISAC and the
Association of Biomolecular Resource Facilities” - but this is your Forum and we want your input so
the majority of the evening will be devoted to open discussion. Refreshments will be provided.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
35
Congress
Overview
Tuesday, 21 May 2013
Special
Lectures
Poster Viewing
700 – 1900
Exhibit Hall GH
Saturday,
18 May
Frontiers Session 2: Discovery
830 – 1000
Ballroom 20D
Monday,
20 May
Sunday,
19 May
Chair: Ryan Brinkman
Cochair: Donat Alpar
830
76
Insights into the Immune System via Single Cell Network Profiling: Towards
Improved Disease Classification and Therapeutic Selection A. Cesano. Nodality Inc.,
South San Francisco.
915
342
Cell-Based Assays in Drug Discovery: Changing the HTS Paradigm H. Djaballah.
Mem. Sloan-Kettering Cancer Ctr.
Coffee Break
Tuesday,
21 May
1000 – 1015
Foyer/Terrace, Upper Level
Concurrent Parallel Sessions
Wednesday,
22 May
1015 – 1145
Parallel 11: Image Cytometry
Room 33AB
1015
77
Multiparameter Image Cytometry Using Surface Enhanced Raman Scattering Tags
and Spectral Imaging E. Liu, E. Duggan and J. Nolan. La Jolla Bioengineering Inst.
1035
78
Phenotyping TILs in Situ: Tissue Cytometric Enumeration of Intra- and ExtraFollicular Foxp3+ Regulatory T Cells in Follicular Lymphoma J. Mansfield, C. van der
Loos, L.S. Nelson, C. Rose, H.E. Sandison S. Usher, J.A. Radford, K.M. Linton and
R. Byers. PerkinElmer, Hopkinton, MA, Amsterdam Med. Ctr. and Univ. of
Manchester.
1055
79
Biophotonic Nanoswitches – Light-Activation of the BH3 Pathway in Cellular
Systems R. Errington, R. Mart, S. Chappell, C. Watkins, M. Wiltshire, A. Jones,
P. Smith and R. Allemann. Schs. of Med., Chem. and Pharm. and Pharmaceut. Sci.,
Cardiff Univ., U.K.
1115
80
Quantitative Co-imaging of Activated Signaling Proteins and Downstream Individual
Transcripts in Breast Cancer Single Cells S. Kwon, M. Nederlof, K. Chin and J. Gray.
Oregon Hlth. & Sci. Univ. and Quantitative Imaging Systs. Inc., Pittsburgh.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Chair: Silas Leavesley
Cochair: Anis Larbi
36
ISAC 2013 Program and Abstracts
Congress
Overview
Parallel 12: Immune Monitoring
Room 32AB
82
The Cytokine Secretion Assay Reveals an Analog to Digital Switch in Cytokine
Expression H-D. Chang, J. Koeck, F. Hatam, M. Bardua, S. Kreher, H. Bendfeldt,
R. Baumgrass and A. Radbruch. DRFZ, Berlin.
1055
83
Determination of Antigen Localization and Trafficking When Targeted to C-Type
Lectin Receptors Using Human Antibody-Antigen Fusion Protein Constructs
Harboring Specificity to CD205 or CD206 J. Tario, Jr., T. Keler and P.K. Wallace.
Roswell Park Cancer Inst., Buffalo and Celldex Therapeut., Needham, MA.
1115
84
Dissection of Anti-CTLA4-Induced Cytotoxic T Cell Responses in Melanoma
P. Kvistborg, D. Philips, S. Kelderman, B. Hemskerk, C. Ottensmeier, D. Speiser,
C. Blank, J. Haanen, and T. Schumacher. Netherlands Cancer Inst., Amsterdam,
Southampton Univ. Hosp., U.K. and Ludwig Cancer Ctr., Lausanne.
Tuesday,
21 May
1035
Monday,
20 May
Standardization of Whole Blood Immune Phenotype Monitoring for Clinical Trials:
Panels and Methods from “The ONE Study” M. Streitz, T. Miloud, M. Kapinsy, M.
Reed, E.K. Geissler, J. Hutchinson, K. Vogt, S. Schlickeiser, A. Kverneland,
C. Meisel H-D. Volk and B. Sawitzki. Charité - Med. Univ. Berlin, Immunotech
S.A.S., Marseille, Beckman Coulter GmbH, Krefeld, Beckman Coulter Inc., Miami
and Univ. Hosp. Regensburg, Germany.
Sunday,
19 May
81
Saturday,
18 May
1015
Special
Lectures
Chair: Mario Roederer
Cochair: Gergely Toldi
Parallel 13: Cell-Derived Microvesicles
Room 31ABC
Wednesday,
22 May
Chair: Mehrnoosh Abshari
Cochair: Phillip Hexley
Flow Cytometric Analysis of Single Lipid Membrane Vesicles S. Stoner and J. Nolan.
La Jolla Bioengineering Inst.
1035
86
Novel Approach for Characterizing Circulating Microparticles Using Imaging Flow
Cytometry J. Lannigan, C. Rudy and U. Erdbrugger. Univ. of Virginia.
1055
87
Study of Potential Markers for Tumor-Derived Mp in an in Vitro Model of Spiked
Whole Blood P. Poncelet, J. Bez, T. Bouriche, and W. Ruf. BioCytex, Marseille and
The Scripps Res. Inst.
1115
88
Extracellular Vesicles from Plasma of Healthy Donors, Characterized by CryoElectron Microscopy, Receptor-Specific Gold Labeling and Flow Cytometry
A. Brisson, N. Arraud, R. Linares, S. Tan and C. Gounou. Univ. of Bordeaux.
Commercial
Tutorials &
Exhibits
85
Poster
Session
1015
Oral Session
Abstracts
Parallel 14: Flow Cytometry Instrument and Software Advances
Room 30CDE
1015
89
Poster Session
Abstracts
Chair: Bruce Edwards
Cochair: Michael Zordan
High Throughput Drug Screening J.P. Robinson, V. Patsekin, P.K. Narayanan and
N. Li. Purdue Univ. and Amgen, Seattle.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
37
Special
Lectures
‘Deep Blue’ Lasers for Detecting Low-Level Cyan Fluorescent Protein Expression:
An Example of Optimizing Excitation Conditions for Maximum Probe Sensitivity
W. Telford, J. Hu-Li, V. Kapoor, N. Hawk, B. Mester, A. Schmidt, R. Kyle, K. Price and
G. Le Gros. NCI and NIAID, NIH and Malaghan Inst. of Med. Res., Wellington, New
Zealand.
1055
91
Saturday,
18 May
Congress
Overview
90
A New Computational Method for Predicting Optimal Reagent-Dye Combinations
in Staining Panel Design for High-Dimensional Flow Cytometry W.A. Moore,
S.W. Meehan, A.B. Kantor, D.R. Parks and L.A. Herzenberg. Stanford Univ., Stanford,
CA and Burnaby, BC.
1115
92
Quantifying the Relationship between Antibody Bound and Signal Observed for
a Large Panel of Dye-Conjugated Antibodies Using Complementary Binding by
Antibody Capture Beads and Use of This Information in Cytogenie Autocolor to
Predict Optimal Multi-color D. Parks, A.B. Kantor, W.A. Moore, S.W. Meehan and
L.A. Herzenberg. Stanford Univ., Stanford, CA and Burnaby, BC.
Sunday,
19 May
1035
Commercial Exhibits
Monday,
20 May
1130 – 1900
Exhibit Hall GH
See pages 76– 89 for full details.
Tuesday,
21 May
President’s Award for Excellence Presentations
Room 28C
12:15 – 13:15
Wednesday,
22 May
Award Finalists
Nicolas Arraud
Kinetics of Annexin A5 Interaction with Model Membranes, determined by Flow Cytometry.
Poster
Session
Silas Leavesley
Automated Intracellular Fret Measurements using Hyperspectral Microscopy and Feature Extraction
Er Liu
Multiparameter Image Cytometry using Surface Enhanced Raman Scattering Tags and Spectral Imaging
Commercial
Tutorials &
Exhibits
Adriano Taddeo
Analysis and Sorting of Antigen-Specific Antibody Secreting Cells in Mixed Cultures using the Affinity
Matrix Technology
Commercial Tutorials
Oral Session
Abstracts
1215 – 1315
Featured Companies
Speaker/Author
Index
Poster Session
Abstracts
DVS Sciences – Room 33AB
Union Biometrica, Inc. – Room 32AB
BD Biosciences – Room 31ABC
Sony Biotechnology, Inc. – Room 30CDE
Tree Star, Inc. – Room 30AB
Miltenyi Biotec GmbH – Room 29CD
Bio-Rad – Room 29AB
See pages 69 – 71 for full details.
38
ISAC 2013 Program and Abstracts
Congress
Overview
Plenary Session 2: Cancer
1330 – 1500
Ballroom 20D
DNA Sequencing Detects Residual Leukemia B. Wood. Univ. of Washington.
1400
94
Quantitative IF – A Molecular Tool for Assay Development and Tissue Quality
Assessment in Cancer V. Neumeister, K. Schalper, A. England, E. Zarella, F. Parisi,
Y. Kluger, D. Hicks and D. Rimm. Yale Univ. Sch. of Med. and Univ. of Rochester Sch.
of Med.
1430
95
Tissue Factor Bearing Microparticles Measured by Impedance-Based Flow
Cytometry Predict Thrombosis in Cancer Patients J. Zwicker. Beth Israel Deaconess/
Harvard Med. Sch.
Sunday,
19 May
93
Saturday,
18 May
1330
Special
Lectures
Chair: Paul Wallace
Cochair: Zbingiew Darzekewicz
Coffee Break
Monday,
20 May
1500 -1545
Exhibit Hall GH
Concurrent Workshop Sessions
Tuesday,
21 May
1545 – 1715
Workshop 11
Room 33AB
96
Design and Application of Receptor Occupancy Assays Used to Measure
Pharmacodynamic Response to Treatment with Biologic Therapies V. Litwin,
C. Green, M. Williams, D. Wunderlich, M. Liang and J. Ferbas. Covance Inc.,
Indianapolis, Amgen Inc., Los Angeles, Genentech, Pfizer Inc. and MedImmune.
Wednesday,
22 May
1545
Poster
Session
Workshop 12
Room 32AB
1545
97
Microvesicle Analysis N. Fisher and J. Lannigan. Univ. of North Carolina and Univ. of
Virginia.
Commercial
Tutorials &
Exhibits
Workshop 13
Room 31ABC
1545
98
Oral Session
Abstracts
Spectral Imaging and Tissue Cytometry S. Leavesley and J. Mansfield. Univ. of South
Alabama and PerkinElmer, Hopkinton, MA.
Workshop 14
Room 30CDE
99
Poster Session
Abstracts
1545
Career Development: Cytometry Still Needs You, but Do You Need Cytometry?
R. Walker and A. Filby. Babraham Inst. and Cancer Res. UK.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
39
Congress
Overview
Workshop 15
Room 30AB
Special
Lectures
1545
100
Trends in Cytometry Instrumentation S. Graves and G. Vacca. Univ. of New Mexico
and Kinetic River Corp., San Francisco.
POSTER SESSION 2
Saturday,
18 May
1715 – 1845
Exhibit Hall GH
Authors of even numbered boards present.
Happy Hour
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
1800 – 1900
Exhibit Hall GH
40
ISAC 2013 Program and Abstracts
Congress
Overview
Wednesday, 22 May 2013
Special
Lectures
Poster Viewing
700 – 1600
Exhibit Hall GH
Saturday,
18 May
Frontiers Session 3: Translation
830 – 1000
Ballroom 20D
830
101
In Pursuit of Immune Tolerance G. Nepom. Immune Tolerance Network, Seattle.
915
102
Integrating Flow Cytometry and Transcriptomics M. Roederer. NIAID, NIH.
Sunday,
19 May
Chair: Gergely Toldi
Cochair: Tomas Kalina
Monday,
20 May
Coffee Break
1000 – 1015
Foyer/Terrace, Upper Level
Tuesday,
21 May
Concurrent Parallel Sessions
1015 – 1145
Parallel 15: Flow Cytometry and Sorting
Wednesday,
22 May
Room 33AB
Chair: Geoffrey Osborne
Cochair: Jessica Houston
1035
104
Hollow Core Photonic Crystal Fiber Laser Sources: Closing in on True Tunable Laser
Sources for Flow Cytometry W. Telford, V. Kapoor, N. Hawk, Y. Wang, F. Gerome and
F. Benabid. NCI, NIH and XLIM Res. Inst., Limoges.
1055
105
Determining Intracellular Protein Localization with Fluorescence Lifetime-Based
Flow Cytometry A. Vaziri Gohar, R. Cao, W. Li, P. Jenkins, J.P. Houston and
K.D. Houston. New Mexico State Univ.
1115
106
A Label-Free Shape-Based Detection of Activated Platelets with Scanning Flow
Cytometry A. Moskalensky, M. Yurkin, A. Konokhova, D. Strokotov, V. Nekrasov,
A. Chernyshev and V. Maltsev. Inst. of Chem. Kinet. and Combustion, SB RAS and
Novosibirsk State Univ., Russia.
Oral Session
Abstracts
A UV-C LED Sheath Fluid Desinfection Module for Flow Cytometric Cell Sorting
T. Kaiser, J. Kirsch, J. Glaab, T. Kolbe, M. Kneissl and H-D. Chang. DRFZ, Berlin,
Ferdinand Braun Inst., Berlin and Tech Univ. Berlin.
Commercial
Tutorials &
Exhibits
103
Poster
Session
1015
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
41
Congress
Overview
Parallel 16: Ligand-Receptor Dynamics
Room 32AB
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
Special
Lectures
Chair: Steve Graves
Cochair: Bruno Paredes
1015
107
Kinetics of Annexin A5 Interaction with Model Membranes, Determined by Flow
Cytometry N. Arraud, C. Gounou and A. Brisson. Univ. of Bordeaux.
1035
108
Novel Flow Cytometry Assay for Real-Time Detection of Molecular Extension of
Lymphocyte Function-Associated Antigen-1 A. Chigaev, Y. Smagley, S. Zhang,
M. Haynes, W. Wang and L. Sklar. Univ. of New Mexico.
1055
109
Discovery of Regulators of Receptor Internalization by High Throughput Flow
Cytometry Y. Wu, P. Tapia, G. Fisher, A. Waggoner, J. Jarvik and L. Sklar. Univ. of
New Mexico and Carnegie Mellon Univ.
1115
110
Studies of Immunological Synapse Formation and Downstream Signaling Events
Using the Flowsight and ImageStream Imaging Flow Cytometers H. Pugsley,
S. Friend, R. Kong, B. Hall, S. Vaidyanathan and D. Basiji. Amnis of EMD Millipore,
Seattle.
Parallel 17: New Probes and Assays
Room 31ABC
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Chair: Joanne Lannigan
Cochair: Rachael Walker
1015
111
Development of Brighter Surface Enhanced Raman Scattering Tags for Multiplexed
Cytometry J. Nolan and E. Duggan. La Jolla Bioengineering Inst.
1035
112
SUPER Dots: The Next-Generation Bio-Labels. J. Zhao and D. Jin. Macquarie Univ.,
Australia.
1055
113
RNA Flow Cytometry for Multiplex Gene Expression Analysis for Specific
Intracellular mRNAs in Individual Cells E. Park, W. Lomas, M.E. Hanley, D. Mittar,
N. Su, Y. Luo and V. Maino. BD Biosciences, San Jose, CA and Adv. Cell Diagnostics
Inc., Haywood, CA.
1115
114
Cytometry of Low-Cell-Count Samples: Chipcytometry for Deep
Immunophenotyping of Cerebrospinal Fluid and Bronchoalveolar Lavage Cells
C. Hennig, A. Mirenska, M. Stangel and G. Hansen. Hannover Med. Sch., Germany.
Parallel 18: Hematological Disorders
Room 30CDE
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Chair: Zofia Maciorowski
Cochair: Derek Davies
42
1015
115
Image Cytometry-Based Detection of Aneuploidy by FISH-IS O. Maguire,
K. Humphrey, E. Wang, A. Block, S. Sait, P. Wallace and H. Minderman. Roswell Park
Cancer Inst., Buffalo.
1035
116
Pilot Investigation of EuroFlow Standardized 8-Color Panel on Different Flow
Cytometry Platforms T. Kalina, M. Nováková, M. Vlková, D. Thürner, E. Mejstrikova,
Q. Lecrevisse and O. Hrusak. Charles Univ. Prague 2nd Med. Fac. and St. Anne’s
Univ. Hosp. and Fac. of Med., Masaryk Univ., Czech Republic and Univ. of
Salamanca, Spain.
ISAC 2013 Program and Abstracts
Use of Imaging Flow Cytometry as an Assay for Sickling Capacity in Patients with
Sickle Cell Anemia L. Samsel, E. van Beers, L. Mendelsohn, R. Saiyed, P. McCoy and
G. Kato. NHLBI, NIH.
1115
118
Kinetic Study of Morphological Changes in Human Lymphocytes during Early Stages
of Apoptosis Using Scanning Flow Cytometry I. Polshchitcina, D. Strokotov and
V. Maltsev. Inst. of Chem. Kinet. and Combustion, SB RAS and Novosibirsk State
Univ., Russia.
Special
Lectures
117
Congress
Overview
1055
Saturday,
18 May
Commercial Exhibits
1130 – 1630
Exhibit Hall GH
Sunday,
19 May
See pages 76– 89 for full details.
Commercial Tutorials
1215 – 1315
Monday,
20 May
Featured Companies
Tuesday,
21 May
EMD Millipore – Room 33AB
Molecular Devices LLC – Room 32AB
BD Biosciences – Room 31ABC
PARTEC – Room 30CDE
Verity Software House – Room 30AB
Life Technologies – Room 29CD
De Novo Software – Room 29AB
Wednesday,
22 May
See pages 72 – 74 for full details.
Plenary Session 3: Stem Cells
1330 – 1500
Ballroom 20D
13:30 119
Poster
Session
Chair: Paul Smith
Cochair: Anne Plant
120
Identification and Targeting of Leukemia Stem Cells M. Guzman. Weill Cornell Med.
Col.
1430
121
The Marylou Ingram Lecture: Genomic and Phenotypic Pedigree of Breast Cancer
Cell Subsets V. Donnenberg, J. Hicks and A. Donnenberg. Univ of Pittsburgh Hillman
Cancer Ctr.
Oral Session
Abstracts
1400
Commercial
Tutorials &
Exhibits
Neural Stem and Progenitor Cells in Human Cortical Development and Evolution
A. Kriegstein, J. Lui and D. Hansen. UCSF.
Coffee Break
Poster Session
Abstracts
1500 – 1545
Exhibit Hall GH
Speaker/Author
Index
ISAC 2013 Program and Abstracts
43
Congress
Overview
POSTER SESSION 3
1500 – 1600
Exhibit Hall GH
Special
Lectures
All authors present.
Authors must remove their posters from boards from 1600 – 1630.
Saturday,
18 May
ISAC BUSINESS MEETING
1615 – 1645
Ballroom 20D
Awards Ceremony
Sunday,
19 May
1645 – 1730
Ballroom 20D
Master of Ceremonies: Paul J. Smith, Awards Committee Chair and Past President
Monday,
20 May
Recognition of New ISAC Scholars
Tuesday,
21 May
Michael Halter
Er Liu
Yiqing Lu
Frank Alexander Schildberg
Joseph Tario, Jr.
Cytometry Part A: 2012 Best Paper Award
Wednesday,
22 May
Single-Cell Mass Cytometry Adapted to Measurements of the Cell Cycle
G.K. Behbehani, S.C. Bendall, M.R. Clutter, W.J. Fanti, G.P. Nolah
Exceptional Student Award Finalists
Poster
Session
Ali Vaziri Gohar
Irina Polshchitcina
Charles Shields IV
Jiangbo Zhao
Commercial
Tutorials &
Exhibits
Nicolas Arraud
Silas Leavesley
Er Liu
Adriano Taddeo
Oral Session
Abstracts
President’s Award for Excellence Finalists
To Be Announced:
Poster Session
Abstracts
Distinguished Service Award
Membership Award
Outstanding Poster Awards
The Fulwyler Award for Innovation Excellence
Speaker/Author
Index
Closing Reception at the Stingaree
1900 – 2300
Tickets required for admittance. See page 11 for full details.
44
ISAC 2013 Program and Abstracts
Congress
Overview
Multimedia and Poster Sessions
Exhibit Hall GH
Special
Lectures
Author presentation and discussion times:
Tuesday, 21 May
700 – 1900
1715 – 1845
Poster Viewing
Poster Session 2: Authors of EVEN numbered poster boards present
Sunday,
19 May
Authors must set up posters on assigned board
Poster Viewing
Poster Session 1: Authors of ODD numbered poster boards present
Saturday,
18 May
Monday, 20 May
700 – 1100
1130 – 1900
1715 – 1845
Monday,
20 May
Wednesday, 22 May
700 – 1600
Poster Viewing
1500 – 1600
Poster Session 3: All authors present
1600 – 1630
All posters must be removed from the boards.
Multimedia Presentations
B2
123
Study of Sensitivity and Specificity of DNA Image Cytometry in Cervical Squamous
Lesions X. Sun, H. Li and M. Zhang. Wuhan Landing Med. High-Tech Co. Ltd. and
Hubei Zhong Shan Hosp., Wuhan, China.
B3
124
Simultaneous Recording of Action Potentials and Calcium Transients from Stem
Cell-Derived Cardiomyocytes: Applications for Cardiotoxicity Testing R. Whittaker,
R. Vega, R. Ingermanson, F. Cerignoli, R. Towart, D. Gallacher, M. Mercola and
J.H. Price. Vala Sciences Inc., San Diego, Ctr. of Excellence for Cardiovasc. Safety
Res., Beerse, Belgium, Sanford-Burnham Med.Res. Inst., La Jolla and UCSD.
Commercial
Tutorials &
Exhibits
Population-Based Study of Automated DNA Image Cytometry as a Screening
Method for Cervical Cancer in Rural Areas of China X. Sun, H. Li and M. Zhang.
Wuhan Landing Med. High-Tech Co. Ltd. and Hubei Zhong Shan Hosp., Wuhan,
China.
Poster
Session
122
Wednesday,
22 May
B1
Tuesday,
21 May
Automated Microscopy
Cell Proliferation and Death
B4
125
High Resolution Cell Cycle and Apoptosis Analysis in a Two Color Fluorescence Plot
O. Herault and C. Vignon. Univ. Hosp. of Tours, CNRS UMR 7292 GICC, Tours.
Oral Session
Abstracts
Cell Sorting and Selection
B5
126
Poster Session
Abstracts
Optimization of Flow Cytometric Detection and Sorting of Transgenic Plasmodium
Parasites by Selection of Optical Filters I. Vorobjev, K. Buchholz, P. Prabhat and
N. Barteneva. Moscow State Univ., Harvard Sch. of Publ. Hlth., Semrock Inc.,
Rochester, NY, Boston Children’s Hosp. and Harvard Med. Sch.
Speaker/Author
Index
“B” references the board number.
ISAC 2013 Program and Abstracts
45
Congress
Overview
Computation and Informatics
Special
Lectures
Diagnostics
Saturday,
18 May
Facility Management
Sunday,
19 May
Flow Cytometry Instrumentation
B6
B7
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
B8
127
128
129
An Event-Level Relational Database for Flow Cytometry Data J. Cavenaugh, A. Straw,
T. Pawlicki, J. Rebhahn and T. Mosmann. Univ. of Rochester.
Multidimensional Data Visualization Tools for Highly Multiparametric Analysis of
Signaling Pathways by Mass Cytometry J. De. Deepath Med., Palo Alto, CA.
Chromocyte: An Online Resource for the Flow Cytometry Community A.G. Pockley.
Nottingham Trent Univ. and Chromocyte Ltd., Sheffield, U.K.
B9
130
Microfabricated Square Channels for Two Dimensional Acoustically Focused Flow
Cytometry T. Woods and S.W. Graves. Univ. of New Mexico.
B10
131
Using the Flexibility of the BD Influx™ Platform for the Development of a Stream
Monitoring and Correction Solution S. Dervish, S. Allen, F. Kao and A. Smith.
Centenary Inst., Sydney.
B11
132
Simplifying Multiparametric Analysis Further on Microcapillary Flow K. Gillis, E.
Santarelli, A. Barican, P. de Borja, D. Luong, A. Khan, J. Clor, R. Pittaro and
R. Lefebvre. EMD Millipore, Hayword, CA.
B12
133
Cytometry and Microscopy Aboard the International Space Station P. Todd, M. Kurk,
N.S. Logan, S. Moyers, J. Vellinger, T. Maleki Jafarabadi and J.F. Leary. Techshot Inc.,
Greenville, IN and Purdue Univ.
Immunology
134
Poster
Session
B13
Apoptosis as Modulating Factor of CD8+ T Lymphocytes in Human Cutaneous
Leishmaniasis R. Nogueira, C. Cunha, A. Gomes-Silva, A.M. Da-Cruz, A. Schubach,
M.I. Pimentel, S. Mendonça, M. Lyra and Á.L. Bertho. Oswaldo Cruz Inst.-Fiocruz
and Evandro Chagas Clin. Res. Inst., Rio de Janeiro.
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Microelectro-Mechanical Systems (MEMS) and Microfluidics
B14
135
Acoustic Manipulation of Liposomes P.P. Austin Suthanthiraraj and S.W. Graves.
Univ. of New Mexico.
B15
136
Microfluidic Image Cytometry: Modernizing in Vitro Cell-Based Assays with
Microfluidic Technology and Image Cytometry T.H. Yoon and J. Park. Hanyang Univ.,
South Korea.
Speaker/Author
Index
Poster Session
Abstracts
Multi-dimensional Image Cytometry
46
B16
137
Development of a Confocal Imaging Based Immunological Synapse Formation
Assay to Visualize Bispecific Antibody-Mediated Tumor Cell Killing S. Ludmann,
M. Weidner, N. Li, C. Afshari, P. Narayanan and K. Keegan. Amgen, Seattle.
B17
138
Discovering New Ligands with Diverse Signaling Pathways for Old Receptors Y. Wu,
P. Tapia, G. Fisher, J.J. Strouse, P. Simons, A. Waggoner, J. Jarvik and L. Sklar. Univ. of
New Mexico and Carnegie Mellon Univ.
ISAC 2013 Program and Abstracts
Quantification of Protein Aggregates with Flowsight Imaging Cytometer C. Probst,
B. Hall and D. Basiji. Amnis, EMD Millipore, Seattle.
B19
140
Optogenetic Coupling for Calcium Transient Analysis and Cardiotoxicity Testing
F. Cerignoli, S. Ray, P. McDonough, M. Mark and J.H. Price. Sanford-Burnham Med.
Res. Inst., La Jolla, Vala Sciences Inc., San Diego and UCSD Sch. of Engin.
Special
Lectures
139
Congress
Overview
B18
Other Biological Applications
141
Automated Imaging of Cell Sorter Aerosol Containment Test Samples: A Dual Bead
Method K.J. Acklin, V. Papanna, K.E. Ruisaard, K. Ramirez and K. Clise-Dwyer. Univ.
of Texas MD Anderson Cancer Ctr.
Saturday,
18 May
B20
Other Technology Advances
142
Sunday,
19 May
B21
Using Videos to Teach Software M. Aranda, E. Hodges, A. Lewis, M. Stadnisky and
A. Treistar. Tree Star Inc., Ashland, OR.
Tissue Cytometry/Morphometry
143
Monday,
20 May
B22
Phenotypic Characterization of Polarized Epithelial Cells in a 3D EpiAirway Culture
Model Using Confocal Microscopy S. Ludmann, K. Jen, A. Pirrone, K. Rohrbach,
N. Li, C. Afshari, E. Trueblood and P. Narayanan. Amgen, Seattle and Thousand Oaks, CA.
Tuesday,
21 May
Poster Presentations
Antigen-Specific Immune Responses
CD4+ T Cells Are Source of Antigen Specific IFN-g Production in Whole Blood of
Patients with Visceral Leishmaniasis O.P. Singh, R. Kumar, S. Gautam, N. Singh,
S. Nylen, D. Sacks and S. Sundar. Banaras Hindu Univ., India, Karolinska Inst. and
NIAID, NIH.
B24
145
Simultaneous Assessment of CMV Specificity and Functional Response CD8+ T Cells
from Bone Marrow Transplant Recipients O. Maguire, G. Chen, K. O’Loughlin, T.
Hahn, P. McCarthy, P.K. Wallace and H. Minderman. Roswell Park Cancer Inst., Buffalo.
Poster
Session
144
Wednesday,
22 May
B23
Automated Microscopy
Ultra High Throughput Image Cytometry Using Time Delay and Integrate CCD
Imaging and Reflective Positioning Autofocus M. Guigli, D. Charlot, B. Azimi,
R. Agustin, G.J. Gemmen, A.L. Kellner and J. Price. Sanford-Burnham Med. Res. Inst.,
La Jolla, UCSD and Vala Sciences Inc., San Diego.
B26
147
Ultra High Throughput Image Cytometry via TDI Scanning G. Gemmen, B. Azimi,
R. Agustin, M. Guigli and J. Price. Vala Sciences Inc., San Diego and SanfordBurnham Med. Res. Inst., La Jolla.
Oral Session
Abstracts
146
Commercial
Tutorials &
Exhibits
B25
148
A Quantitative Flow Cytometry Assay Applied to Receptor Occupancy
Measurements Helps for Preparation and Pharmacodynamic Monitoring of Clinical
Trials for Targeted Drugs P. Poncelet, M-L. and M. Moulard. BioCytex, Marseille and
DSAR, Sanofi, Vitry-sur-Seine.
B29
150
Cell Type-Specific Analysis of Oncogenesis D. Galbraith, N. Weng, T. Doetschman,
R. Lasken and R. Grindberg. Univ. of Arizona and J. Craig Venter Inst., San Diego.
ISAC 2013 Program and Abstracts
Speaker/Author
Index
B27
Poster Session
Abstracts
Biomarkers
47
Congress
Overview
151
Analysis of Immunohistological Images of Stromal Cell Populations in Ultrasound
Guided Biopsies of Synovium to Help Predict Patient Outcomes in Rheumatoid
Arthritis D. Hardie, J. Turner, K. Raza, C. Buckley and A. Filer. Univ. of Birmingham,
U.K.
Special
Lectures
B31
152
A Flow Cytometry-Based Method for Enumeration of Foxp3+ Regulatory T Cells in
Blood Samples Collected and Stabilized in Cyto-Chex BCT Suitable for Use in Central
Laboratories R. Janani, G. Fesseha and T. Robins. Quest Diagnotics Lab., Valencia, CA.
B32
153
Novel Method for the Identification of Endothelial Microparticles Using ImageBased Flow Cytometry and CDd144 A. Venable, R. Williams and B. McFarlin. Univ.
of North Texas, Denton.
B33
154
Global Implementation of Flow Cytometry Instrument Standardization by Covance
Central Laboratory Services L. Du, V. Glutz, V. Holl, V. Litwin, M. Edinger,
J. Hildmann and J. Batchelder. Covance (Asia) Pte Ltd., Singapore, Covance (Geneva)
Inc., Covance (Indianapolis) Inc. and BD Biosciences, San Jose, CA.
B34
155
A Cross-Platform Validation Study of EGFR-Activating Mutations in Non-small Cell
Lung Carcinoma Cells S. Bokhari, W-R. Lie and T. Baginski. EMD Millipore Corp./
Merck KGaA, St. Charles, MO.
B35
156
A Flow Cytometric Approach to Monitoring Cell Health and Productivity of a
Recombinant Human Cell Line in Bio-Pharmaceutical Development M. Gottlieb,
J.C. Chuang and F. Boldog. Shire HGT, Lexington, MA.
B36
157
Implementation of a Flow Cytometric Eosinophil CD11b Expression Assay for
Clinical Application R. Wnek , M. Tseng, T. Natasha, D. Gallagher, C. Cilissen,
R. Weiner and D. Wu. Merck , Rahway, NJ.
B37
158
A Micromethod for Bead-Based Microarray Immunoassays Enabling Clinical
Translational Research Studies C. Baker, S. Secor-Socha, D. Roumanes, T. Mosmann
and S. Quataert. Univ. of Rochester.
B38
159
Lightening Up the Cellular-Level Reactive Oxygen Species Using Upconversion
Sensitized Ruthenium Biosensors J. Zhao, R. Zhang, L. Zhang, Y. Lu, E.M. Goldys
and D. Jin. Macquarie Univ., Australia.
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
B30
Biopharmaceutical Applications
160
Commercial
Tutorials &
Exhibits
B39
A High-Throughput Flow Cytometry-Based Method for the Detection of AntigenSpecific T Lymphocyte Activation in Non-human Primates for Pre-clinical Drug
Development Assessments M. Bernard, G. Rao, J. Loffredo, A. Suri, H. Haggerty and
W. Freebern. Bristol-Myers Squibb, New Brunswick, NJ and Princeton, NJ.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Cell Proliferation and Death
48
B40
161
Differential Homing and Senescene of CD8+ T-Lymphocytes from Elderly Humans
O. Onyema, I. Bautmans, M. De Waele, J. Aerts and T. Mets. Vrije Univ. and Univ.
Ziekenhuis, Brussels.
B41
162
Induced Germination of B. atrophaeus Spores by Singlet Oxygen-Sensitizing
Cationic Oligo-Phenylene Ethynylenes H. Pappas and D. Whitten. Univ. of New
Mexico.
B42
163
Automated Quantification of Budding Saccharomyces cerevisiae Using an Image
Cytometry Method L. Chan, D. Laverty, A. Kury, D. Kuksin, A. Pirani and K. Flanagan.
Nexcelom Bioscience LLC, Lawrence, MA.
ISAC 2013 Program and Abstracts
B45
166
Bradykinin Functions in Bone Marrow Metatastis H. Ulrich, C. Lameu and
M. Ratajczak. Univ. of São Paulo and Univ. of Louisville.
B46
167
Detection and Evaluation of Cellular Phenomena in the Breast Cancer Cell Line
Resistance to Cisplatin J. Markovic, B. Krunic, N. Apostolova, S. Bañuls and
F.V. Pallardo. Univ. of Valencia, Spain.
B47
168
Improved Click Chemistry Demonstrating EdU Cell Proliferation with GFP
Expressing Cells and R-PE Based Immunophenotyping C. DeMarco, J.A. Bradford,
S. Clarke, R. Deveny, K. Gee, S. Grecian and U. Singh. Life Technologies, Eugene,
OR.
B48
169
Investigating Mitochondrial Changes during Autophagy and Apoptosis Using
Microcapillary Cytometry K. Gillis, J. Clor and K. Tyagarajan. EMD Millipore,
Hayward, CA.
B49
170
Measure of Cell Cycle with the Tali Image Instrument Using a Broad Range of Dyes
T. Lopes, L. Roh and M. Garcia. Fed. Inst. of Technol., Lausanne.
Tuesday,
21 May
Characterization of C22:0 and Saturated Very Long Chain Fatty Acids (C24:0;
C26:0)-Induced Cell Death by Microscopical and Flow Cytometric Methods on
Human Neuronal Cells (SK-N-BE) A. Zarrouk, H. Iddir, M. Yousfi, T. Nury,
C. Gondcaille, M. Hammami and G. Lizard. Univ. of Monastir Fac. of Med., Tunisia
and Univ. of Bourgogne, INSERM, Fac. of Sci. Gabriel, Dijon, France.
Monday,
20 May
165
Sunday,
19 May
B44
Saturday,
18 May
Probing the Impact of Bystander Cells on Irradiated Cells in Mixed Co-cultures
B. Gerashchenko and R. Howell. R.E. Kavetsky Inst. of Exptl. Pathol., Oncol. and
Radiobiol., Kyiv, Ukraine and New Jersey Med. Sch. Cancer Ctr., Newark.
Special
Lectures
164
Congress
Overview
B43
Cell Sorting and Selection
B52
173
Comparison of Proliferation Markers CD71, Ki-67, and Pyronin Y in Combination
with Hoechst 33342 N. Hanson, J. Brown, P. Lopez and M. Schober. New York Univ.
Langone Med. Ctr.
B53
174
High Throughput Chip-Based Cellular Sorting via Acoustically Enhanced
Magnetophoresis L. Gao, C.W. Shields IV, D.M. Murdoch, B.B. Yellen and G.P. Lopez.
Duke Univ., Res. Triangle Material Res. Sci. and Engin. Ctr. and Sch. of Med., Duke
Univ.
B54
175
Microvesicle Detection and Cell Sorting V. Toxavidis, J. Tigges and K. Groglio. Beth
Israel Deaconess Med. Ctr.
B55
176
Use of Near-IR Detection in Flow Cytometry M. Bigos, D. Parks and T. Schmidt.
Stanford Univ.
B56
177
Assessing Sample Behavior in the Context of Sorter Performance Using a Dispersion
Index J. Trotter and S. Iyer. BD Biosciences, San Jose, CA.
B57
178
Performance Study of the Closed Piezo-Based Cell Sorter (CyFlow® Sorter) J. Klose,
D. Köhler and V. Ost. Partec GmbH, Münster.
ISAC 2013 Program and Abstracts
Speaker/Author
Index
Single Cell Sorting for Cancer Stem Cells Enrichment in Hepatocellular Carcinoma
E. Trombetta, F. Colombo, S. Mazzucchelli, A. Cattaneo, D. Prati, P. Rebulla and
L. Porretti. IRCCS Fndn. Ca’ Granda Policlin., Milan and Hosp “”A. Manzoni””
Lecco, Italy.
Poster Session
Abstracts
172
Oral Session
Abstracts
B51
Commercial
Tutorials &
Exhibits
Advances in Fluorescence Decay Analysis: New Techniques for Use in Cytometers
of All Shapes and Sizes R. Cao, M.A. Naivar and J. Houston. New Mexico State Univ.
and DarklingX, Los Alamos.
Poster
Session
171
Wednesday,
22 May
B50
49
Congress
Overview
179
Study of Platelet-Derived Microparticles Using the BD FACSVerse™ Flow Cytometer
L. Yu and Y. Wang. BD Biosciences, San Jose, CA.
B59
180
Importance of Quality Control to Follow-Up and Compare Instrument Performance
for Microparticle Analysis by Flow Cytometry: Correlation to Microparticle Count
N. Bailly, P. Poncelet, B. Devalet, S. Robert, R. Lacroix, J-M. Dogné, F. DignatGeorges, B. Chatelain and F. Mullier. CHU Mont-Godinne, Yvoir, Belgium, BioCytex,
Marseille, Univ. de la Méditerranée, France and Univ. of Namur, Belgium.
B60
181
Accurate Detection of Counting Beads for Cell-Derived Microparticle Enumeration
by Flow Cytometry N. Fisher, M. Mooberry, S. Javardi, R. Zucker and N. Key. Univ. of
North Carolina at Chapel Hill, Stratedigm Inc., San Jose, CA and U.S. EPA, Research
Triangle Park, NC.
Sunday,
19 May
B61
182
Identification and Analysis of Circulating Endothelial Microparticles as a Potential
Biomarker of Drug-Induced Vascular Injury S. Sokolowski, A. Shen, L. Obert, T.
Wisialowski, M. Lawton, P. Nugent, T. Swanson, S. Portugal, C. Rief and B. Enerson.
Pfizer Inc., Groton, CT.
B62
183
Flow and Imaging Cytometry Characterization of Microparticles: Analysis and
Sorting on the Basis of Size and Fluorescence N. Barteneva, E. Fasler-Kan, L. Duckett
and I. Vorobjev. Boston Children’s Hosp., Univ. of Basel, BD Biosciences Inc., San
Jose, CA and Moscow State Univ.
Tuesday,
21 May
Saturday,
18 May
Special
Lectures
B58
Monday,
20 May
Cell-Derived Microvesicles
Poster
Session
Wednesday,
22 May
Clinical Trials
B63
184
The Applications of Acridine Orange in Cytometry F. Samani, P. Khosravani, E. Jan
Zamin and M. Ebrahimi. Royan Inst. for Stem Cell Biol. and Technol., Tehran.
B64
185
Simultaneous Restorations of Foxp3+ Treg, Type 1-Like Treg and B Cells by Anti-TNF
Therapy for IBD Z. Li, S. Vermeire, D. Bullens, M. Ferrante, K. Van Steen, M. Noman,
P. Rutgeerts, J. Ceuppens and G. Van Assche. Catholic Univ. of Leuven and Univ. of
Liege, Belgium.
B65
186
An Approach to Qualifying Flow Cytometry Panels J. Hill, E. Dearstyne, T. Beckett,
N. Purdue and S. Apone. Dendreon, Seattle.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Computation and Informatics
50
B66
187
Creating Standards for InstrumentXML and PanelXML N. Ostrout, J. Katz,
M. Stadnisky, J. Quinn and A. Treister. Fluorish LLC, FlowJo LLC and Tree Star Inc.,
Ashland, OR.
B67
188
Variance-Stabilized Meta-Clustering in Flow Cytometry A. Azad, B. Rajwa and
A. Pothen. Purdue Univ.
B68
189
Gating the Gate Makers: How to Decide Whose Gates Are Bright, and Whose Are
Dim in a High-Throughput Manner J. Quinn, J. Almarode, M. Stadnisky, I. Taylor and
A. Treister. Tree Star Inc. and FlowJo LLC, Ashland, OR.
B69
190
Generative Modeling of F-Actin in Cells to Understand Drug-Induced Cytoskeletal
Changes J. Kinser, T. Turbyville, K. Reilly, J. Beutler and S.J. Lockett. Sch. of Phys. and
Comput. Sci., George Mason Univ. and Frederick Natl. Lab., MD.
B70
191
A Shared Standard for Cytometry and Pathology R. Leif and S. Leif. Newport
Instruments, San Diego.
ISAC 2013 Program and Abstracts
Automated Flow Cytometry Data Analysis K. Feher and T. Kaiser. Univ. of Potsdam
and DRFZ, Berlin.
B72
193
Automated Flow Cytometry Data Analysis with the OpenCyto Framework J. Ramey,
G. Finak, M. Jiang, J. Taghiyar, S. DeRosa, R. Brinkman and R. Gottardo. Fred
Hutchinson Cancer Res. Ctr., Seattle and British Columbia Cancer Agcy., Vancouver.
Special
Lectures
192
Congress
Overview
B71
Cytometry in Resource Poor Settings
194
Reliable and Accurate CD4 T Cell Count and CD4 Percent of the New Portable Flow
Cytometer Cyflow Minipoc M. Nasi, S. De Biasi, E. Bianchini, L. Gibellini, M. Pinti,
T. Scacchetti, T. Trenti, V. Borghi, C. Mussini and A. Cossarizza. Univ. of Modena
and Reggio Emilia, NOCSAE Baggiovara, Modena and Azienda Ospedaliero-Univ.
Polyclin. of Modena, Italy.
Saturday,
18 May
B73
Sunday,
19 May
Diagnostics
Determining Reference Bead Concentration and Fluorescence Intensity for
Quantitative Flow Cytometry at 660 nm and 760 nm P. DeRose, A. Gaigalas and
L. Wang. NIST, Gaithersburg, MD.
B77
198
Characterization of Two Human CD4+ Lymphocyte Preparations for Quantitative
Flow Cytometry L. Wang, M. Wang, H-J. He, M. Misakian, I. Turko and K. Cole. NIST,
Gaithersburg, MD.
B78
199
Assessment of Myeloid Nuclear Differentiation Antigen in Myelodysplastic
Syndrome and Acute Myeloid Leukemia K.T. Soh and P.K. Wallace. Univ. at Buffalo,
SUNY and Roswell Park Cancer Inst., Buffalo.
B79
200
Six Color-Single Tube Analysis of Minimal Residual Disease in Paediatric B Lineage
Acute Lymphoblastic Leukemia on Paired Mid-induction Peripheral Blood and Bone
Marrow Samples: Can Peripheral Blood Replace Bone Marrow Aspirate Sample?
M.U. Sachdeva, K. Bommannan, P. Bose, N. Varma, D. Bansal and R.K. Marwaha.
Postgrad. Inst. of Med. Educ. & Res., Chandigarh, India.
B80
201
Salivary Cytomics — A Useful Clinical Diagnostic Tool for Dental Professionals
Z. Chen and F.Y.S. Hou. Peking Univ. Sch. of Stomatol. and Marquette Univ.,
Milwaukee.
B81
202
Transfix®/EDTA Stabilization of Leukocytes in Cerebrospinal Fluid for Flow
Cytometric Screening of Patients with Suspected Leukemic Conditions T. Almond,
D. Harrison, M. Crawford and U. Johansson. Caltag Medsystems Ltd., Buckingham
and Bristol Royal Infirm., U.K.
B82
203
Development of 8 Color Panel for Lymphoma Diagnostics with Minimal
Compensation Requirements I. Vorobjev and O. Khoudoleeva. Moscow State Univ.
and GeneTechnol., Moscow.
ISAC 2013 Program and Abstracts
Speaker/Author
Index
197
Poster Session
Abstracts
B76
Oral Session
Abstracts
Flow Cytometry Detection of LAMP2 Protein in Danon Disease — A Rare X-Linked
Cardiomyopathy O. Pelak, J. Sikora, L. Krol, F. Majer, L. Dvorakova, H. Vlaskova,
T. Honzik, T. Palece, M. Kubanek and T. Kalina. Charles Univ. in Prague and Gen.
Univ. Hosp. and St. Anne’s Univ. Hosp. Brno, Czech Republic.
Commercial
Tutorials &
Exhibits
196
Poster
Session
B75
Wednesday,
22 May
Investigation of Protein-Coated Particle Aggregation Using Scanning Flow
Cytometry A. Polshchitsin, V. Nekrasov, A. Chernyshev and V. Maltsev. Inst. of Chem.
Kinet. and Combustion, SB RAS, Novosibirsk, Russia.
Tuesday,
21 May
195
Monday,
20 May
B74
51
Congress
Overview
Special
Lectures
B83
204
Analysis of Skeletal Muscle: Correlated Quantification of Mitochondrial Metabolic
Enzymatic Activities and Fiber Type-Specific Biomarkers P. McDonough, D. Reiner,
T. Kostrominova and R. Haas. Vala Sciences Inc., San Diego, WM&G Consulting,
Imperial Beach, CA, Sch. of Med., Northwest, Univ. of Indiana and UCSD.
205
Saturday,
18 May
Extended NK Cells Phenotyping in Patients with Acute Myeloid Leukemia
G. Bouvier, F. Orlanducci, C. Fauriat, E. Gautherot, F. Montero Julian, C. Arnoulet and
D. Olive. Beckman Coulter Life Sci. and Inst. Paoli Calmettes, Marseille.
B85
206
Correlation of Oxidant Status and Molecular Damage with Disease Activity Score
in Patients with Rheumatoid Arthritis S. Kundu, S. Datta, P. Ghosh, A. Ghosh, S.
Chattopadhyay and M. Chatterjee. Inst. of Post Grad. Med. Educ. & Res., Kolkata and
Bhabha Atomic Res. Ctr., Mumbai.
B86
207
Significant Antigens for Detection of Minimal Residual Disease in Patients with
Mantle Cell Lymphoma Using Flow Cytometry Approach J. Chovancova, M. Doubek
and J. Mayer. Masaryk Univ. Brno, Central European Inst. of Technol. and Fac. Hosp.
Brno, Czech Republic.
B87
208
Detection of Elevated Monocyte Proinflammatory Cytokine Responses by Flow
Cytometry in Previously Cryopreserved Samples from HIV+ Individuals at Risk
for Cardiovascular Disease E. Jalbert, N. Parikh, T. Seto, D. Chow, L. Ndhlovu, C.
Shikuma and J. Barbour. Hawaii Ctr. for HIV/AIDS, Honolulu and The Queen’s Med.
Ctr., Univ. of Hawaii.
Tuesday,
21 May
Monday,
20 May
B84
Sunday,
19 May
Disease Progression Monitoring
DNA Damage and Repair
Poster
Session
Wednesday,
22 May
B88
209
Quantitative Imaging Analysis of Replication - Vis-a-Vis DNA Damage-Sites in Cells
Exposed to DNA Targeting Anticancer Drugs and Oxidative Stress J. Dobrucki,
K. Berniak, P. Rybak, T. Bernas, A. Waligórska, M. Zarebski, E. Biela, H. Zhao and
Z. Darzynkiewicz. Jagiellonian Univ., Poland, Nencki Inst. of Exptl. Biol., Polish
Acad. of Sci., Warsaw and New York Med. Col.
B89
210
Establishment of Flow Cytometry Shared Resource at Children’s Research Institute
N. Loof. Children’s Res. Inst. at Univ. of Texas Southwestern.
Commercial
Tutorials &
Exhibits
B90
211
Algorithm for Calculating the Utilization and Efficiency of Sorting Service
A. Petrunkina. Univ. of Cambridge and Univ. of Vet. Med. Hannover.
B91
212
Identifying Challenges, Opportunities, and Strategies for Core Operations T. Fallows
and H. Lorenz. iLab Solutions, Boston.
Oral Session
Abstracts
Facility Management
B92
213
The Purdue Cytometry Email Discussion List J.P. Robinson and B. Rajwa. Purdue
Univ.
Poster Session
Abstracts
Flow Cytometry Instrumentation
214
Detection of the Fluorescence Lifetime of Green Fluorescent Protein Expressed in
Yeast Cells with a Time-Resolved Flow Cytometry F. Crawford, B. Sands, P. Jenkins,
R. Cao, W. Peria, R. Brent and J. Houston. New Mexico State Univ. and Fred
Hutchinson Cancer Res. Ctr., Seattle.
Speaker/Author
Index
B93
52
ISAC 2013 Program and Abstracts
218
The Importance of Area Scaling with FACS DIVA Software D. Haviland and
A. Hazen. Methodist Hosp. Res. Inst., Univ. of Texas Hlth. Sci. Ctr. at Houston.
B98
219
Nano-View Version II: An Improved Novel Approach to Microparticle Cell Sorting
V. Toxavidis, J. Tigges and K. Groglio. Beth Israel Deaconess Med. Ctr.
B99
220
8 Way Fluorescence-Activated Cell Sorting on the BD Influx™ S. Dervish, F. Kao,
S. Allen and A. Smith. Centenary Inst., Sydney.
B100
221
A High Throughput Flow Cytometric Assay for Rapid Quantitation and Detection of
Mouse IgG R. Danielzadeh and M. Krusemeier. Charisela Technol. Inc., Menlo Park,
CA and UCSF.
B101
222
Detection of Brother of the Regulator of Imprinted Sites Expression in Peripheral
Blood Neutrophils by Flowcytometry in Benign and Malignant Breast Lesions
N. El Sharkawy, W.M. Radwan, E.S. Eissa, S.H. Kandil and A.M. Kamel. NCI, Cairo
Univ. and Fac. of Med., Menofeya Univ., Egypt.
B102
223
Enhanced Far-Red Fluorescence Sensitivity in CCD Camera-Based Cytometers
Increases Threshold Antibody Titration B. McFarlin, K. Clise-Dwyer, K.E. Ruisaard,
K.J. Acklin and A. Venable. Univ. of North Texas, Denton and Univ. of Texas,
Houston.
B103
224
Withdrawn.
B104
225
Sorted or Agonized? H. Ulrich, I. Andrae, L. Henkel, D. Busch and M. Schiemann.
Tech Univ. Munich.
B105
226
Concentration Measurements Using Acoustic Cytometry J. Bradford, B. Dubbels,
A. Anderson and Y-Z. Zhang. Life Technologies, Eugene, OR.
B106
227
Low Cost Precision Pulsed LED for Flow Cytometer Calibration in Statistical
Photoelectron Units J. Wood. Wake Forest Univ.
B107
228
Improved Light Scatter Detection for Small Particle Analysis and Counting
D. Houck, B. Cao and D. Lindseth. BD Biosciences, San Jose, CA.
B108
229
Optimal Baseline PMT Voltages for Multicolor Staining: Optimizing CST Settings for
Cellular Samples B. McLaughlin. Univ. of California, Davis.
Oral Session
Abstracts
B97
Commercial
Tutorials &
Exhibits
Acoustically Enhanced Flow Cytometry for Remote Plankton Monitoring D.M. Kalb,
R.J. Olson, H.M. Sosik, M.E. Piyeasena and S.W. Graves. Univ. of New Mexico and
Woods Hole Oceanographic Instn.
Poster
Session
217
Wednesday,
22 May
B96
Tuesday,
21 May
Utilizing Plasmon Surface Resonance for Flow Cytometry M. Buescher, A. Esslinger,
J. Krieg, C. Peth and M. Nagel. Miltenyi Biotec, Bergisch Gladback, Germany.
Monday,
20 May
216
Sunday,
19 May
B95
Saturday,
18 May
Polarizing Light-Scattering Profile – Advanced Characterization of Non-spherical
Particles with the Scanning Flow Cytometry D. Strokotov, I. Polshchitcina,
A. Moskalensky, V. Nekrasov, A. Chernyshev and V. Maltsev. Inst. of Chem. Kinet.
and Combustion, SB RAS and Novosibirsk State Univ., Russia.
Special
Lectures
215
Congress
Overview
B94
Hematological Disorders
230
Important Role of Flow Cytometry Study in Diagnosis of Angioimmunoblastic T-Cell
Lymphoma X. Zhang. Geisinger Hlth. Syst., Wilkes-Barre, PA.
B110
231
Withdrawn.
Poster Session
Abstracts
B109
Speaker/Author
Index
ISAC 2013 Program and Abstracts
53
Special
Lectures
Novel Approach Using Imaging Cytometry to Monitor Red Blood Cell Surface
Area Loss and Splenic Retention M. Nguyen, I. Safeukui, P.A. Buffet, G. Deplaine,
S. Perrot, V. Brousse, A. Ndnour, O. Mercereau-Puijalon, P.H. David, G. Milon and
N. Mohandas. Inst. Pasteur, INSERM/UPMC, AP-HP Pitie-Salpetriere Hosp., AP-HP,
Necker Hosp., Paris and New York Blood Ctr.
B112
233
Saturday,
18 May
Congress
Overview
232
Combined Cell- and DNA-Based Analysis of Genetic Abnormalities in Multiple
Myeloma D. Alpar, B. Kajtar, D. de Jong, S. Savola, P. Jakso, L. Kereskai, L. Pajor and
K. Szuhai. Univ. of Pecs, Hungary, Leiden Univ., Netherlands and MRC-Holland,
Amsterdam.
B113
234
Plasma Cell Heterogeneity in Normal Bone Marrow J. van Velzen, M. Minnema and
A. Bloem. Univ. Med. Ctr. Utrecht, Netherlands.
B114
235
Separating Platelets and RBCs by FSC Amplitudes M. Krockenberger. Abbott
Hematol., Santa Clara, CA.
Sunday,
19 May
B111
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
High Content Analysis
B115
236
Application of 5 Multivariate Classification Methods to Compare Conventionally
Analyzed Multidimensional Flow Cytometry Data A. Donnenberg, V.S. Donnenberg
and D. Normolle. Univ of Pittsburgh Sch. of Med. and Grad. Sch. of Publ. Hlth.
B116
237
Modeling Subcellular Distribution from Chemical Structure D. Sullivan, E. Cesanek,
G.R. Rosania and R.F. Murphy. Carnegie Mellon Univ., Vassar Col. and Univ of
Michigan.
B117
238
Manipulating 30-50 Parameters in Real Time: High Content Analysis of Flow
Cytometry Data J.P. Robinson. Purdue Univ.
B118
239
A Novel High Content Microscopic Method for Mitochondrial Morphometry
A. Leonard, C. Beeson and B. Rohrer. Med. Univ. of South Carolina.
B119
240
CyTOFAnalyzer: A High Content Analysis Pipeline Developed via the PlateAnalyzer
Environment J.P. Robinson, V. Patsekin and B. Rajwa. Purdue Univ.
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
High Throughput Instrumentation
B120
241
Mega-Multiplexing Suspension Arrays J. Lu, Y. Lu, J. Zhao, E.M. Goldys, R.C. Leif,
J.A. Piper, J.P. Robinson and D. Jin. Macquarie Univ., Australia, Newport Instruments,
San Diego and Purdue Univ.
B121
242
Development of FAP Technology: Fluorogen Activated Protein in High Throughput
Cytometry D. Gebhard, S. Hallowell, E. Benvenuti and R. Doyonnas. Pfizer Inc.,
Groton, CT.
B122
243
A Mix-and-Read Cell-Based Assay for Antibody Screening against Epithelial Growth
Factor Receptor D. Caracino, W.P. Bowen, D. Onley, P. Wylie and T. Cope. TTP
Labtech, Cambridge, MA and Melbourn, U.K.
Speaker/Author
Index
Poster Session
Abstracts
High Throughput Screening
54
B123
244
Development of a Multiplex Oligonucleotide Ligation-PCRr Assay for the Detection
of Shiga Toxin Producing E. coli in the Beef Chain T. Woods, S.W. Graves and
A. Deshpande. Univ. of New Mexico and Los Alamos Natl. Lab.
B124
245
Single-Cell Nanotoxicity of Nanobarcoded Superparamagnetic Iron Oxide
Nanoparticles Quantified by High-Throughput Scanning Image Cytometry J. Leary
and T. Eustaquio. Purdue Univ. and Natl. Ctr. for Toxicol. Res., FDA, Jefferson, AR.
ISAC 2013 Program and Abstracts
247
Development of a Multiplexing, High-Throughput Technique for Initial Cell
Screening J. Tasset, P. Hexley, C. Robinson, A. Osterburg, C. Fu and G. Babcock.
Univ. of Cincinnati, Shriners Hosps. for Children, Cincinnati and Wright Patterson Air
Force Base, Dayton, OH.
B127
248
Differences in Whole Blood Light Scattering from Multiple Laser Excitation Sources:
A Rapid No-Lyse, No-Wash Method Using the Attune® Acoustic Focusing Cytometer
with Red or Violet Laser Option A. Dickson and J. Bradford. Life Technologies,
Eugene, OR.
B128
249
Complex Cell Based Assays with a Novel Imaging Cytometry System O. Sirenko,
S. Boege, J. Hesley, D. Henderson, A. Cohen, M. Starodynov, E. Cromwell and
P. Comita. Molecular Devices, Sunnyvale, CA.
Sunday,
19 May
B126
Saturday,
18 May
Flow Cytometry as a Drug Screening Platform R. Jepras, P. Shah, M. Patel, E. Sutton
and S. Ludbrook. Glaxosmithkline, Stevenage, U.K.
Special
Lectures
246
Congress
Overview
B125
Image Processing and Analysis
Detecton of Internalized Exosomes by Tumor Cells Using Amnis ImageStreamX
P. Simms, C. Franzen, V. Volgina and G. Gupta. Loyola Univ. Chicago, Maywood.
B130
251
Cellometer Image Cytometry as a Complementary Analysis Tool to Flow Cytometry
for Visual Verification of Gated Cell Populations L. Chan, D. Kuksin, C. Kuksin and
J. Qiu. Nexcelom Bioscience LLC, Lawrence, MA and Univ. of Massachusetts
Amherst.
B131
252
Effects of Flow Cytometry on the Physical Appearance of Flow Cytometry
Professionals C.K. Becker, M. Becker, P.R. Becker and B. Becker. Phoenix Flow Systs.
Inc., San Diego.
Tuesday,
21 May
250
Monday,
20 May
B129
Wednesday,
22 May
Immune Monitoring
Functional Defects in the Immune Cells Responses in Erdheim-Chester Disease
W. Tsai, K. Davis, J. Estrada-Veras, R. Wang, B. Gochuico, W. Gahl and M. Gadina.
NIAMS and NHGRI, NIH.
B134
255
Tumor Exome Analysis Reveals Neo-antigen-Specific T Cell Reactivity in an
Ipilimumab-Responsive Melanoma P. Kvistborg, N. van Rooij, M. van Buuren,
D. Philips, A. Velds, M. Toebes, L. van Dijk, S. Behjati, H. Hilkmann, D. el Atmioui,
M. Nieuwland, M. Stratton, R. Kerkhoven, C. Kesmir, J. Haanen and T. Schumacher.
Netherlands Cancer Inst., Amsterdam, Wellcome Trust Sander Inst., Cambridge and
Utrecht Univ., Netherlands.
B135
256
Altered Mitochondrial Functional Response to Activation in T Cells of the Neonate
G. Meszaros, A. Kaposi, B. Gyarmati and B. Vasarhelyi. Semmelweis Univ.,
Hungarian Acad. of Sci. and Uzsoki Street Hosp., Budapest.
B136
257
Assays to Measure Natural Killer Cell Function in Cynomolgus Macaques
C. Donovan, S. Haskett, C. Homiski and C. Kamperschroer. Pfizer Drug Safety R&D,
Groton, CT.
B137
258
Rapid and Semi-automated Monitoring of Antigen-Specific T Cell Immunity
M. Herber, M. Niemöller, K. Lange, S. Weber-Lohmann, S. Höher-Peters, M.
Assenmacher and A. Richter. Miltenyi Biotec GmbH, Bergisch-Gladbach.
ISAC 2013 Program and Abstracts
Speaker/Author
Index
254
Poster Session
Abstracts
B133
Oral Session
Abstracts
Investigation of Immune System Involvement in Prosthetic Implant Failure with
Polychromatic Flow Cytometry P. Court, D. Ebreo, S. Donell and S. Carding. Inst. of
Food Res., Norwich and Norfolk and Norwich Univ. Hosp., U.K.
Commercial
Tutorials &
Exhibits
253
Poster
Session
B132
55
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
B138
259
Using Image-Based Flow Cytometry to Simultaneously Measure Granulocyte
Phagocytosis and Oxidative Burst: A Beginner’s Guide R. Williams, A. Venable and
B. McFarlin. Univ. of North Texas, Denton.
B139
260
Following Reconstitution of CMV Immunity in Allogeneic Hematopoietic Cell
Transplantation Patients K. Jacobsen, L. Brix, D. Pan, C. Halgreen, T. Hahn,
P. McCarthy and P.K. Wallace. Immudex, Copenhagenk and Roswell Park Cancer
Inst., Buffalo.
B140
261
Mass Cytometry for Multiparametric Immunophenotyping of Seasonal Flu Vaccine
Recipients M. Leipold, M. Davis and H. Maecker. Stanford Univ.
B141
262
Multi-Color Flow Analysis of Subsets of PBMC for TLR Ligand Stimulation S. Chitta,
H-K. Lee, P. Quintel, G. Singh, J. Rosernberg and S. Singh. Imgenex Corp., San
Diego.
B142
263
The Prevalence of Intracellular Galectin-1-Expressing Lymphocytes in Umbilical
Cord Blood in Comparison with Adult Peripheral Blood G. Toldi, S. Kollár, J. Rigó, Jr.,
T. Tulassay and B. Vásárhelyi. Semmelweis Univ., Hungary.
B143
264
Simultaneously Measuring 18 Human Cytokines on a Conventional Flow Cytometer
Y. Song and S. Luo. YSL Bioprocess Develop. Co., Pasadena.
B144
265
Technical Advancements Allowing Improved Selection of True, Viable, Regulatory
T Cells J. Tigges, V. Toxavidis and K. Groglio. Beth Israel Deaconess Med. Ctr.
B145
266
Identification and Functional Characterization of Four Subsets of Renal
Mononuclear Phagocytes in Healthy and Diseased Kidney X. Wang, Q. Cao, Y. Wang
and D. Harris. Westmead Millennium Inst., Australia and Univ. of Sydney.
B146
267
Quality Assurance for Bone Marrow Aspirate Specimens from Non-human Primates
C. Porretta, R. Siggins II, S. Cormier and G. Bagby. LSU Hlth. Sci. Ctr., New Orleans.
B147
268
18-Color Flow Cytometry: Immunophenotyping Cellular Infiltration during
Flavivirus Encephalitis in the Mouse T. Ashhurst, C. van Vreden, M. Karimi
Azardaryany, K. Lundsten, S. Allen, S. Dervish, F. Kao, A. Smith and N. King. Univ. of
Sydney and BioLegend, San Diego.
B148
269
The Regulatory Role of NLRC5 in MHC I Expression in B Lymphocytes A. Veres,
L. Mátyus, S. Benko and A. Jenei. Univ. of Debrecen, Hungary.
B149
270
Modulating in Vivo T-Cell Activation: 15 Color Immunophenotyping, Cytokine
Analysis, and Cellular Redistribution K. Lundsten, M. Tam, J. Ampudia, N. Urbina,
J. Ransom and G. Lay. BioLegend, San Diego.
B150
271
A Longitudinal Flow Cytometry–Based Study on Phenotypic Changes of
Cryopreserved Murine T Cells Using the BD FACSVerse™ System Y. Wang, S. Cai,
W. Feng and R. Jana. BD Biosciences, San Jose, CA and Stanford Univ. Sch. of Med.
B151
272
Defining CD4+ T-Cell Subsets Using Probability State Modeling M. Inokuma,
J. Trotter, E. Hill, B. Hunsberger, M. Munson, D. Herbert, C. Bray, S. Ghanekar,
V. Maino and C.B. Bagwell. BD Biosciences, San Jose, CA and Verity Software House,
Topsham, ME.
B152
273
Immunophenotyping in Clinical Translational Studies Using Brilliant Violet Dye
Conjugates D. Roumanes, C. Baker, S. Secor-Socha, Y. Qi, A. Dunham, T. Mosmann
and S. Quataert. Univ. of Rochester Med. Ctr.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Immunology
56
ISAC 2013 Program and Abstracts
Congress
Overview
Infectious Diseases
B153
274
Special
Lectures
A Flow Cytometric Method for Intracellular Detection of HIV-1 P24 Core Antigen
Used to Investigate the Prevention of HIV-1 Transfer from Dendritic Cells to CD4 T
Cells by Neutralizing Antibodies V. Holl, B. Su, A. Lederle, M. Peressin, V. Glutz,
M. Lambotin and C. Moog. Covance, Geneva and INSERM U748, Strasbourg.
Live Cell Imaging/Tracking
B155
276
Determining RNA Expression at the Single Cell Level in Live Cells Using Flow
Cytometry D. Weldon, A. Ko, Y. Williams, L. White, L. Armstrong and V. Koong.
EMD Millipore, Temecula, CA.
B156
277
Simultaneous Tracking of Cell Type, Viability, Proliferation and Expression of
Intracellular Proteins in Co-cultures of Leukemia and HS-5 Stroma Cells Using
Multiparameter Flow Cytometry K. Piwocka, P. Podszywalow-Bartnicka,
M. Brewinska-Olchowik, L. Bugajski and M. Kusio-Kobialka. Nencki Inst. of Exptl.
Biol., Warsaw.
Monday,
20 May
Multiplexing Analysis of Cell Proliferation and Cellular Functions Using a New
Multicolor Panel of Fluorescent Cell Proliferation Dyes Z. Diwu, Q. Zhao, Y. Wu
and J. Liao. AAT Bioquest Inc., Sunnyvale, CA.
Sunday,
19 May
275
Saturday,
18 May
B154
Microbiology and Aquatic Sciences
Scanning Flow Cytometry for Static and Dynamic Characterization of E. coli Cells
A. Konokhova, M. Yurkin and V. Maltsev. Inst. of Chem. Kinet. and Combustion,
SB RAS and Novosibirsk State Univ., Russia.
B158
279
Moflo Astrios™ Forward Scatter: Cell Sorting of Nano and Large Phytoplankton
Simultaneously with High Purity C. Ross, A. Dean and R. Morris. Beckman Coulter
Life Sci. Div., Fort Collins, CO.
B159
280
Development of a Plankton Cell Sorter Utilized with the Amnis ImageStream
G. Wiegand. Estuary Biophotonics, Baltimore, MD.
Wednesday,
22 May
278
Tuesday,
21 May
B157
Poster
Session
Microelectro-Mechanical Systems (MEMS) and Microfluidics
B161
282
Chip Based Impedance Flow Cytometer with Integrated Acoustophoretic Sample
Preconditioning C. Grenvall, C. Antfolk, C. Zoffmann Bisgaard and T. Laurell. Lund
Univ., Sweden and FOSS Analytical A/S, Denmark.
B162
283
Microfluidic Fluorescence-Activated Cell Sorter Employing ‘Space-Time’ Coding
and On-Chip Piezoelectric Actuator. M. Arámbula, S.H. Cho, D. Johnson, R. Alon,
P. Buerki, P. Poonka, K. Chuang, Y-H. Lo, Z. Olson and J. Morachis. NanoCellect
Biomed. Inc., San Diego, UCSD and Natl. Instruments, Lake Forest, CA.
B163
284
Optofluidic Flow Cytometer Employing Color-Space-Time Coding: On-Chip
Multiple Fluorescence Differentiation S.H. Cho, P. Poonka, K. Chuang, P. Buerki,
M. Arambula, Z. Olson, J. Hanks, Y-H. Lo and J. Morachis. NanoCellect Biomed. Inc.,
San Diego, Natl. Instruments Corp., Austin, TX and UCSD.
Poster Session
Abstracts
Human Organ-on-a-Chip BioMEMSs Devices for More Rapid Testing of New
Multimodal in Vivo Diagnostic Imaging and Therapeutic Strategies J. Leary, P-A. Vidi,
C. Cooper and S. Lelievre. Purdue Univ.
Oral Session
Abstracts
281
Commercial
Tutorials &
Exhibits
B160
Speaker/Author
Index
ISAC 2013 Program and Abstracts
57
Congress
Overview
Multi-dimensional Image Cytometry
B164
285
Special
Lectures
Rapid Method for Evaluating Micronuclei Formation Using ImageStreamX
A.N. White, A. Sullivan, S. Thornton and S. Pfuhler. Cincinnati Children’s Hosp. Med.
Ctr. and Procter & Gamble, Mason, OH.
B165
286
High-Throughput Image Analysis Software Applied to High-Content Neuronal
Screens D. Logan and A. Carpenter. Broad Inst. of Harvard and MIT.
B166
287
PerFix-nc (No Centrifuge Assay): The New Intracellular No-Wash Assay with
Extended Capabilities F. Malergue, L. Khemici and F.A. Montero-Julian. BeckmanCoulter Immunotech, Marseille.
B167
288
Development of Novel Metal-Chelating Polymers for Mass Cytometry D. Majonis,
X. Lou, O.I. Ornatsky, V. Baranov and S.D. Tanner. DVS Sciences Inc., Markham and
Richmond Hill, ON.
B168
289
A Novel Rapid Apoptosis Assay Based on Thiol Redox Status L. Johansen, S. Kjærulff
and M. Skindersø. Chemometec, Allerod, Denmark.
B169
290
New Fluorophore for Violet Laser Excitation J. Bradford, W. Zhou, B. Dubbels,
B. Bone, A. Anderson and K. Gee. Life Technologies, Eugene, OR.
B170
291
Brilliant UltraViolet™ Dyes: A New Set of High Sensitivity Fluorescence Reporters
for Multicolor Flow Cytometry with a Uv Laser B. Gaylord, Y. Liang, F. Uckert,
B. Leonard, H. Li, L. Tran, G. Bartholomew, Y. Chen, F. Luan, S. Widmann and
A. Stall. BD Biosciences, San Diego.
B171
292
Monitor Caspase 3/7 Activity without Cell Fixation: A Novel Apoptosis Reagent
from Molecular Probes® B. Seredick, J.A. Bradford and M. Olszowy. Life
Technologies, Eugene, OR.
B172
293
Reactive Oxygen Probes — A Broad Range of Colors with Easier Labeling and
Compatibility with Fixation: Novel CellROXx® Reagents from Molecular Probes®
B. Seredick, B. Bone and M. Olszowy. Life Technologies, Eugene, OR.
B173
294
Barcoding with Lipophilic Dyes Can Further Increase the Throughput of Flow
Cytometry Screening O. Sharif, J. Gilligan, P. Anderson, C. Trussell, E. Ainscow and
J. Joslin. Novartis (GNF), San Diego.
B174
295
Ptychography — Label-Free Imaging of Dividing Cells Using Quantitative Phase
Information P. O’Toole, K. Hogg and J. Marrison. Univ. of York, U.K.
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
New Probes and Assays
New Software Development
Oral Session
Abstracts
B175
296
High-Fidelity Data Transfer: The Secret to Pipelining and Automated Cytometric
Analysis M. Stadnisky, J. Quinn, J. Almarode and A. Treister. Tree Star Inc., Ashland,
OR.
Speaker/Author
Index
Poster Session
Abstracts
Other Biological Applications
58
B176
297
The Dipole Potential Influences the Clustering of ErbB Proteins T. Kovacs, A. Szabo,
J. Szollosi and P. Nagy. Univ. of Debrecen, Hungary.
B177
298
Flow Cytometric Measurement of the Chromosomal DNA Content and the Genome
Size of the Tasmanian Devil B.L. Ng, E. Murchison and D. Adams. The Wellcome
Trust Sanger Inst., Cambridge, U.K.
ISAC 2013 Program and Abstracts
B179
300
Using of Combination Cytophotometrical Method for the Determination of Dry
Mass, Glycogen and DNA Contents of Hepatocytes in Normal and Cirrhotic
Rat Liver A. Chestnova, N. Bezborodkina and B. Kudryavtsev. Inst. of Cytol., St.
Petersburg, Russia.
B180
301
Comparison of Viability and Functionality of Sorted Human Dendritic Cells Using
Two Types of Flow Cells M. Jaimes, D. Gorgone, D. Ellemore, D. Soni, P. Norton,
D. Vrane, S. Waheed, J. Crane and K. Pennebaker. BD Biosciences, San Jose, CA.
Saturday,
18 May
Flow Cytometric Standarization for the Analysis of Microparticles V. Toxavidis,
J. Tigges and K. Groglio. Beth Israel Deaconess Med. Ctr.
Special
Lectures
299
Congress
Overview
B178
Other Clinical Applications
303
The Effect of Nucleotide Supplementation on Atlantic Salmon Intestine J. Bierla,
W. Ladno, M. Kamaszewski, T. Ostaszewska, D. Martinez-Puig, C. Chetrit, E. Borda
Casas and R. Zabielski. Med. Univ. of Warsaw, Warsaw Univ. of Life Sci. and
Bioiberica, Barcelona.
B183
304
Preliminary Results in Tissue Cytometry Investigation of Apelin Influence on Young
Rats Digestive System J. Bierla, H. Antushevich, M. Kapica, B. Pawlina, A. Kuwahara
and R. Zabielski. Med. Univ. of Warsaw, Polish Acad. of Sci., Univ. of Life Sci. in
Lublin, Poland, Univ. of Shizuoka, Japan and Warsaw Univ. of Life Sci.
B184
305
Preliminary Results of Ion Channels Expression in Pyramidal Cells of Prefrontal
Cortex 20-Day-Old Rats. J. Bierla, G. Witkowski, W. Ladno, I. Wieczorkowska,
B. Greszta and P. Szulczyk. Med. Univ. of Warsaw Fac. of Pharm.
Wednesday,
22 May
B182
Tuesday,
21 May
A Flow Cytometric Study on DEMRON-Mediated Radioprotection in Mice
Z. Sinkorova, L. Zarybnicka, L. Navratil and P. Castulik. Univ. of Defence, Czech Tech
Univ. and Masaryk Univ. Brno, Czech Republic.
Monday,
20 May
302
Sunday,
19 May
B181
Other Technology Advances
Simultaneous Analysis of Total and Phospho Proteins in Single Cells for Accurate
Assessment of Intracellular Cell Signaling Events M. Santos, M. Bader, P. deBorja,
A. Barican, D. Luong, R. Lefebvre and M. Hsu. EMD Millipore Corp., Temecula and
Hayward, CA.
B187
308
SWOFF — The Unrecognized Sibling of FMO M. Kapinsky. Beckman Coulter Inst.,
Nittendorf, Germany.
B188
309
Evaluation of Multiplexed RNA Flow Cytometry in HIV-Infected Cells M.B. Hanley,
E. Park and V. Maino. BD Biosciences, San Jose, CA.
B189
310
Tumor Histoids: Scalable Production of Living Human Mini-tumors for High
Throughput Drug Screening M. Ingram, G. Techy, S.A. Imam, J. Nolan and B. Ward.
Huntington Med. Res. Insts., Pasadena, CA and La Jolla Bioengineering Inst.
B190
311
Single Cell Analyses of Natural Killer Cell Development with Fluidigm Microfluidic
Devices: A Work in Progress D. Redelman, V. Lombardi, L. Peri and J. Townsend.
Univ. of Nevada Sch. of Med., Reno.
ISAC 2013 Program and Abstracts
Speaker/Author
Index
307
Poster Session
Abstracts
B186
Oral Session
Abstracts
The Feasibility of Using Tunable Thin-Film Optical Filters for Excitation - or EmissionScanning Hyperspectral Microscopy P. Favreau, T. Rich, A. Stringfellow, D. Alvarez,
P. Prabhat and S. Leavesley. Univ. of South Alabama and Semrock Inc., a Unit of IDEX
Corp., Rochester, NY.
Commercial
Tutorials &
Exhibits
306
Poster
Session
B185
59
Congress
Overview
Special
Lectures
Bridging the Gap between Plate Reader Assays and High Content Imaging J. Hesley,
S. Boege, B. Wade, J. Dzubay, O. Sirenko, J. Itatani and P. Comita. Molecular Devices
LLC, Sunnyvale, CA.
B192
313
EdU Cell Proliferation Improved Compatibility with GFP and Other Fluorescent
Proteins S. Clarke, H. Frend, J. Sordet-Dessimoz, U. Singh and K. Gee. Life
Technologies, Eugene, OR, Cambridge Univ., U.K. and Fed. Inst. of Technol.,
Lausanne.
Saturday,
18 May
312
Rare Event Detection
B193
314
Progress towards Using Lanthanide Nanoparticles as Isotope Tags for Mass
Cytometry P. Cao, A. Halupa, L. Tong, G. Zhao, P. Chattopadhyay, M. Roederer,
M. Winnik and M. Nitz. Univ. of Toronto and NIAID, NIH.
Sunday,
19 May
B191
B194
315
RNA Flow Cytometry for the Analysis of Rare Tumor Cells Present in Blood
C. Lomas, D. Mittar, W. Zhu, J. Lang and E. Park. BD Biosciences, San Jose, CA and
Norris Comprehen. Cancer Ctr., Univ. of Southern California.
316
Linolenic Acid Counteracts TNF Negative Effects on Skeletal Muscle Cells L. Teodori,
F. Carotenuto, M.C. Albertini, M. Rocchi, D. Coletti, A. Costa, M. Minieri and
Di Niardo. ENEA-Frascati, Rome, Fndn. San Raffaele Pisana, Rome, Univ. of Rome
Tor Vergata, Univ. of Urbino “Carlo Bo”, Univ. Pierre et Marie Curie, Paris 6, Univ. of
Rome Sapienza and Link Campus Univ. of Rome.
B196
317
Wednesday,
22 May
Functionalization of Decellularized Scaffold for Skeletal Muscle Tissue Engineering
A. Costa, P. Aprile, P. Aulino, B. Perniconi, S. Adamo, D. Coletti and L. Teodori.
Sapienza Univ. of Rome, Tor Vergata Univ. of Rome, ENEA-Frascati, Rome and Univ.
Pierre et Marie Curie, Paris 6.
B197
318
Exploiting Vasopressin Signaling in Muscular Atrophy P. Aprile, A. Costa, B.M.
Scicchitano and S. Adamo. Tor Vergata Univ. of Rome, ENEA-Frascati and Sapienza
Univ. of Rome.
Signal Transducton
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Tuesday,
21 May
B195
Poster
Session
Monday,
20 May
Regenerative Medicine
B198
319
PerFix-EXPOSE (Phospho Epitopes Exposure Kit): A New Fast and Easy Procedure for
Cell Signaling Analysis F. Malergue, V. Mallet, C. Scifo, A. Van Agthoven and
F.A. Montero-Julian. Beckman-Coulter Immunotech, Marseille.
B199
320
Quantitative Microscopic Analysis Reveals Cell Confluence Regulated Divergence of
PDGFR-Initiated Signaling Pathways with Logically Streamlined Cellular Outputs
A. Szoor, L. Ujlaky-Nagy, J. Szollosi and G. Vereb. Univ. of Debrecen, Hungary.
Small Molecule Discovery
Poster Session
Abstracts
B200
321
Development of a Cytomics-Based Mitochondrial Signaling Assay for Hazard
Identification of Small Molecule Drug Candidates N. Li, Y. He, H. Ingle,
J.P. Robinson, J. Davisson, H. Hamadeh, C. Afshari and P. Narayanan. Amgen,
Seattle and Thousand Oaks, CA and Purdue Univ.
Speaker/Author
Index
Solid Tumors
B201
60
322
Cyclooxygenase-Independent Chemoprevention of Colorectal Cancer by Nonsteroidal Anti-inflammatory Drugs V. Vaish, and S. Sanyal. Panjab Univ., India.
ISAC 2013 Program and Abstracts
324
The Effect of Hypoxia on the Efficiency of Elisidepsin T. Varadi, A. Kiraly, T. Hajdu,
J.M. Molina-Guijarro, J. Szollosi, C. Galmarini and P. Nagy. Univ. of Debrecen,
Hungary and PharmaMar, Madrid.
B204
325
Integrative Analysis of Genomic and Gene Expression Data of Paired Primary and
Metastatic Melanoma M. Balazs, L. Vizkeleti, S. Ecsedi, G. Emri, V. Koroknai, T. Kiss
and R. Adany. Med. and Hlth. Sci. Ctr., Univ. of Debrecen, Hungary.
B205
326
Selective Internalization of a Novel Recombinant Human Granzyme B by
Membrane Hsp70 Positive Tumor Cells and Its Cytotoxic Consequences: A Novel
Therapeutic Approach for Cancer? A.G. Pockley, G. Foulds, M. Gehrmann, B. Doss
and G. Multhoff. Nottingham Trent Univ., Univ. of Sheffield, Tech. Univ. Munich and
German Res. Ctr. for Envrn. Hlth. Munich.
Sunday,
19 May
B203
Saturday,
18 May
TGFb1 Increased Migration Characteristics and Induced Different Stemness in A549
Cell Fractions Sorted for CD133 Surface Expression and Side Population Phenotype
G. Pirozzi, V. Tirino, R. Camerlingo, E. Irollo, R. Montella, F. Paino, G. Sessa,
M.V. Carriero, N. Normanno and G. Rocco. INT Fndn. G Pascale and Second Univ.
of Naples.
Special
Lectures
323
Congress
Overview
B202
Monday,
20 May
Standards and Calibration
328
Comparison of LED and Microsphere Methods for Estimating Photoelectron Scales
in Flow Cytometers and Use of LED Signals to Compare Efficiencies of PMTs
D. Parks, E. Chase, M. Bigos and W.A. Moore. Stanford Univ. and Cytek Develop.
Inc., Freemont, CA.
B208
329
Microparticle Analysis by High-Sensitivity Flow Cytometry: Out-of-Routine
Detection below the Standardized 0.3µm-eq FSC Cut-Off Is Feasible and Reveals
More Small Vesicles P. Poncelet, S. Robert, R. Lacroix and F. Dignat-George.
Biocytex, VRCM, INSERM UMR-S1076 and La Conception Hosp., AP-HM, Marseille.
B209
330
Performance Benchmarking the Detection Sensitivity of Image Cytometers Using
Fluorescent Glass M. Halter, P. DeRose, G. Cooksey, A.L. Plant and J. Elliott. NIST,
Gaithersburg, MD.
B210
331
A Strategy for Comparing Antibody-Based Measurements Using a Cell-Based
Reference Standard in Flow and Image-Based Cytometry G. Cooksey, J. Elliott and
A. Plant. NIST, Gaithersburg, MD.
B211
332
Characterization of Fluorescence Detection Resolution Limit in Flow Cytometer
M. Yan and A. Zhong. BD Biosciences, San Jose, CA.
Commercial
Tutorials &
Exhibits
B207
Poster
Session
A Simple Spreadsheet-Guided Method for Evaluating Cytometer Measurement
Linearity and Generating Correction Tables for More Accurate Results D. Parks,
M. Bigos and W.A. Moore. Stanford Univ.
Wednesday,
22 May
327
Tuesday,
21 May
B206
Oral Session
Abstracts
Stem Cells
Multicolour Flow Cytometry Panel for Identification of Human Mesenchymal Stem
Cells I.L. Pieper, J.C. Bishop, S. Sultan, C.A. Thornton and G. Morgan. Col. of Med.,
Swansea Univ., U.K. and Cell Therapy Ltd., Cardiff, U.K.
B213
334
ProtocolNavigator – Interpreting Provenance in Stem Cell Cytometry I. Khan,
A. Fraser, M-A. Bray, P. Smith, P. Stephens, A. Sloan, A. Carpenter and R. Errington.
Sch. of Med., Cardiff Univ., U.K. and Broad Inst. of MIT and Harvard.
ISAC 2013 Program and Abstracts
Speaker/Author
Index
333
Poster Session
Abstracts
B212
61
Congress
Overview
B214
335
Special
Lectures
Vg9Vd2 T Cell Cytotoxicity against Oral Tumors Is Enhanced in the Presence of
Monoclonal Antibody B11F12 A. Anand and S. Chiplunkar. Adv. Ctr. for Treatment
Res. and Educ. in Cancer, Tata Mem. Ctr., Mumbai.
B215
336
Effect of Silibinin on Stemness Properties in 3D Model of Breast Cancer Cells
P. Abdollahi, M. Ebrahimi and N. Motamed. Fac. of Sci., Univ. of Iran and Royan Inst.
for Stem Cell Biol. and Technol., Tehran.
B216
337
Phosphorylated CrkL Level in Association to P-Glycoprotein (Pgp) Activity Identifies
Imatinib Sensitive Chronic Myeloid Leukemia Patient Samples F.C. Vasconcelos,
G.N. Moraes, A.L. Mencalha and R.C. Maia. Brazilian Natl. Cancer Inst., Rio de
Janeiro.
Sunday,
19 May
Tissue Cytometry/Morphometry
Monday,
20 May
Tools: Chemical Probes and Fluorescent Proteins
Tuesday,
21 May
Saturday,
18 May
Therapeutics
Toxicology
B217
B218
339
FRET as a Spectroscopic Ruler Revisited with Multiple Fluorophores T. Rente,
J. Szollosi, L. Matyus and A. Jenei. Univ. of Debrecen, Hungary.
Detection of Silver and Titanium Dioxide Nanoparticles Using Flow Cytometry Light
Scatter and Darkfield Microscopy R. Zucker, L. Degn, J. Ortenzio, and W. Boyes.
U.S. EPA, Research Triangle Park.
Wednesday,
22 May
340
Tissue Cytometry Based on Chipcytometry: Strengths, Limitations and Applications
C. Hennig and A. Mirenska. Hannover Med. Sch., Germany.
Vaccines
Poster
Session
B219
338
Late-Breaking Abstracts
341
The Effect of Alternative Oxygen Environments on Immunotherapeutic Dendritic
Cell Vaccines J. Tario, Jr., C. Choi and P. Wallace. Roswell Park Cancer Inst., Buffalo.
B221
343
Plateanalyzer: Gate-Free Comparison of Phenotypes for Automated High
Throughput Flow Cytometry J.P. Robinson, V. Patsekin, E. Foo, J. Sturgis and
B. Rajwa. Purdue Univ.
B222
344
Plateanalyzer: Tracking Signaling Pathways from Multivariate Flow Cytometry
Listmode Data J.P. Robinson, V. Patsekin and J. Sturgis. Purdue Univ.
B223
345
A Single Laser Flow Cytometer: Simplifying Multicolor Immunophenotyping Using
Brilliant Violet Dyes J. Rohrer, L. Duckett, B. Gaylord, D.T. Sasaki, A. Stall,
A.A. Nguyen and J. Vidal. BD Biosciences Inc.
B224
346
Particle Characterization of Therapeutic Protein Formulations: A Comparison of
Flow and Imaging Cytometry with Industry Standard Technologies. K. Kelley, N. Ball,
T. Dillon and J. Ferbas. Amgen Inc.
B225
347
Intact Cell-Based Analysis and Screening by Combination of Flow Cytometry and
Fluorescence Lifetime Measurement M. Suzuki, K. Koike, I. Sakata, N. Nemoto,
T. Sakai and K. Nishigaki. Grad. Sch. of Sci. and Engin., Saitama Univ., Japan.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
B220
62
ISAC 2013 Program and Abstracts
B230
352
Orientation of Erythrocytes for Flow Cytometry O. Jakobsson, C. Grenvall and
T. Laurell. Lund Univ., Sweden.
B231
353
Flow Cytometric Sorting of Spermatozoa as a New Strategy to Improve the Quality
of Spermatozoa in Assisted Reproductive Medicine S. Forte, G. Sartorius, F. Pletscher,
M. De Geyter, H. Zhang and C. De Geyter. Univ. Hosp. of Basel and Univ. of Basel.
B232
354
Resolution of FOXP3+ Treg Cells Is Facilitated by Use of CD6 and PerFix-nc Kit
F. Monsonis, J. Tung, J. Quintana, C. Garcia Santana and W. Godfrey. Beckman
Coulter.
B233
355
VersaComp Antibody Capture Beads — A Versatile and Advanced Compensation
Bead Product W. Godfrey, L. Yang, M. Rogers and J. Tung. Beckman Coulter.
B234
356
Reasons to Stop Collecting List Mode Data M. Naivar. DarklingX LLC.
B235
357
Hepatobiliary Transporters in Drug Safety Assessment R. Morgan. Amgen, Thousand
Oaks, CA.
B236
358
Novel Flow Cytometry Method for the Detection of Neutrophil Activation and
Degranulation in Peripheral Human Whole Blood D. Polancec, M. Vrancic, S.
Dupont, K. Oreskovic and V. Erakovic Haber. Fidelta , Croatia, Galapagos SASU,
France and Univ. of Rijeka, Croatia.
B237
359
An LED Pulser for Standardized Measurements of Fluorescence and Simplified Panel
Development S. Perfetto, P. Chattopadhyay, R. Nguyen, D. Ambrozak, M. KuKuruga,
M. Roederer and J. Wood. NIAID, NIH, USDA, Bethesda and Wake Forest Univ.
B238
360
Dual-Color Dynamic Live Cell-Based Assays for Autophagy C. Chen, T. Gaige,
I. Zubonja and P. Hung. EMD Millipore.
B239
361
The S3™ Prodrop™ System, a Novel Approach to Automated Drop Delay
Measurement and Monitoring C. Oxford, K. Kroeger, M. Ma, A. Vandergaw, D. Fox
and S. Hunter. Bio-Rad Labs. and Propel Labs.
B240
362
4D Analysis of Akt Signaling in Breast Cancer K. Chin, M. Nederlof, M. CrapsterPregont, S. Kwon, S. Boddapati, K. Iljin, D. Sudar, R. Baehner, E. Barklis and J. Gray.
Oregon Hlth. & Sci. Univ., UCSF, Quatitative Imaging Systs. LLC and Lawrence
Berkeley Natl. Lab.
B241
363
Reduced Sample Loss by Remedial Engineering of BD Fortessa and LSR II Analyzers
J. Hendrikx, M. Kweens, T. Baumgartner and C. O’Donnell. Mem. Sloan-Kettering
Cancer Ctr.
ISAC 2013 Program and Abstracts
Speaker/Author
Index
Multi-step Quality Control for a Multidimensional Cytometric Data Set of Clinical
Importance S. Melzer, J. Bocsi, A. Szabó, A. Mittag, I. Dähnert and A. Tárnok. Univ.
of Leipzig.
Poster Session
Abstracts
351
Oral Session
Abstracts
B229
Commercial
Tutorials &
Exhibits
ZAP-70 and IgVH Mutation Status in Indian Patients with Chronic Lymphocytic
Leukemia R. Gupta, L. Rani, N. Mathur, S. Vishnubhatla, A. Sharma, L. Kumar and
V. Raina. All India Inst. of Med. Sci.
Poster
Session
350
Wednesday,
22 May
B228
Tuesday,
21 May
Cytomics – Importance of Multimodal Analysis of Cell Function and Proliferation in
Biomonitoring M. Boumhras, B. Nasser, T. Nury, M. Cherkaoui-Malki and G. Lizard.
Univ. of Hassan I Fac. of Sci. and Technol., Morocco and Univ. of Bourgogne, Dijon,
France.
Monday,
20 May
349
Sunday,
19 May
B227
Saturday,
18 May
Assessment of Neutrophil Activation Antigen Up-Regulation in Whole Blood on the
BD FACSVerse™ Cytometer Y. Zeng, B.R. Lee, M. Paulsen, J. Thorpe, L. Zhu and
J.H. Allaert. BD Biosciences.
Special
Lectures
348
Congress
Overview
B226
63
Congress
Overview
364
High Frequency of T Memory Stem Cells Precedes T Cell Immune-Reconstitution
Following Human Bone Marrow Transplantation E. Lugli, A. Roberto, L. Castagna,
S. Gandolfi, M. Roederer and D. Mavilio. Humanitas Clin. and Res. Ctr., Milan and
NIAID, NIH.
Special
Lectures
B243
365
A New, Automated Approach to Quality Control of High-Throughput Flow
Cytometry Data P. Chattopadhyay and C. Fletez-Brant. NIAID, NIH.
B244
366
Genedata Screener Cell Population Extension: Interactive Processing of Single-Cell
Data for Plate-Based High-Throughput Flow Cytometry J. Tupy, P. Aiello and
O. Leven. Genedata, Switzerland.
B245
367
MoFlo Astrios Enhanced Forward Scatter: Simultaneous Sorting of Micro- and
Macro-Samples A. Dean, C. Ross, B. McCarty and R. Morris. Beckman Coulter Life
Science.
Sunday,
19 May
B246
368
Flow Cytometric Measurement of the CD11b Activated Epitope Expression on
Human Neutrophils in Whole Blood as a Tool in Drug Development Process
D. Polancec, M. Vrancic, S. Dupont, K. Oreskovic and V. Erakovic Haber. Fidelta,
Croatia, Galapagos SASU, France and Univ. of Rijeka, Croatia.
Monday,
20 May
B247
369
Systematic Screening of Ovarian Cancer Cell Lines by Multicolour Flow Cytometry
Reveals Tremendous Heterogeneity S. Sopper, M. Bösch, A. Zeimet, D. Reimer and
D. Wolf. Med. Univ. Innsbruck and Univ. Clin. Bonn.
B248
370
The New Flow Cytometry Research Group of the Association of Biomolecular
Resource Facilities A. Bergeron, A. Box, S. Chittur, M. Cochran, M. DeLay, P. Lopez,
M. Meyer, T. Neubert, H. Pletcher and S. Tighe. Geisel Sch. of Med. at Dartmouth,
Stowers Inst. for Med. Res., Univ. at Albany SUNY, Univ. of Rochester Med. Ctr.,
Cincinnati Children’s Hosp. Med. Ctr., NYU Ofc. of Collaborative Sci., Univ. of
Pittsburgh, NYU Sch. of Med. and Univ. of Pennsylvania.
B249
371
New Method for High-Throughput Analysis and Sorting of Live Intact Plant
Protoplasts R. Pulak and M. Malinouski. Union Biometrica Inc.
B250
372
Nine Color Flow Cytometric Characterization of Disease Progression in MRL/MpJFaslpr/j Mice W. Schott, D. Schultz, A. O’Neill, S. Grindle, E. Taylor, K. Snow and
M. Strobel. The Jackson Laboratory.
B251
373
Using Imaging Flow Cytometry and Fish-In Suspension Method to Enumerate
Human X Chromosomes by PCR-Generated Fluorescent Probes W. Wojciechowski
and T. Bushnell. Univ. of Rochester Med. Ctr.
B252
374
High-Resolution Flow Cytometric Analysis of Human Immune Populations by a
Competitive Clustering Algorithm T. Mosmann, J. Rebhahn, I. Naim, J. Cavenaugh,
G. Sharma, J. Weaver, D. Roumanes and S. Quataert. Univ. of Rochester Med. Ctr.
and Univ. of Rochester.
B253
375
FluoroFinder.com: A Free Flow Cytometry Resource to Simplify Polychromatic Panel
Design R. Marcus, T. Powell, B. O’Connor, M. D’Souza, D. Mack, G. Veltri,
J. Lannigan, S. Coquery and B. Palmer. FluoroFinder LLC, Univ. of Colorado Denver
Sch. of Med., Natl. Jewish Hlth. and Univ. of Virginia Sch. of Med.
B254
376
Unsolved Challenges in Acoustic Focusing of Nanoparticles M.E. Piyasena and
S.W. Graves. Univ. of New Mexico.
B255
377
Sorter Interface for High Resolution Confocal Imaging and Micro-Scale Live-Cell
Matrix Experiments A. Donnenberg, V.S. Donnenberg, E.M. Meyer and C. Baty. Univ.
of Pittsburgh Sch. of Med. and Univ. of Pittsburgh.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Saturday,
18 May
B242
64
ISAC 2013 Program and Abstracts
B257
379
Microfluidic Cell Sorters Enabled by Standing Surface Acoustic Waves T.J. Huang.
Penn State.
B258
380
Parasite-Induced Immune Modulation of Dendritic Cells Identified Using Flow
Cytometry J. Roberts, I. Sutherland, R. Shaw and A. Pernthaner. Hopkirk Res. Inst.,
AgResearch, New Zealand.
Saturday,
18 May
Evaluation of Flow Cytometry High-Throughput Cell Cycle Analysis on the Analyzers
BD LSR Fortessa, BD Accuri C6, and the BD FACS Canto II D. Kratochwil-Otto,
R. Maranchuk and A. Holme. Fac. of Med. and Dent., Univ. of Alberta.
Special
Lectures
378
Congress
Overview
B256
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
65
Congress
Overview
COMMERCIAL TUTORIALS
Special
Lectures
Sunday, 19 May
Cost Effective Options for Optimizing
Your Lab
Sunday,
19 May
Saturday,
18 May
Cytek Development
4059 Clipper Court
Fremont, CA 94538
Phone: 804-234-8110
Email: rlannigan@cytekdev.com
Web: www.cytekdev.com
1245 – 1345 - Room 33AB
Presenter: Lisa Nichols, PhD
Beckman Coulter Life Sciences
250 S. Kraemer Boulevard
Brea, CA 92821
Phone: 714-342-9072
Email: donna.gorman@beckman.com
Web: www.beckmancoulter.com
1245 – 1345 - Room 32AB
Presenter: Mathias Streitz
Michael Kapinsky
Objective Flow Cytometry is a key component in
immune monitoring. The flexibility and open nature
of this technology makes comparison of results a
major challenge in multicenter clinical studies such
as the international trial conducted by The ONE
Study consortium (www.onestudy.org). In order to
assure consistency of results across participating
laboratories, flow cytometry panel design and sample
preparation procedures were designed to meet distinct
requirements of robustness and reproducibility.
Methods Multicolor panels, containing up to 9
colors, were constructed based on a set of rules
related to optical properties, such as dye selection,
based on brightness and emission spectra as well as
taking the various types of known expression patterns
into account.
Conclusions The ONE Study panels could generally
be useful for all laboratories performing immune
monitoring for either multi-centre or local clinical
trials. Multicolor panel construction needs to take
into consideration the entire spectral and biological
scenario related to a given panel, instead of selecting
antibody-fluorochrome combinations based on single
conjugate properties.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
What quality control measures can be used to ensure
that cytometers are performing optimally? And,
what options exist to expand your lab’s cytometry
capabilities in the most cost-effective manner? Cytek
Development is an established company capable of
providing quality service and upgrades that optimize
cytometers in order to keep pace with today’s
experiments. This workshop introduces our latest
offerings including a cross-platform application for
evaluating cytometer performance and a new addition
to our menu of custom upgrade options. Cytek’s Q&b
method offers the ability to quantify performance on
a variety of cytometers, provides absolute Q and b
measurements (instead of relative), and calculates
the resolution limit for each channel in MEFL units.
We will discuss the relative advantages of the various
Q and b cytometer validation programs. Cytek has
also expanded its custom upgrade options for BD
cytometers. We have sourced a compact 20mW 355
nm CW UV laser that is equal in performance to other
typically used lasers. The compact size of this new
laser allows it to be installed in FACSCalibur and LSR
II models. We have integrated this option into our
upgrades and will present data demonstrating their
application.
The ONE Study – Novel Approaches
in Multicolor Panel Design and
Intracellular Preparation Chemistry
66
ISAC 2013 Program and Abstracts
Presenter: Kamala Tyagarajan, PhD
Beckman Coulter Life Sciences
250 S. Kraemer Boulevard
Brea, CA 92821
Phone: 714-342-9072
Email: donna.gorman@beckman.com
Web: www.beckmancoulter.com
Poster
Session
1245 – 1345 - Room 29 CD
Presenters: Vasilis Toxavidis
John Tigges
Dr. Robert Sleiman,
Mr. Timothy Reed
Commercial
Tutorials &
Exhibits
There is great interest in both medical and scientific
communities in submicron cell-derived particles,
termed microparticles or microvesicles. Examples
of tissues thatshed fragments of their plasma
membrane (or microparticles) into the circulation
include platelets, endothelial cells, leukocytes, and
erythrocytes. There is increasing evidence that these
submicron fragments have important physiological
roles. Although many techniques have been used
with limited success for the identification and
characterization of microparticles, flow cytometry
remains the most accurate and reproducible.
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
MoFlo AstriosEQ – The New Standard
in Forward Scatter and Fluorescence
Dynamic Range Performance: A Case
Study on Microparticles
Wednesday,
22 May
The Muse™ Cell Analyzer is a compact, affordable,
revolutionary cell analyzer based on microcapillary
cytometry that greatly simplifies cytometric analysis by
using a guided touch screen interface and dedicated
software modules to provide quantitative cellular data.
The workshop will discuss the novel miniaturized
flow technology utilized in the Muse™ Cell Analyzer,
and also showcase data on the expanding range of
optimized Muse™ assays available. A wide range
of cell health assays allows for the rapid, single-step
measurement of cell count and viability, provide
information on cell cycle distribution and allow
for characterization of apoptosis and death using
Annexin V binding, caspase activity or mitochondrial
depolarization. Immunology assays on the Muse™
platform allow for the identification and enumeration
of CD4 T cells, CD8 T cells or B cells in whole
blood or PBMC samples and also allow for obtaining
information on activation status of lymphocytes
based on CD69 or CD25 expression levels. Data
from recent Cell Signalling assays on the platform
will also be discussed. The combination of easy to
perform assays with the simple, dynamic interface
of the Muse™ Cell Analyzer can greatly empower
researchers to obtain cytometric data with ease.
Designed for the detection of up to three RNA
transcripts using flow cytometry, QuantiGene® Flow
RNA Assay is an in situ hybridization assay that
offers robust detection of RNA in individual cells and
retains compatibility with antibody surface staining
for simultaneous detection of protein. Incorporating
dual oligonucleotide probe design with branched
DNA signal amplification, this novel chemistry
provides unique transcript expression in specific cell
populations to develop biosignature profiles highly
applicable in studying immune response.
Tuesday,
21 May
1245 – 1345 - Room 30CDE
Presenter: Sue Reynolds
Monday,
20 May
EMD Millipore
17 Cherry Hill Drive
Danvers, MA 01932
Phone: 1-800-645-5476
Email: emdinfo@merckgroup.com
Web: www.emdmillipore.com
1245 – 1345 - Room 30AB
Sunday,
19 May
The Muse™ Cell Analyzer: A Versatile
Platform for Simplified Cellular Analysis
eBioscience, an Affymetrix company
10255 Science Center Drive
San Diego, CA 92121
Phone: 888-999-1371
Email: info@ebioscience.com
Web: www.ebioscience.com
Saturday,
18 May
1245 – 1345 - Room 31ABC
A Novel RNA ISH Assay for Flow
Cytometry
Special
Lectures
Sony Biotechnology, Inc.
2100 South Oak Street
Champaign, IL 61820
Phone: 217-328-9396
Fax: 217-328-6292
Email: sales@sonybiotechnology.com
Web: www.sonybiotechnology.com
Congress
Overview
Biosafe Sorting Solutions from Sony
Biotechnology for Better, Faster, Easier
Cell Sorting
67
Congress
Overview
Traditionally however, the most challenging element
has been the availability of sufficient forward scatter
(FSC) resolution to delineate target populations from
background signal and instrument noise.
Sunday,
19 May
Saturday,
18 May
Special
Lectures
The Beckman Coulter new MoFlo AstriosEQ
(AstriosEQ) sets a new standard in forward scatter
and fluorescence dynamic range performance for
cell sorting – delivering enhanced functionalityand
sensitivity. Accommodating as standard, a 488 nm
200 mW diode laser with a conditioned flat-top shape
beam profile, the AstriosEQ offers a patent pending
FSC assembly capable of discriminating 200 nm to 30
µm particles on the same dynamic range. Designed
for researchers who desire high productivity with
more analytical capability, the AstriosEQ combines
nano-scatter resolution with 5 decade fluorescence
sensitivity on a 4-9 decade scale for the detection of
submicron cell-derived particles.
Fluidigm
7000 Shoreline Ct.
S. San Francisco, CA 94080
Phone: 650-266-6033
Web: www.fluidigm.com
12:45 – 13:45 - Room 29 AB
Tuesday,
21 May
Monday,
20 May
Therefore, we propose a patent pending innovative
Forward Scatter detection module that will not only
improve accuracy, but also allow for validation
of the process by recovery of microparticles. The
current study will present independent testing of the
AstriosEQ demonstrating use of its two-parameter
head-on PMT-based FSC optical assembly technology
to identify and recover microparticles for downstream
analysis.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
For Research Use Only. Not for use in diagnostic
procedures.
68
ISAC 2013 Program and Abstracts
Congress
Overview
Tuesday, 21 May
Union Biometrica, Inc.
84 October Hill Road
Holliston, MA 01746
Phone: 508-893-3115
Fax: 508-893-8044
Email: sales@unionbio.com
Web: www.unionbio.com
1215 – 1315 - Room 32AB
Presenters: Brice Gaudilliere
Gabi Fragiadakis
Presenter: Rock Pulak
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
This tutorial demonstrates that the CyTOF can
be used to detect dynamic immune changes in a
clinically relevant setting. Mass cytometry-based
immune monitoring of patients undergoing surgery
thus holds the promise to identify patients at risk
for aversive perioperative outcomes and to develop
strategies to improve surgical recovery. These
efforts may be particularly relevant in patients with
advanced diseases (e.g. cancer), surgeries with high
complication rates (e.g. infection after colo-rectal
surgery), or specific vulnerabilities (e.g. extreme of
ages).
Tuesday,
21 May
The tutorial will focus on the optimization of
experimental conditions for metal-labeled antibody
staining with the CyTOF® system, and data analysis of
endogenous intracellular signaling activity in patients’
samples using Cytobank, a platform specifically
designed for the analysis of both fluorescence and
mass cytometry data, and SPADE, a hierarchical
clustering algorithm that enables the visualization of
signaling events across the hematopoietic continuum.
Finally we will describe how these methods enabled
the system-wide analysis of endogenous signaling
events in patients undergoing surgery.
Union Biometrica extends the utility of flow cytometry
to samples that are too large or too fragile for
traditional flow instruments. This technology provides
a method for analysis and dispensing, increasing
throughput and replacing manual sorting. The
BioSorter™ flow cytometer is designed to provide
the flexibility for working with samples ranging in
sizes from 1 to 1500 microns. This very broad range
of sizes includes many interesting types of cells,
cell clusters and even whole organisms. We present
some new applications of the BioSorter, including
the use of large delicate cells such as adipocytes and
plant protoplasts, cell clusters such as mammalian
neurospheres, plant calli and fungal pellets, and cells
encapsulated within hydrogel particles. These samples
can be analyzed and dispensed intact owing to the
low sample pressures, low shear forces and the gentle
sorting mechanism. Union Biometrica has developed
an autosampler, LP Sampler™ that automatically loads
samples from multiwell plates to the BioSorter for
analysis and resorting, and also the VAST BioImager,
a simplified flow-based platform for image acquisition
and analysis. Union Biometrica continues to develop
research tools for large particle analysis.
Monday,
20 May
A critical component of immune monitoring is
analyzing the dynamics of the immune system in
response to perturbation. This tutorial examines
the phenotypic and signaling single-cell responses
in patients undergoing surgery, a clinically relevant
immune perturbation. Using mass cytometry we are
able to characterize this response at the systems level
with single-cell resolution.
Sunday,
19 May
1215 – 1315 - Room 33AB
Saturday,
18 May
DVS Sciences
639 N. Pastoria Avenue
Sunnyvale, CA 94085
Phone: 408-900-7224
Email: Nicole.ovadia@dvssciences.com
Web: www.dvssciences.com
Flow Cytometry for Large Delicate
Cells, Cell Clusters, Encapsulated Cells,
and Small Model Organism
Special
Lectures
Mass Cytometry Reveals an
Endogenous Immune Response to
Physiological Perturbation in Humans –
Optimization and Design
Speaker/Author
Index
ISAC 2013 Program and Abstracts
69
Congress
Overview
Biosafety and Sorting Solutions from
BD Biosciences
Saturday,
18 May
Special
Lectures
BD Biosciences
2350 Qume Drive
San Jose, CA 95131
Phone: 1-877-232-8995
Email: answers@bd.com
Web: www.bdbiosciences.com
1215 – 1315 - Room 31ABC
Presenters: Gil Reinin
Janet Horta
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
In the last few years there has been an increased
customer awareness of the biosafety risk from aerosols
produced in a clog situation while cell sorting,
especially when sorting unfixed human cells. This
concern was validated by aerosol generation studies
where the data demonstrated that aerosol droplets
produced from a stream clog could potentially be
a safety risk if the proper biosafety measures were
not taken. Biosafety measures include a proper risk
assessment of samples being run, wearing proper PPE,
always using an aerosol management system, and if
required, the use of a BSC that can pass international
biosafety standards such as the NSF-49 standard for
product, personnel, and environmental protection.
As a result of these guidelines a greater percentage of
sorters are being purchased with biosafety cabinets.
In this seminar we will review recent biosafety
guidelines for sorting and discuss solutions for
meeting these guidelines for BD Biosciences sorters.
Commercial
Tutorials &
Exhibits
Poster
Session
Innovative Spectral Analysis
Technology and Novel Applications
for Cellular Analysis from Sony
Biotechnology
FlowJo Ninja Skills
Tree Star, Inc.
340 A Street
Suite 101
Ashland, OR 97520
Phone: 800-366-6045
Fax: 541-482-3153
Email: jasper@treestar.com
Web: www.flowjo.com
1215 – 1315 - Room 30AB
Presenters: Nicholas Ostrout, PhD
John Quinn, PhD
Become a FlowJo Ninja! Many users aren’t aware of
all the amazing features, interesting short cuts, and
awesome options to help improve and streamline
your data analysis in FlowJo. Now with version 10
of FlowJo, this list has grown even longer! Come
hone your data analysis skills, learn new techniques,
and become a master of level 10 FlowJo. Of course
becoming a first degree ninja shouldn’t be enough,
so earn your second degree and stay around for
Fluorish. We will be showcasing the redesigned panel
builder! Do you have what it takes to be a master
panel designer? If it has been a while since you last
saw Fluorish, the website and panel builder now
have a growing list of new features that will make
your life (ok, maybe just your life in flow cytometry)
much easier. Quickly locate reagents from more
than 10 major antibody suppliers, manage a core,
grab the instrument configurations, create a Fluorish
lab to share/manage your lab inventory, and order
everything you need for your flow experiments from
one spot.
Oral Session
Abstracts
Sony Biotechnology, Inc.
2100 South Oak Street
Champaign, IL 61820
Phone: 217-328-9396
Fax: 217-328-6292
Email: sales@sonybiotechnology.com
Web: www.sonybiotechnology.com
Speaker/Author
Index
Poster Session
Abstracts
1215 – 1315 - Room 30CDE
70
ISAC 2013 Program and Abstracts
Presenter:TBD
Bio-Rad presents the all new S3(™) Cell Sorter. In
partnership with Propel Labs, the S3 cell sorter makes
cell sorting easy to use and accessible to researchers
with several automated features. Using state of the
art technology, ProDrop(™) automates the drop
delay procedure providing accurate and consistent
droplet deflection. The S3 cell sorter is fully featured
with an onboard temperature control using Peltier
solid state system, completely enclosed fluidic and
a convenient, compact space saving design- all
without compromising in performance or sensitivity.
Created with researchers in mind, an affordable 1-4
fluorescent parameter system and equipped with up
to two lasers, this system is designed to accommodate
everyday experiments amongst cell biologist.
Complimentary to systems currently in flow core labs,
offload 1-4 color experiments onto the S3 cell sorter
that researchers can use on their own with minimal
training.
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Optimization of a novel microchip-based cell
sorting valve has recently been combined with flow
cytometry analytic capabilities to create a novel
closed cell sorting platform. The microchip-based
valve is a MEMS (Micro Electro Mechanical System)
that delivers the robustness, precision and speed of
silicon chip products combined with a magnetic
actuation mechanism for high-speed cell sorting. The
system uses typical fluorescence gating selection logic
to actuate the valve which directs cells to one of two
microfluidic paths in a closed single-use cartridge.
Thus, in a typical workflow, an input sample
containing 10e7-9 cells is processed into a sample
highly enriched for the desired cell population(s) and
a sample depleted for such cells. All the cells remain
in the cartridge and can be recovered for subsequent
analysis or processing, including re-sorting. The
cells remain in buffer or media undiluted by sheath,
and cell viability appears unchanged by the sorting
process. The purity of the recovered sort sample
depends on the input cell density and the frequency
of desired cells as predicted by Poisson statistics.
Characterization of the platform’s performance will
be presented in the context of several relevant cell
purification protocols including isolation of human
T regulatory cells (T regs), tetramer-positive human
T cells and circulating tumor cells (CTCs) from
peripheral blood in an experimental murine model of
pancreatic ductal carcinoma.
1215 – 1315 - Room 29AB
Sunday,
19 May
Presenters: John Foster, President, Owl
biomedical, Inc.
Jack Dunne, Executive Vice
President, Science & Technology,
Owl biomedical, Inc.
Bio-Rad
2000 Alfred Nobel
Hercules, CA 94547
Phone: 510-741-4494
Email: melissa_ma@bio-rad.com
Web: www.bio-rad.com
Saturday,
18 May
1215 – 1315 - Room 29CD
The S3 Cell Sorter – Cell Sorting Made
Simple
Special
Lectures
Miltenyi Biotec GmbH
Friedrich-Ebert-Strasse 68
51429 Bergisch Gladbach
Germany
Phone: 49 2204 83060
Email: annabelk@miltenyibiotec.de
Web: www.miltenyibiotec.com
Congress
Overview
Fluorescence-Activated Cell Sorting
without Droplets
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
71
Congress
Overview
Wednesday, 22 May
Special
Lectures
Studies of T-Cell/Antigen Presenting
Cell Conjugation Using Imaging Flow
Cytometry
Saturday,
18 May
EMD Millipore
17 Cherry Hill Drive
Danvers, MA 01932
Phone: 1-800-645-5476
Email: emdinfo@merckgroup.com
Web: www.emdmillipore.com
Sunday,
19 May
1215 – 1315 - Room 33AB
Presenter: Haley Pugsley, PhD
Molecular Devices LLC
1311 Orleans Drive
Sunnyvale, CA
Phone: 1-800-635-5577
Email: info@moldev.com
Web: www.moleculardevices.com
1215 – 1315 - Room 32AB
Presenter: Evan F. Cromwell, PhD, Director of
Assay Development
A continuing trend in drug discovery and
development is the use of complex cell-based
models that are able to provide more biologically
relevant & predicative assays. We present new
advancements in plate reader and imaging cytometry
systems that enable multiplexed analysis of important
biological outputs. The system has been designed for
environments from basic research labs to medium/
high throughput screening cores, and the intuitive user
interface allows non-imaging specialists to get up and
running quickly. We will present examples of use of
the system for cells-based assays including plate QC
and models for testing anti- inflammatory compounds
and in vitro toxicity assessment.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Imaging flow cytometry combines the speed,
sensitivity, and phenotyping abilities of flow cytometry
with the detailed imagery and functional insights of
microscopy. This unique combination enables a broad
range of applications that would be impossible using
either technique alone. This seminar will explore
two studies of the T-cell/Antigen Presenting Cell
(APC) Immune Synapse performed on the Amnis®
FlowSight® and ImageStreamX Mark II imaging flow
cytometers. In the first study we will demonstrate
image-based parameters that were used to assess the
frequency of T-cell/APC conjugates with an organized
immune synapse in an objective and statistically
significant manner. In the second study we evaluate
the specific location of the adhesion and signaling
molecules LFA-1 and Lck within the immunological
synapse complex in T-cells when presented with SEB,
as well as the activation of the T cells in contact with
APC and with organized immunological synapse by
measuring the nuclear localization of NFkB in the T
cell.
Complex Cell Based Assays Using
SpectraMax® i3 Multi-Mode Plate
Reader and SpectraMax® MiniMax™
Imaging Cytometer
72
ISAC 2013 Program and Abstracts
Taking Flow Cytometry to the Limits
PARTEC
Am Flugplatz 13
Goerlitz
Saxony, Germany 02828
Phone: 49 0 3581 87460
Fax: 49 0 3581 874670
Email: mail@partec.com
Web: www.partec.com
Presenters: Dr. Roy Overton
Dr. Danny Koehler
Advances in technology have made multi-color flow
cytometry more accessible to researchers worldwide.
Taking flow cytometry to the limits - Partec Nano/
ViroFlow™ & Nano/ViroSort™: Detection and
sorting of viruses and other nanoparticles. - Sorting
neurons, cardiomyocytes and other fragile cells gently
and safely with the Partec CyFlow® Sorter. - Highresolution DNA analysis.
Today scientists choose dyes based on the capabilities
of the instrumentation available, the number of
surface receptors on the cells they are studying, their
brightness and spillover value.
•How the impact of optical design of the cytometer
on signal detection
Verity Software House
45A Augusta Road
PO Box 247
Topsham, ME 04086
Phone: 207-729-6767
Fax: 207-729-5443
Email: sales@vsh.com
Web: www.vsh.com
1215 – 1315 - Room 30AB
•Importance of standardization of instruments and
optimization of instrument setup to data quality
Presenters: Dr. Beth Hill
Mark Munson
•Importance of the use of appropriate controls
Poster
Session
•Methodology for choosing fluorochromes for
optimum multicolor panels – with data from 6 to
18 colors
Wednesday,
22 May
Topics include
GemStone: High-Dimensional Analysis
Principles and Applications
Tuesday,
21 May
The tutorial will present a methodology for designing
multicolor experiments, including demonstrations
of how effective use of fluorochromes allows for the
identification of cell populations with lower receptor
density than was previously possible. A wide range of
data using this methodology will be presented.
Monday,
20 May
Presenter: Robert Balderas, Vice President,
Biological Sciences
Sunday,
19 May
1215 – 1315 - Room 30CDE
Saturday,
18 May
1215 – 1315 - Room 31ABC
Special
Lectures
BD Biosciences
2350 Qume Drive
San Jose, CA 95131
Phone: 1-877-232-8995
Email: answers@bd.com
Web: www.bdbiosciences.com
Congress
Overview
Advances in Technology Have Made
Multi-color Flow Cytometry More
Accessible to Researchers Worldwide
Commercial
Tutorials &
Exhibits
This first part of this presentation is designed to
introduce the principles of multi-dimensional data
modeling via a common example, with a closing
presentation on current applications. The unique
capabilities of GemStone’s Probability State Modeling
will be presented in a familiar format, with perhaps
an unfamiliar twist. There will be opportunities for
discussion, questions, and review.
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
73
Congress
Overview
Special
Lectures
The Latest Advances in Cytometry
Powered by Molecular Probes® Life
Technologies™ Presents: Discovery
Using the Attune® Acoustic Focusing
Cytometer, Molecular Probes®
Reagents, and Evos® Cell Imaging
Sytems
Sunday,
19 May
Saturday,
18 May
Life Technologies
5791 Van Allen Way
Carlsbad, CA 92008
Phone: 760-603-7200
Email: technical-molecularprobes@lifetech.
com
Web: www.probes.com
1215 – 1315 - Room 29CD
Monday,
20 May
Presenter: Julie K. Jang, Department of
Pathology, Keck School of Medicine,
University of Southern California
De Novo Software
3250 Wilshire Boulevard, Suite 803
Los Angeles, CA 90010
Phone: 213-814-1240
Fax: 213-814-1511
Web: www.denovosoftware.com
1215 – 1315 – Room 29AB
Presenters: David Novo (De Novo Software)
and Peter Li (Nexcelom Biosciences)
Digital Microscopy and Image Cytometry are rapidly
evolving technologies in biomedical research and
drug discovery. However, with imaging, managing
your analysis results and images is a daunting task.
Nexcelom’s image cytometry solutions automate the
time-consuming counting procedures for hundreds
of different cell types, enabling scientists to focus
less on the process and more on the research results.
Nexcelom’s products are currently being used in the
labs of leading pharmaceutical companies, Biotech
organizations, universities, and research institutions.
By combining the power of image cytometry with
De Novo Software’s FCS Express 4 Cytometry
software you can now manage your multi-parametric
data image cytometry data sets with a proven flow
cytometry styled analysis. With FCS Express 4
Cytometry you combine data processing and image
analysis with report building in a single package.
Eliminate the redundant data processing steps
you now perform in Excel, and create PowerPoint
presentation with a click of a button. Work with your
images and multi-parametric data dynamically in FCS
Express and then simply export a report in the format
you desire.
Nexcelom’s Vision CBA is now compatible with
FCS Express Cytometry to provide users a unique
analysis solution for simple to complex image analysis
experiments. Join the Nexcelom Biosciences and De
Novo Software teams to learn how to turn your image
cytometry data into results.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Life Technologies’™ Attune® Acoustic Focusing
Cytometer is the first-of-its-kind cytometry system to
use sound waves to precisely control the focusing of
particles and cells. The same scientists who designed
and built Attune® inspire true discovery in flow
cytometry with the newest flow cytometry reagents
and selection tools from Molecular Probes® - reagents
and tools for flow cytometry that take significant steps
forward in the analysis of cellular function, far beyond
immunophenotyping. The EVOS® line of Cell Imaging
Systems are designed to work together – from the initial
cell culture check (for viability and morphology) to
more complex analyses such as time lapse, image
tiling and stitching. The EVOS® systems make cell
imaging accessible to almost every lab application
and budget. Molecular Probes® integrated solutions
will help you design and execute your flow cytometry
and cell imaging experiments. Late breaking data from
both platforms will be presented.
Getting the Most Out of Your Image
Cytometry Data with the Nexcelom
Vision CBA and FCS Express Cytometry
74
ISAC 2013 Program and Abstracts
Congress
Overview
EXHIBITOR SHOWCASE
Monday, 20 May
Special
Lectures
Exhibit Hall GH
Novel Polymer Fluorochromes and their
Application in Multicolor Flow Cytometry
Large, Multidimensional Data Sets Analysed in
Seconds!
1215 – 1225 - Exhibit Hall
1255 – 1305 - Exhibit Hall
Polymer dye technology from Sirigen, a BD
Biosciences company, has opened the door to the
development of novel dyes that are four to ten times
brighter than conventional dyes, with equivalent
background — a breakthrough in the field. For
cells that have few receptors on the surface, bright
reagents are essential in resolving these dim cells
from others in a sample. The characteristics of the
Sirigen polymer dyes enable them to achieve much
brighter fluorescence signals than traditional organic
fluorescent dyes or even phycobiliproteins such as
PE or APC. This means that they are effective for
identifying cell populations with a broader range of
receptor density than previously possible. Come learn
about the latest advances in polymer dye development
from BD Biosciences.
SOPHE represents a breakthrough in processing flow
cytometer generated data sets.
•Analyse large data sets with 20 + parameters or
dimensions and hundreds of thousand cells.
•Divide the data into clusters without requiring
prior knowledge of the number of clusters.
The SOPHE algorithms find these otherwise hidden
relationships and report the characteristics of each
such cluster. The SOPHE method delivers results in
a few seconds. The algorithms on which SOPHE is
based are patent protected by PCT/AU2012/000252
“Multidimensional cluster analysis”
Poster
Session
Commercial
Tutorials &
Exhibits
handyem aims to democratize cytometry by providing
day-to-day autonomy and agility to life science
researchers with simple, highly efficient instruments at
a fraction of the cost of today’s solutions. handyem’s
HPC-100 Personal Cytometer, with its F3 microchip
platform, brings simplicity, accessibility and ease of
use to life science researchers and core lab managers.
This short presentation of the HPC-100 will draw the
main highlights of the solution.
The algorithms in SOPHE can:
Wednesday,
22 May
1235 – 1245 - Exhibit Hall
Data sets generated can have 10 or even 20
independent dimensions, and each dimension in any
combination can generate sets of clusters. Current
software systems cannot do this level of cluster
analysis, and where they attempt it, it is incomplete,
slow, and computationally inefficient.
Tuesday,
21 May
Flow Cytometry in your Hands®
The development of flow cytometer hardware has
been advancing faster than the ability to analyse the
data generated by the instrumentation with its ever
increasing number of variables or markers contained
within the data sets.
Monday,
20 May
handyem
Sunday,
19 May
NewSouth Innovations
Saturday,
18 May
BD Biosciences
A brief demonstration of the software will be
presented and an opportunity for discussion with one
of the key developers Dr John Zaunders.
Oral Session
Abstracts
BioStatus
DRAQ7™ - A Unique Far-Red Real-Time
Viability Monitor
Poster Session
Abstracts
1315 – 1325 - Exhibit Hall
ISAC 2013 Program and Abstracts
Speaker/Author
Index
DRAQ7(tm) is a patented far-red fluorescing
anthraquinone-derived viability dye that extends the
opportunities for multi-colour flow cytometry and
image-based assays. It allows facile combination with
commonly used orange/red mitochondrial health probes
for temporal interrogation of apoptosis and has been
validated to permit new real-time viability monitoring.
75
Congress
Overview
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
Special
Lectures
Exhibitor Listing
CompanyBooth(s)
CompanyBooth(s)
AAT Bioquest, Inc........................................... 625
Abcam............................................................ 614
ALPCO Diagnostics........................................ 715
Bangs Laboratories......................................... 612
Bay Bioscience Co., Ltd.................................. 501
BD Biosciences.............................................. 420
Beckman Coulter, Inc..................................... 602
BioLegend...................................................... 601
Bio-Rad Laboratories...................................... 619
Biostatus Limited............................................ 406
BioTek Instruments, Inc.................................. 909
Blue Sky Research.......................................... 438
Brooks Automation Ltd................................... 212
Caltag Medsystems......................................... 414
Cedarlane....................................................... 733
Chroma Technology Corp............................... 810
Cobolt............................................................ 629
Coherent, Inc.................................................. 620
Cytek Development, Inc................................. 313
Cytobank Inc.................................................. 442
CYTOGNOS................................................... 721
De Novo Software.......................................... 329
DVS Sciences Inc........................................... 530
eBioscience, an Affymetrix company.............. 723
EMD Millipore................................................ 814
Etaluma.......................................................... 222
EXBIO Praha a.s............................................. 538
Flow Contract Site Laboratory, LLC................. 404
Fluidigm......................................................... 631
GE Healthcare................................................ 808
Hamamatsu Corporation................................ 440
handyem........................................................ 719
Hellma USA, Inc............................................ 520
iLab Solutions................................................. 713
IMGENEX Corporation................................... 840
Innova Biosciences......................................... 502
IntelliCyt Corporation..................................... 907
ISAC............................................................... 742
Life Technologies............................................ 624
Market Tech.................................................... 213
Melles Griot................................................... 634
Miltenyi Biotec GmbH................................... 512
Molecular Devices, LLC................................. 802
Newport Corporation..................................... 504
NewSouth Innovations Pty Ltd........................ 323
Nexcelom Bioscience..................................... 333
Novus Biologicals........................................... 804
OSELA Inc...................................................... 605
OXXIUS Lasers............................................... 506
PARTEC.......................................................... 830
Pavilion Integration Corporation..................... 729
Phoenix Flow Systems, Inc............................. 630
Photop Technologies Inc................................. 211
Propel Labs..................................................... 623
Semrock......................................................... 632
Sony Biotechnology Inc.................................. 303
SouthernBiotech............................................. 311
Spherotech, Inc.............................................. 416
Stratedigm, Inc............................................... 522
Thermo Fisher Scientific................................. 633
Tonbo Biosciences.......................................... 513
Toptica Photonics Inc..................................... 505
Tree Star, Inc................................................... 611
TTP Labtech Ltd.............................................. 325
Union Biometrica, Inc.................................... 711
Verity Software House.................................... 402
Vortran Laser Technology, Inc......................... 319
Wiley............................................................. 842
Woodside Logic............................................. 321
Xitogen Technologies Inc........................ 516, 615
Exhibit Hours
Speaker/Author
Index
Monday, 20 May Tuesday, 21 May Wednesday, 22 May
76
1130 – 1900
1130 – 1900
1130 – 1630
ISAC 2013 Program and Abstracts
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
77
ISAC 2013 Program and Abstracts
Congress
Overview
CYTO 2013 EXHIBITS
Monday – Wednesday, 20 – 22 May
Exhibit Hall GH
Congress
Overview
Exhibiting Companies
Special
Lectures
Disclaimer
Saturday,
18 May
Participation in the Exhibits Program does not constitute an endorsement by the International
Society for Advancement of Cytometry (ISAC) of the claims, products or services offered.
AAT Bioquest, Inc.
625
Sunday,
19 May
520 Mercury Drive
Sunnyvale, CA 94085
Phone: 408-733-1055
Fax: 408-733-1304
Email: support@aatbio.com
Web: www.aatbio.com
Monday,
20 May
Tuesday,
21 May
1 Kendall Square, Suite B2304
Cambridge, MA 02139
Phone: 617-577-4277
Fax: 617-225-3938
Email: elise.arsenault@abcam.com
Web: www.abcam.com
Poster
Session
Wednesday,
22 May
Abcam sells high-quality, research-grade antibodies
and associated protein research tools. Comprehensive
datasheets, together with expert customer support and
fast delivery, continue to make Abcam the researcher’s
choice. Abcam’s catalogue also includes a growing
range of non-antibody products, such as proteins,
peptides, lysates, immunoassays and other kits and
biochemicals --- all available at www.abcam.com.
715
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
26-G Keewaydin Drive
Salem, NH 03079
Phone: 800-592-5726
Fax: 603-898-6854
Email: nsliney@alpco.com
Web: www.alpco.com
ALPCO is a provider of life science research tools
for academic, pre-clinical and clinical use. Our
comprehensive portfolio includes immunoassays for
a variety of therapeutic areas of interest including
Cytokines and Cell Signaling. We recently expanded
our product catalog with the addition of Flow
Cytometry reagents. Stop by our booth to learn more,
or visit www.alpco.com.
Bangs Laboratories, Inc. produces fluorescence
microsphere standards to help the research and
clinical community conduct studies and evaluate data
in an easier, more precise manner. Our products set
the standard for quality control and quantitation in
the fields of flow cytometry, fluorescence microscopy,
image analysis, and other fluorescence analytical
applications. Superior Customer and Technical Service
complement Bangs’ extensive product offerings.
Bay Bioscience Co., Ltd.
501
2-5, Minatojima Minamimachi 5-chome,
Chuo-ku
Kobe, 650-0047
Japan
Phone: 81 78 304 5881
Fax: 81 78 304 5889
Email: DaveCoder@BaybioscienceUSA.com
Web: www.baybio.co.jp
Bay Bioscience designs, develops, and manufactures
high performance cell sorting and analysis systems.
The company’s current products, the JSAN and JSAN
Swift combine high performance and affordability in
a way previously not available. Configure a system to
exactly suit your needs, including true UV excitation.
Please visit the company website at www.baybio.
co.jp The JSAN: desktop sorting at an affordable price.
Categories Cell separation products Data analysis
systems and software Flow cytometry and cell sorting
equipment
Speaker/Author
Index
Poster Session
Abstracts
612
9025 Technology Drive
Fishers, IN 46038
Phone: 317-570-7020
Fax: 317-570-7034
Email: karen@bangslabs.com
Web: www.bangslabs.com
Abcam614
ALPCO Diagnostics
Bangs Laboratories
78
ISAC 2013 Program and Abstracts
420
2000 Alfred Nobel Drive
Hercules, CA 94547
Phone: 510-741-4494
Email: melissa_ma@bio-rad.com
Web: www.bio-rad.com
100 Tigan Street
Winooski, VT 05404
Phone: 802-655-4040
Fax: 802-655-7941
Web: www.biotek.com
BioTek is a leader in the design, manufacture, and sale
of microplate instrumentation and software. BioTek’s
instrumentation, like the just-released Cytation3 Cell
Imaging Multi-Mode Microplate Reader, a system that
combines automated microscopy and Hybrid multimode detection, is used to aid in the advancement
of life science research, facilitate the drug discovery
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
909
Oral Session
Abstracts
World-Class Antibodies, Proteins, Assays and
Research Solutions. Complete Brilliant Violet™
Antibody Conjugates for the Violet Laser: BV510™,
BV711™, BV785™. Personalized Multicolor Flow
Cytometry Panel Design. New LEGENDScreen™
Human Cell Screening (PE) Kits. Request Bulk
Cytokines & Chemokines for Bioassay. Ultra-LEAF™
(Low Endotoxin, Azide-Free) Antibodies. New ELISA
Kits: IL-35, Active TGF-ß1.
BioTek Instruments, Inc.
Commercial
Tutorials &
Exhibits
9727 Pacific Heights Boulevard
San Diego, CA 92121
Phone: 858-455-9588
Fax: 858-455-9587
Email: info@biolegend.com
Web: www.biolegend.com
Our latest product - DRAQ7™ - is the far-red cell
impermeant DNA dye
DRAQ7™ only enters cells with compromised
membranes: dead, damaged, and apoptotic cells for
enumeration and sorting.
DRAQ7™ is ready-to-use, permits multi-colour
analysis and does not interfere with GFP, Annexin
V and mitochondrial health probes (just like
DRAQ5™!).
DRAQ7™ shows negligible toxicity for use in longterm, real-time assays and can be used as a viability
reporter in RNAi knock-downs and toxicity assays.
Poster
Session
BioLegend601
56 Charnwood Road, Shepshed
Loughborough LEICS LE12 9NP
United Kingdom
Phone: 44 1509 558163
Fax: 44 1509 651061
Email: roy@biostatus.com
Web: www.biostatus.com
Wednesday,
22 May
Research Applications
Offering the broadest range of cellular analysis
systems in the world, Beckman Coulter provides a
variety of reagents, instruments and software to meet
the diverse needs of today’s research laboratories.
406
Tuesday,
21 May
Clinical Applications
Beckman Coulter provides a comprehensive line of
flow cytometry solutions that lead the charge against
life’s most challenging diseases. Our instruments,
sample preparation systems and family of antibodies
offer an unparalleled continuum of cellular testing.
Biostatus Limited
Monday,
20 May
250 S. Kraemer Boulevard
Brea, CA 92822
Phone: 714-961-3260
Web: www.beckmancoulter.com
Sunday,
19 May
602
Discover Bio-Rad’s automated, easy-to-use benchtop
S3 cell sorter that is affordable to researchers. Bio-Rad
continues to provide a wide variety of workflow
solutions in instrumentation and reagents for flow
cytometry, transfection, cell counting, droplet digital
PCR, conventional and real-time PCR, amplification
reagents and primers, cancer biomarkers,
electrophoresis, blotting-systems, chromatography,
and imaging.
Saturday,
18 May
BD Biosciences, a segment of Becton, Dickinson and
Company, is one of the world’s leading businesses
focused on bringing innovative tools to life science
researchers and clinicians. Its product lines include:
flow cytometers, cell imaging systems, monoclonal
antibodies, research reagents, diagnostic assays, and
tools to help grow tissue and cells.
Beckman Coulter, Inc.
619
Special
Lectures
2350 Qume Dr.
San Jose, CA 95131
Phone: 877-232-8995
Fax: 408-954-2009
Email: answers@bd.com
Web: www.bdbiosciences.com
Bio-Rad Laboratories
Congress
Overview
BD Biosciences
79
Congress
Overview
Special
Lectures
process, provide rapid, cost-effective industrial
analysis and to enable sensitive and accurate
quantification of a wide range of molecules across
diverse applications.
Blue Sky Research
438
Saturday,
18 May
1537 Centre Pointe Drive
Milpitas, CA 95035
Phone: 408-941-6003
Email: cmackrodt@blueskyresearch.com
Web; www.blueskyresearch.com
Sunday,
19 May
Brooks Automation Ltd.
212
Monday,
20 May
Wednesday,
22 May
Tuesday,
21 May
Brooks Life Science Systems provides a wide range of
automated solutions including the Celigo®Imaging
Cytometer. With both brightfield and fluorescence
imaging capabilities, Celigo enables whole well
automated, label-free cell counting, single cell
detection and clonal expansion as well as spheroid
analysis. Celigo’s fluorescence functionality provides
rapid image acquisition and analysis. With unrivaled
simplicity and ease of use, Celigo is the fastest
imaging cytometer available today.
414
Commercial
Tutorials &
Exhibits
Poster
Session
Whiteleaf Business Centre 11, Little Balmer
Buckingham Buckinghamshire MK18 1TF
United Kingdom
Phone: 01280 827460
Email: samc@caltagmedsystems.co.uk
Web: www.cytomark.co.uk/
Oral Session
Abstracts
Caltag Medsystems is a UK based manufacturer and
distributor of flow cytometry products. The Cytomark
Division of Caltag MedSystems is a manufacturer
of blood stabilisation products and stabilised whole
Blood Controls. TransFix® - A Cellular Antigen
Stabilisation Reagent. TransFix® is used to stabilise
whole blood, plus Cerebrospinal Fluid (CSF), Bone
Marrow and Fine Needle Aspirates. Cytomark whole
Blood Controls are used to verify absolute cell counts
and instrument set up.
810
10 Imtec Lane
Bellows Falls, VT 05101
Phone: 802-428-2500
Fax: 802-428-2525
Email: sales@chroma.com
Web: www.chroma.com
Optical components provide precise color separation
and signal purity for applications such as fluorescence
microscopy, flow cytometry, confocal or multiphoton
microscopy. BP/LP/SP * Multiband * Notch * Dichroic
Mirrors * Polychroic Mirrors * UV/VIS/NIR * AR
Coatings * Hot/Cold Mirrors * ND/AG/AL Mirrors *
Laser Grade and more, engineered and manufactured
by a team of employee-owners committed to bringing
you the finest optical filters, filter sets and optics
solutions. Chroma Technology, 800-824-7662.
Cobolt629
Vretenvagen 13
SE-171 54 Solna
Sweden
Phone: 46 8 54591230
Fax: 46 8 54591231
Email: info@cobolt.se
Web: www.cobolt.se
Cobolt AB (Stockholm, Sweden) has, since year
2000, been committed to development and supply
of innovative laser products that meet or exceed
the market’s expectations concerning performance,
quality and robustness. Through continuous
technology development, customer orientation
and an ISO-certified quality management system,
Cobolt has become a preferred supplier of lasers to
major manufacturers of analytical instrumentation
equipment and leading research labs.
Speaker/Author
Index
Poster Session
Abstracts
1210 Turrentine Street
Burlington, NC 27215
Phone: 336-513-5135
Fax: 336-513-5138
Email: cindy.greer@cedarlanelabs.com
Web: www.cedarlanelabs.com
Chroma Technology Corp
Northbank
Irlam, Manchester M44 5AY
United Kingdom
Phone: 44 (0) 161 722-2000
Web: www.brooks.com
Caltag Medsystems
Cedarlane733
80
ISAC 2013 Program and Abstracts
620
De Novo Software
442
639 N. Pastoria Avenue
Sunnyvale, CA 94085
Phone: 408-900-7224
Fax: 408-900-7224
Email: nicole.ovadia@dvssciences.com
Web: www.dvssciences.com
Poster Session
Abstracts
DVS Sciences Inc. is an analytical equipment and
reagents development company that produces and
markets Instruments and reagents for mass cytometry.
Multiplex analysis capability of the CyTOF® system
enables new insights into the functional complexity of
biological systems at the single cell level.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
530
Oral Session
Abstracts
Cytobank Inc. provides scientific solutions built
on Cytobank, a cloud-computing platform focused
on single cell technologies. We are a team of
scientist-entrepreneurs from Stanford University with
expertise in cytometry, cell signaling, drug discovery,
informatics and software development. We are the
engine behind landmark papers in the growing field
of high dimensional cytometry and have partnerships
with key vendors in the field.
DVS Sciences Inc.
Commercial
Tutorials &
Exhibits
800 West El Camino Real, Suite 180
Mountain View, CA 94040
Phone: 650-265-7806
Fax: 650-204-6866
Email: nikesh@cytobank.org
Web: www.cytobank.org
De Novo Software specializes in producing high
quality cytometry data analysis software. Our flagship
product, FCS Express 4, is a full feature solution
for Flow Cytometry data. FCS Express 4 Image
Cytometry introduces the same support and flexibility
for imaging data files. FCS Express combines a user
friendly modern interface with powerful analysis
tools, visualization capabilities and sophisticated
presentation features, making it the tool of choice for
thousands of researchers needing quick results from
their data.
Poster
Session
Cytobank Inc.
3250 Wilshire Boulevard, Suite 803
Los Angeles, CA 90010
Phone: 213-814-1240
Fax: 213-814-1240
Email: info@denovosoftware.com
Web: www.denovosoftware.com
Wednesday,
22 May
Cytek provides the flow cytometry tools scientists
need at prices they can afford accompanied by cost
effective service programs. Customize a high-powered
cytometer from a menu of industry standard platforms
at a fraction of the cost of a new system. Or, select an
upgrade package to expand the capabilities and extend
the useful life of your existing cytometer. Finally, our
comprehensive service plan options allow you to select
coverage that aligns with your needs and resources.
329
Tuesday,
21 May
4059 Clipper Court
Fremont, CA 94538
Phone: 510-657-0102
Fax: 510-657-0151
Email: cytekdev@cytekdev.com
Web: www.cytekdev.com
Our mission is to offer complete solutions in the
diagnosis of haematological diseases by flow
cytometry. Our vision is to create a paradigm shift
in the way of doing clinical diagnosis using flow
cytometry.
Monday,
20 May
313
Cytognos S.L. is a biotechnology company based
in Salamanca (Spain) dedicated to the design
and development of new reagents, software and
techniques that provide innovative solutions in the
flow cytometry field.
Sunday,
19 May
Cytek Development, Inc.
Pol. La Serna, Nave 9, KM. 0
Santa Marta de Tormes
Salamanca 37900
Spain
Phone: 34623125067
Email: sara.sanchez@cytognos.com
Web: www.cytognos.com
Saturday,
18 May
Coherent Inc. is one of the world’s leading suppliers
of laser-based solutions offering reliability, cost,
and performance advantages for the widest range
of commercial and scientific research applications.
Founded in 1966, Coherent designs, manufactures
and markets laser systems and components, laser
beam measurement and control equipment as well
as leading-edge beam forming and beam guidance
systems for manufacturers and scientific researchers
across the globe.
Cytognos721
Special
Lectures
5100 Patrick Henry Drive
Santa Clara, CA 95054
Phone: 408-764-4168
Email: rhea.igarta@coherent.com
Web: www.coherent.com
Congress
Overview
Coherent, Inc.
81
Congress
Overview
eBioscience, an Affymetrix
company723
Special
Lectures
3420 Central Expressway
Santa Clara, CA 95051
Phone: 408-731-5000
Email: laurie_durlester@affymetrix.com
Web:www.eBioscience.com
Sunday,
19 May
Saturday,
18 May
eBioscience, an Affymetrix company, develops and
manufactures over 13,000 high-quality antibodies,
recombinant proteins, immunoassays and multiplex
assays at ISO-certified facilities worldwide for research
and clinical use that accelerate scientific discovery in
the areas of immunology and oncology.
EMD Millipore
814
Monday,
20 May
290 Concord Road
Billerica, MA 01821
Phone: 781-533-6000
Fax: 978-715-1393
Email: elaine.fitzpatrick@emdmillipore.com
Web: www.emdmillipore.com
Wednesday,
22 May
Tuesday,
21 May
EMD Millipore is the Life Science division of Merck
KGaA of Germany, supporting research, development
and production of biotech and pharmaceutical drug
therapies. We support customers in cellular analysis,
network elucidation and functional genomics with
platforms for cell counting, simplified cell health
assessment, benchtop flow cytometry and imaging
flow cytometry.
Poster
Session
Etaluma222
Commercial
Tutorials &
Exhibits
1914 Palomar Oaks Way Suite 150
Carlsbad, CA 92008
Phone: 760-298-2355
Email: cshumate@etaluma.com
Web: www.etlauma.com
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Etaluma Inc. is focused on bringing research-grade
microscopy to a spectrum of users by decreasing the
cost and complexity of these devices and encouraging
the open-source development of application. Etaluma
offers three models of inverted digital microscopy; the
true color Model 400, the single 488nm fluorescence
Model 500, and the new 3-color fluorescence Model
600. Etaluma is actively looking to provide hardware
to reagent and kit manufacturers who want to expand
their market into the economics of smaller labs.
82
EXBIO Praha a.s.
538
Nad Safinou II 341
Vestec 25242
Czech Republic
Phone: 420 261 090 595
Email: samkova@exbio.cz
Web: www.exbio.cz
EXBIO Antibodies is a dynamic biotechnology
company focused on design, development and
manufacture of highest quality flow cytometry
products (antibodies and kits) for IVD and RUO
application, as well as on custom services at
affordable prices. Besides world-wide distribution
of its products under EXBIO label, the company
sales many antibody reagents also on OEM basis.
These products are widely recognized and appear
in catalogues of large number of immunochemical
retailers.
Flow Contract Site Laboratory, LLC 404
13029 NE 126th Place
Kirkland, WA 98034
Phone: 425-821-3900
Fax: 425-821-3925
Email: info@fcslaboratory.com
Web: www.fcslaboratory.com
Flow Contract Site Laboratory (FCS Lab) is a
specialized Contract Research Organization (CRO)
which offers expert services focused on GLP
and non-GLP flow cytometry assays for research
and development, nonclinical and clinical drug
development. The management of our organization
has resolved, as part of our diversification objective,
to seek out partners who are interested in utilizing
our capabilities to reduce existing work load while
increasing flexibility and expanding their capabilities.
Fluidigm631
7000 Shoreline Court Suite #100
South San Francisco, CA 94080
Phone: 650-266-6033
Email: tammy.kim@fluidigm.com
Web: www.fluidigm.com
Fluidigm develops, manufactures, and markets
life science systems based on integrated fluidic
circuits (IFCs). This technology furthers research
by minimizing costs and enhancing sensitivity for
applications such as single-cell gene expression,
high-throughput SNP genotyping, and next-generation
sequencing. Single-cell gene expression profiling has
recently emerged as a powerful method to uncover
ISAC 2013 Program and Abstracts
808
440
840
11175 Flintkote Avenue
San Diego, CA 92121
Phone: 858-642-0978
Fax: 858-642-0937
Email: jrosenberg@imgenex.com
Web: www.imgenex.com
Oral Session
Abstracts
IMGENEX Corporation develops and commercializes
reagents for human biology and disease for
diagnostics or potential therapy. Products include a
market leading portfolio of antibodies, reagents and
LUCPorter™ Reporter Cell Lines for Innate Immunity,
Tregs, Th17 cells, Dendritic Cells, and Inflammatory/
Immune Signaling Pathways for flow cytometry,
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
IMGENEX Corporation
Commercial
Tutorials &
Exhibits
handyem develops and manufactures innovative
flow cytometry solutions based on its patented,
F3FiberFlowFluidics® microfluidics technology.
handyem aims to democratize cytometry by providing
day-to-day autonomy and agility to life science
researchers with simple, highly efficient instruments at
a fraction of the cost of today’s solutions. handyem’s
HPC-100 Personal Cytometer, with its F3 microchip
platform, brings simplicity, accessibility and ease of
use to life science researchers.
iLab Solutions provides web-based research
management services to scientific institutions.
iLab’s research management and product selection
tools enable researchers to share knowledge, track
resources, manage budgets, and make smarter
purchasing decisions. iLab also supports the work
flow within core facilities to streamline request
management, improve usage tracking and reduce
non-compensation. iLab serves over 50 leading
research institutions across North America and
Europe.
Poster
Session
360-2750 Einstein Street
Quebec QC G1P 4R1
Canada
Phone: 418-650-5999
Email: ngagnon@handyem.com
Web: www.handyem.com
Ten Post Office Square
Boston MA, 02109
Phone: 617-297-2805
Email: info@ilabsolutions.com
Web: www.ilabsolutions.com
Wednesday,
22 May
handyem719
713
Tuesday,
21 May
360 Foothill Road
Bridgewater, NJ 08807
Phone: 908-231-0960
Fax: 908-231-1539
Email: usa@hamamatsu.com
Web: www.hamamatsu.com
iLab Solutions
Monday,
20 May
Hamamatsu Corporation
Hellma Analytics is the world’s leading manufacturer
of custom flow channels for Cytometry and a
reliable partner both for scientists and for instrument
manufacturers. Sophisticated equipment and
trained staff guarantees quality flow channels from
prototyping to large scale production. Flow channel
dimensions start as low as 50μm. State of the
art features like cones to optimize hydrodynamic
focusing, thin windows to fit large NA objectives,
application of focusing lenses etc can be integrated at
the highest precision.
Sunday,
19 May
GE Healthcare Life Sciences provides tools and
technologies, solutions and expertise for drug
discovery, biopharmaceutical manufacturing and
cellular technologies that will enable research scientists
worldwide to be more productive, effective, and
creative. Our focus is to support you from idea to result.
80 Skyline Drive
Plainview, NY 11803
Phone: 516-939-0888
Fax: 516-939-0555
Email: info@hellmausa.com
Web: www.hellmausa.com
Saturday,
18 May
800 Centennial Avenue, PO Box 1327
Piscataway, NJ 08855-1327
Phone: 732-457-8000
Fax: 877-295-8102
Email: cs-us@ge.com
Web: www.gelifesciences.com
520
Special
Lectures
GE Healthcare
Hellma USA, Inc.
Congress
Overview
heterogeneity in cell populations. In response to this,
Fluidigm has developed a streamlined and automated
workflow for capturing and analyzing single cells. The
new C1™ Single-Cell AutoPrep System isolates single
cells starting with low cell number input. For more
information, visit our website at www.fluidigm.com
83
Congress
Overview
Special
Lectures
immunofluorescent, or luminescent read-outs
respectively. IHC-Pro™ antibodies are validated for
immunohistochemistry and focused in Cancer and
Immunopathology.
the ISAC booth to learn more about the society, meet
society representatives such as the executive director
or the editor-in-chief of Cytometry Part A, and discover
how membership can help you advance your career.
Innova Biosciences
Life Technologies
502
Saturday,
18 May
Innova Biosciences, Babraham Hall, Babraham
Cambridge CB22 3AT
United Kingdom
Phone: 044 1223 496 171
Email: brian.carpenter@innovabiosciences.com
Web: www.innovabiosciences.com
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
At the core of Innova’s business are LightningLink® and InnovaCoat®, innovative technologies
that simplify antibody, protein and oligonucleotide
labeling for use in R&D applications, drug discovery
and the development of diagnostic kits. The easy to
use, one step procedure allows scientists to label an
antibody with 30 seconds hands-on time, saving both
time and money. Antibody recovery is 100% and the
technology is fully scalable without any deterioration
in the performance of the labeled antibody.
IntelliCyt Corporation
907
Wednesday,
22 May
9620 San Mateo Boulevard NE
Albuquerque NM 87113
Phone: 505-345-9075
Fax: 866-782-3140
Email: ltrinkle@intellicyt.com
Web: www.intellicyt.com
Commercial
Tutorials &
Exhibits
Poster
Session
IntelliCyt Corporation develops and markets screening
solutions for Drug Discovery and Life Sciences
Research. Our HTFC Screening System with new
ForeCyt software provides superior speed, sensitivity
and dynamic range for screening in 96 or 384 well
formats. Our MultiCyt Screening Kits and robotic
support combine to make this flow cytometry-based
platform a complete solution for screening.
ISAC742
Poster Session
Abstracts
Oral Session
Abstracts
9650 Rockville Pike
Bethesda, MD 20814
Phone: 301-634-7454
Email: isac@isac-net.org
Web: www.isac-net.org
Speaker/Author
Index
The International Society for Advancement of
Cytometry (ISAC) is a scientific and educational
organization that leads the way in development
of cytometry, transfer of new methodologies, and
exchange of cutting-edge scientific and technical
information in quantitative cell sciences. Please visit
84
624
5791 Van Allen Way
Carlsbad, CA 92008
Phone: 760-603-7200
Fax: 760-602-6500
Email: tech_support@invitrogen.com
Web: www.invitrogen.com
Life Technologies Corporation (NASDAQ: LIFE) is a
global biotechnology company dedicated to moving
science forward to improve life in meaningful ways
for everyone. Our premier brands are the most cited,
most trusted in the life sciences industry: Invitrogen™,
Applied Biosystems®, Gibco®, Molecular Probes®,
Novex®, TaqMan®, Ambion®, and Ion Torrent™.
Market Tech
213
1500 Green Hills Road, Suite 109
Scotts Valley, CA 95066
Phone: 831-461-1101
Fax: 831-461-1136
Email: info@markettechinc.net
Web: www.markettechinc.net
Market Tech is a leading distribution and sales
company offering a wide variety of Gas, Solid State,
Diode and Fiber Lasers covering UV, visible and IR
spectrum. We also offer laser power meters, light
and color measurement instruments, optics and fiber
optics products for life science, medical, aerospace,
quality control and research applications.
Melles Griot
634
2051 Palomar Airport Road, 200
Carlsbad, CA 92011
Phone: 760-438-2131
Fax: 760-438-5208
Email: mglasers@idexcorp.com
Web: www.cvimellesgriot.com
Melles Griot designs and manufactures solid-state
lasers, gas lasers and laser-based light engines. By
working seamlessly with the customer from start to
finish, we deliver high-value engineered solutions
for Analytical Instrumentation, Bio-Instrumentation,
and Ophthalmic and Medical Imaging applications.
Melles Griot provides the perfect balance of
performance, reliability, and manufacturability for
OEM applications worldwide.
ISAC 2013 Program and Abstracts
512
University of New South Wales
Sydney NSW 2052
Australia
Phone: 612 9385 9813
Email: r.baker@nsinnovations.com.au
Web: www.nsinnovations.com.au/
Nexcelom Bioscience
8100 Southpark Way, A-8
Littleton, CO 80120
Phone: 303-730-1950
Fax: 303-730-1966
Email: kim@novusbio.com
Web: www.novusbio.com
Novus Biologicals is a privately held, Coloradobased antibody and proteomics company. Novus
provides the research community with over 186,000+
antibodies, proteins, peptides, RNAi, and research
support products. Novus is a top tier proteomics
company because of their high quality products,
reliable customer service and technical support, as
well as the Novus Guarantee+. Visit www.novusbio.
com to learn more.
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
804
Oral Session
Abstracts
Newport, the world’s largest photonics company
provides innovative solutions and industry leading
product brands to multiple markets including the
photonics and photovoltaic industries. Our combined
broad product portfolio which includes: Corion®,ILX
Lightwave™, New Focus™, Ophir, Oriel®
Instruments, Richardson Gratings™, and SpectraPhysics® Lasers enables us to better partner with our
customers to provide complete photonic solutions to
make, manage and measure light.
Novus Biologicals
Commercial
Tutorials &
Exhibits
1791 Deere Avenue
Irvine, CA 92606
Phone: 949-863-3144
Fax: 949-253-1680
Email: sales@newport.com
Web: www.newport.com
Nexcelom’s Cellometer line of simple-to-use cell
counters automate manual cell counting procedures
by obtaining accurate counts, viability, and cell
sizes in less than 30 seconds & only 20uL of sample.
Fluorescence detection capabilities enable fast &
simple determination of GFP transfection rates, PIviability, & direct counting of WBCs without lysing.
Poster
Session
504
360 Merrimack Street, Bldg. 9
Lawrence, MA 01843
Phone: 978-327-5340
Fax: 978-327-5341
Email: ddemers@nexcelom.com
Web: www.nexcelom.com
Wednesday,
22 May
Newport Corporation
333
Tuesday,
21 May
Molecular Devices® is a leading provider of highperformance bioanalytical measurement systems
for life science research, and biotherapeutic and
pharmaceutical development. Our platforms for
imaging cytometry, high content imaging, microscopy
automation, image analysis, and microplate reader
applications, enable scientists to improve productivity
and effectiveness, accelerating research and discovery.
Together through life sciences.
Monday,
20 May
1311 Orleans Drive
Sunnyvale, CA 94089
Phone: 408-747-3545
Fax: 408-548-6431
Email: candace.anderson@moldev.com
Web: www.moleculardevices.com
Sunday,
19 May
802
NewSouth Innovations are showcasing the outcome
of research conducted in cluster identification for
flow cytometry data. The software, named SOPHE,
represents a breakthrough in processing flow
cytometry data analysis. The SOPHE algorithms
do not need any prior knowledge of the number
of dimensions or clusters and it can find otherwise
hidden relationships in multidimensional data. See us
at booth 323; we would be happy to demonstrate the
SOPHE system.
Saturday,
18 May
Miltenyi Biotec is a worldwide operating company
offering comprehensive systems and services for
life sciences, biomedical research and cellular
therapies. The product portfolio includes state-ofthe-art instrumentation and reagents for cellular and
molecular magnetic separation, cellular cultivation
and flow cytometric analysis.
Molecular Devices, LLC
323
Special
Lectures
Friedrich-Ebert-Strssa. 68
Bergisch-Gladbach 51429
Germany
Phone: 49 2204 830630
Email: annabelk@miltenyibiotec.de
Web: www.miltenyibiotec.com
NewSouth Innovations Pty Ltd
Congress
Overview
Miltenyi Biotec GmbH
85
Congress
Overview
OSELA Inc.
605
Special
Lectures
218 Brunswick
Pointe-Claire QC H9R 1A6
Canada
Phone: 514-426-2262
Email: tmarzilli@oselainc.com
Web: www.oselainc.com
Pavilion Integration Corporation
Sunday,
19 May
Saturday,
18 May
Osela is a specialized manufacturer of laser
illumination systems for industrial applications in
machine vision, life sciences and research. Our TOP
HAT BEAM SHAPER efficiently transforms a laser
beam (single mode and multimode) into at high
uniform top hat profile with no high frequency noise.
It’s compact, achromatic and easy to integrate into
existing optical systems.
OXXIUS Lasers
506
Tuesday,
21 May
Monday,
20 May
4, rue Louis de Brogile
Lannion 22300
France
Phone: 33 6 74 15 09 18
Email: pvoluer@oxxius.com
Web: www.oxxius.com
Wednesday,
22 May
Oxxius manufactures innovative solid-state lasers
and systems for Life Science and Measurement
applications.
- LaserBoxx monolithic DPSS, available at 532, 553
and 561nm from 25 up to 300mW in very compact
industry standard package (100x40x32mm).
Poster
Session
- LaserBoxx Laser Diode modules at 405nm, 445nm,
473nm, 488nm, 515nm, 638nm.
- LBX-4C Cost effective and modular 4 wavelengths
combiner for laborary use.
Commercial
Tutorials &
Exhibits
Partec830
Oral Session
Abstracts
Am Flugplatz 13
Goerlitz, Saxony 02828
Germany
Phone: 49 3581 87 46 0
Fax: 49 3581 87 46 70
Email: mail@partec.com
Web: www.partec.com
Speaker/Author
Index
Poster Session
Abstracts
Partec is a worldwide leading pioneer, developer and
manufacturer of flow cytometry systems in a wide
range of applications in healthcare, immunology, cell
biology, microbiology, biotechnology, agrosciences,
plant breeding, aquaculture and in pharmaceutical,
food and beverage industries. Partec offers also
innovative solutions for gel electrophoresis, PCR,
fluorescence and transmitted light microscopy. With
86
own subsidiaries and specially trained distributors,
Partec is active in more than 100 countries,
worldwide.
729
2380-F Qume Drive
San Jose, CA 95131
Phone: 408-453-8801
Fax: 408-453-8802
Email: sales@pavilionintegration.com
Web: www.pavilionintegration.com
Phoenix Flow Systems, Inc.
630
6790 Top Gun Street, Suite 1
San Diego, CA 92121
Phone: 858-453-5095
Fax: 858-453-2117
Email: ckb@phoenixflow.com
Web: www.phoenixflow.com
Stop by to build up our egos by feigning interest in
our products for: DNA analysis, MultiCycle, Apoptosis
reagents, Apo-BrdU, Sheath concentrates, Cheap
Sheath or Cell Detachment solutions, Accutase &
Accumax while really only wanting to pick our brain
about micro-brewery locations.
Photop Technologies Inc.
211
253 Fuxin East Road
Fuzhou Fujian 350014
China
Phone: 86 591 8805 2884
Email: jenny.liu@photoptech.com
Web: www.photoptech.com
Photop Technologies, subsidiary of II-VI Corp.
(NASDAQ:IIVI), is a leading photonics designer and
integrated manufacturing company on Fiber Optics,
Precision Optics, Projection and Display Optics, DPSS
Laser, Crystal Materials, and other Photonics Products.
Propel Labs
623
131 E. Lincoln Avenue, Suite 200
Fort Collins, CO 80524
Phone: 970-295-4570
Fax: 970-372-5664
Email: info@propel-labs.com
Web: www.propel-labs.com
Propel Labs, the developer of the Avalon, and now
our partner Bio-Rad Laboratories, offers S3 the high
performance bench-top cell sorter. The S3 is an easy
to use 1-2 laser, 2-way sorter, with sort speeds >30k/
ISAC 2013 Program and Abstracts
Stratedigm, Inc.
522
19 Great Oaks Boulevard, Suite 10
San Jose, CA 95119
Phone: 408-884-4029
Fax: 408-351-7700
Email: ibalakshyna@stratedigm.com
Web: www.stratedigm.com
Commercial
Tutorials &
Exhibits
Stratedigm manufactures the SE520, S1000 and
S1000Ex high-performance, bench-top flow
cytometers upgradeable from one to four lasers
with 14 Colors. Patented high-efficiency optics
support user-configurable detector assignment and
user-changeable filters for maximum performance.
Our A600 High Throughput Auto Sampler offers
a full automation solution compatible with all of
Stratedigm’s analyzers. Our systems provide the
best sensitivity, flexibility and reliability - truly, flow
cytometers without compromise.
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Spherotech manufactures a variety of microparticles
for flow cytometers. These particles are used for
calibration, alignment, multiplexing, compensation,
absolute counting and drop delay determination.
Specifically, the calibration, alignment, and drop
delay particles are used extensively for QC and long
term performance tracking. In addition, Spherotech
has particles for confocal fluorescence microscopy.
Poster
Session
Sony Biotechnology Inc. is dedicated to helping the
cytometry community of life scientists, researchers,
laboratory professionals, & institutions achieve the best
scientific results possible. We offer a comprehensive
line of flow cytometry products, including our sy3200
& ec800 sorter/analyzer platforms & an extensive
portfolio of fluorochrome conjugated antibodies &
support reagents (>8000). We will also be showcasing
Sony Corporation’s new advances in flow cytometry
technology the SH800 and SP6800.
27845 Irma Lee Circle, Unit 101
Lake Forest, IL 60045
Phone: 847-680-8922
Fax: 847-680-8927
Email: service@spherotech.com
Web: www.spherotech.com
Wednesday,
22 May
2100 South Oak Street
Champaign, IL 61820
Phone: 217-979-1414
Email: sales@i-cyt.com
Web: www.i-cyt.com
416
Tuesday,
21 May
303
Spherotech, Inc.
Monday,
20 May
Sony Biotechnology Inc.
Since 1982, SouthernBiotech has been dedicated
to the production of the world’s highest quality
secondary antibodies for research use. Now, we
offer a broad range of polyclonal and monoclonal
antibodies, including, fluorochrome and enzyme
conjugates, Low Endotoxin/Azide Free preparations,
F(ab’)2 fragments, purified immunoglobulins and
collagens.
Sunday,
19 May
Semrock manufactures hard-coated optical
filters that set the standard for biomedical &
analytical instrumentation. Include the acclaimed
VersaChrome® tunable bandpass filters, highperformance fluorescence & Raman spectroscopy
filters & unique laser optics. Based on VersaChrome
filters, Fresco™ tunable filter system provides a
unique combination of high throughput & spectral
flexibility for fluorescence microscopy & other
spectral applications.
160 Oxmoor Boulevard
Birmingham, AL 35209
Phone: 205-945-1774
Fax: 205-945-8768
Email: info@southernbiotech.com
Web: www.southernbiotech.com
Saturday,
18 May
3625 Buffalo Road
Rochester, NY 14624
Phone: 585-594-7000
Fax: 585-594-3898
Email: semrock@idexcorp.com
Web: www.semrock.com
SouthernBiotech311
Special
Lectures
Semrock632
Congress
Overview
sec, purity > 99%, a customized software platform
and integrated sample cooling. Optional features
for operator protection include a Class I and Class
II Biosafety Cabinet. Propel Labs also offers MoFlo
and CyAn upgrades including Co-Lase Towers and
Nanoview for small particle detection.
87
Congress
Overview
Thermo Fisher Scientific
633
Special
Lectures
46360 Fremont Boulevard
Fremont, CA 94538
Phone: 510-979-5000
Email: al.bru@thermofisher.com
Web: www.thermofisher.com
Sunday,
19 May
Saturday,
18 May
Thermo Scientific Cyto-Cal and Cyto-Plex particles
are designed to optimize flow cytometry performance.
Cyto-Cal particles are ideal for calibration and setup, and for monitoring flow and optical stability.
Cyto-Plex particles enable simultaneous detection
and quantitation of multiple analytes within a single
sample. Thermo Fisher Scientific is your single source
for all your particle technology needs. Call our
customer and technical support team at 1-800-2323342 and visit Booth #633 for details.
Monday,
20 May
Tonbo Biosciences
513
Tuesday,
21 May
4940 Carroll Canyon Road
San Diego, CA 92121
Phone: 858-218-6626
Fax: 858-888-7301
Email: sammee@tonbobio.com
Web: www.tonbobio.com
Wednesday,
22 May
Tonbo Biosciences is a company built to serve your
needs for flow cytometry reagents. Our goal is to
do things differently, to fashion a better experience
for you by providing the highest quality, high
performance reagents at the best available prices.
Poster
Session
Same Clones – established clones you trust and use
every day
Outstanding Quality – manufactured in the USA for
the highest quality and performance
Commercial
Tutorials &
Exhibits
Exceptional Value – our products are priced at 30% to
50% less than your lowest priced supplier
Toptica Photonics Inc.
505
Oral Session
Abstracts
1286 Blossom Drive
Victor, NY 14564
Phone: 585-657-6663
Email: lisa.gray@toptica-usa.com
Web: www.toptica-usa.com
Speaker/Author
Index
Poster Session
Abstracts
TOPTICA is the world leader in diode laser and
ultrafast technology for industrial and scientific
markets. We offer the widest range of single mode
tunable light in the 190 to 2900nm and 0.5-2.5THz
spectral region with various accessories to measure,
characterize, stabilize and analyze light. With our
“Passion for Precision”, TOPTICA delivers!
88
Tree Star, Inc.
611
340 A Street, Building 1, Suite 203
Ashland, OR 97520
Phone: 800-366-6045
Fax: 541-482-3153
Email: jake@treestar.com
Web: www.flowjo.com
FlowJo is the next generation of flow cytometry
analysis software. It handles your most ambitious
projects with a high-level drag-and-drop user
interface. Based on a patented experiment-based
analysis paradigm, FlowJo intelligently handles
protocols containing multiple tubes (any FCS files)
with different samples stained with different reagent
sets. Visit flowjo.com for the full story.
TTP Labtech Ltd
325
1 Kendall Square, Suite B2303
Cambridge, MA 02139
Phone: 617-494-9794
Fax: 617-494-9795
Email: sales@ttplabtech.com
Web: www.ttplabtech.com
Would you like to multiplex your homogeneous beadbased assays for pennies per well? TTP Labtech is a
global developer and manufacturer of high quality,
innovative automated laboratory equipment for
pharmaceutical and biotech research. Our portfolio
of products minimize assay volumes, reduce material
handling costs, and put the discovery tools back in
the hands of the scientist. Visit booth #325 to find out
more.
Union Biometrica, Inc.
711
84 October Hill Road
Holliston, MA 01746
Phone: 508-893-3115
Email: thofhuis@unionbio.com
Web: www.unionbio.com
Union Biometrica Large Particle Flow Cytometers
automate the analysis, sorting and dispensing of
objects too big/fragile for traditional cytometers, e.g.,
large cells / cell clusters, cells in/on beads and small
model organism. Choose between 4 COPAS models
and the new BioSorter with interchangeable modules
to cover the full 10-1500µm range.
ISAC 2013 Program and Abstracts
402
279 Campus Drive
Stanford, CA 94305
Phone: 650-723-5054
Fax: 650-725-8564
Email: johnjm@stanford.edu
Web: www.woodsidelogic.com
516, 615
218 Xinghu Road C7-501
Suzhou Industrial Park Jiangsu 215123
China
Phone: 86 051265928665
Email: yong.chen@xitogen.com
Tuesday,
21 May
We’re newcomers to flow cytometry instrumentation.
We strive to make flow cytometers available to
everyone who studies particles. Flow cytometers
should be easy to use and affordable, but we
don’t believe that compromising quality and
performance will get us there. Because we are Xitogen
Technologies - “excellence is in our genes.” Let’s work
together. Come to Booth 615 and become one of the
first to embrace a disruptive flow cytometer. Starting
from $25k, upgradable to 4 lasers, 14FLs.
Wednesday,
22 May
Poster
Session
Vortran Laser Technology, Inc. is the newest and
fastest growing provider of the highest quality laser
solutions. Our products range from OEM laser
modules up to fully integrated photonic sub-systems
maximizing performance and profitability for our
customers. The experience and capabilities of our
engineering will expand your product performance
while reducing costs allowing you to focus on the
tasks you do best, not laser integration and optical
alignment. Leave that to our design and expertise.
Xitogen Technologies Inc.
Monday,
20 May
21 Goldenland Court #200
Sacramento, CA 95834
Phone: 916-283-8208
Email: sthrone@vortranlaser.com
Web: www.vortranlaser.com
Sunday,
19 May
319
Integrated computer-based support for flow and other
cell based studies: Use CytoGenie + AutoColor ?
CytoHub ? EverTrieve ? AutoComp ? ClusterGenie
? DiffGenie ? CytoNote in Irisnote to plan analyses,
collect, label and securely store data, do fluorescence
compensation, sequentially choose axes to guide
automated subset (cluster) identification, compare
datasets, and integrate with other studies in a
comprehensive well-indexed laboratory notebook
cloud system.
Saturday,
18 May
Verity Software House, an industry leader in flow
cytometry software development, offers a unique
combination of innovative software for flow cytometry
and unparalleled technical and customer support.
ModFit LT, WinList, and GemStone: an unbeatable
combination. Please stop by and see us in booth
402, and learn more about high-dimensional data
analysis with GemStone at our Commercial Tutorial
on Wednesday. Without Verity, it’s just software.
Vortran Laser Technology, Inc
938
Special
Lectures
45A Augusta Road, PO Box 247
Topsham, ME 04086
Phone: 207-729-6767
Fax: 207-729-5443
Email: sales@vsh.com
Web: www.vsh.com
Woodside Logic
Congress
Overview
Verity Software House
Wiley842
Commercial
Tutorials &
Exhibits
111 River Street
Hoboken, NJ 07030
Phone: 201-748-5851
Email: clkelly@wiley.com
Web: www.wileyblackwell.com
Oral Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Poster Session
Abstracts
Wiley-Blackwell is the international scientific,
technical, medical and scholarly publishing business
of John Wiley & Sons, with strengths in every major
academic and professional field and partnerships with
many of the world’s leading societies. Wiley-Blackwell
publishes over 1,400 peer-reviewed journals as well
as 1,500+ new books annually in print and online,
as well as databases, major reference works and
laboratory protocols. For more information, please
visit www.wileyblackwell.com.
89
Congress
Overview
Special
Lectures
Oral Abstracts
1
2
Apoptosis, Autophagy and DNA Damage
Cytometer Performance Characterization and
Standardization
Saturday,
18 May
William Telford1, Zbigniew Darzynkiewicz2
1
National Cancer Institute, National Institutes of Health,
Bethesda, MD, United States, 2Brander Cancer Research
Institute, New York Medical College, Valhalla, NY, United
States
Sunday,
19 May
Tutorial Objectives: In the first part of the Tutorial (Telford),
flow cytometric methods to assess apoptosis will be thoroughly
reviewed. Students will learn how to select the appropriate assay,
the technical details necessary for assay success, and combining
multiple assays for multiparametric analysis of cell. The tutorial will
take a very practical approach, and actual cytometry data will be
analyzed as part of the program. Autophagy, a phenomenon both
related to and distinct from cell death will also be covered.
Monday,
20 May
In the second part of the Tutorial (Darzynkiewicz), detection of
DNA damage by flow and image cytometry will be covered, both as
a component of apoptosis and as a means of analyzing the effects
of pharmacological, toxicological and environmental insults to live
cells. Detection of both DNA damage itself and of phosphoproteins
critical for DNA organization and structure that serve as markers for
DNA damage will be particularly emphasized.
Wednesday,
22 May
Tuesday,
21 May
After completing the Tutorial, the student should be able to:
(1) Have a working knowledge of flow cytometric assays for
apoptosis, autophagy and DNA damage.
(2) Be able to select an appropriate apoptosis and DNA damage
assay for their experimental system, procure the necessary
reagents and carry out the assay.
(3) Combine multiple apoptosis assays to develop powerful systems
for studying the complex signaling and progression of apoptosis.
Commercial
Tutorials &
Exhibits
Poster
Session
Tutorial Details or Outline:
Apoptosis and Autophagy (led by William Telford)
1) Types of cell death
2) Types of assays
- DNA damage and loss (DNA binding dyes, TUNEL)
- Cell membrane alterations and damage (DNA dyes, annexin V)
- Caspase activation
3) Combining multiple apoptosis assays for more information
4) Choosing the right apoptosis assay
5) Critical parameters for analyzing apoptosis
6) Data analysis - artifacts and pitfalls
7) Analysis of autophagy by flow cytometry
Tutorial Objectives: Cytometers make optical measurements of
particles. Using flow cytometers as the primary example, this
tutorial will provide an understanding of the measurement process
and how to critically and objectively characterize the instrument’s
ability to make fluorescence and light scattering measurements.
The use of standard instrument performance criteria and testing
materials can allow minimal performance requirements to be
established for an assay and assure that results on different
instruments in different laboratories get comparable results when
running the same sample. Authoritative standards (traceable to
a standards setting organization) are generally not available for
cytometry. Students will learn about the use and limitations of
commercially available standard materials and some options for
laboratory prepared standards.
Tutorial Details or Outline:
1. Measurement characteristics that flow, scanning and static
imaging cytometers have in common.
2. Characteristics of photomultiplier, solid state, and imaging
detectors.
3. How to characterize critical instrument performance factors
including: linearity, resolution, electronic noise, sensitivity
(resolution at low signal levels), dynamic range. Reference
methods and practical approaches to characterizing these
factors are discussed.
4. How detailed characterization helps to troubleshoot problems.
5. Standardizing performance of one or more cytometers.
6. Status of authoritative calibration materials and practical
alternatives.
7. Future possibilities for widely available standard performance
criteria and materials for testing.
3
Approaches in Image-Based High Content Screening
Susanne Heynen-Genel1, Jeffrey H. Price2,3
Conrad Prebys Center for Chemical Genomics, SanfordBurnham Medical Research Institute, La Jolla, CA, United
States, 2NCI-Designated Cancer Center, Sanford-Burnham
Medical Research Institute, La Jolla, CA, United States, 3Vala
Sciences Inc., San Diego, CA, United States
1
Tutorial Objectives: This tutorial will cover current approaches
in image-based high content screening and provide examples.
After attending this tutorial, the participant should have a good
understanding of the currently available image-based screening
methods and their advantages and limitations.
Tutorial Details or Outline:
1. Basics of Image-Based High Content Screening
2. High-Throughput Imaging Assay Approaches
3. Advanced Methods for Image-Based Assays
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
DNA damage (led by Zbigniew Darzynkiewicz)
1) Detection and types of DNA damage in cells
2) Significance of DNA damage in both apoptotic and nonapoptotic cells
3) Role of pharmacological, toxicological and environmental
insults in inducing DNA damage
4) Detection of histone-associated phosphoproteins as markers of
DNA conformational changes and damage
5) Correlation of the above events with apoptosis and apoptosisassociated signalling events (i.e. caspase activity)
Robert Hoffman
BD Biosciences, San Jose, CA, United States
90
ISAC 2013 Program and Abstracts
6
Biosafety: Risk Assessment and SOP Development
Proliferation Tutorial: Cell Cycle Progression and
Division – Unraveled by Flow Cytometry
High Throughput and High Content Screening for
Flow Cytometry
Oral Session
Abstracts
3. Cell Division
We will discuss key assumptions involved using cell tracking
dyes to monitor cell division based on decreasing dye intensity in
successive daughter generations. Common problems encountered
for each of the two major tracking dye types will be illustrated.
• Covalent protein binding dyes (e.g. CFSE, CellTrace Violet, etc.)
• Lipophilic membrane intercalating dyes (e.g. PKH26, CellVue
Claret, etc.)
• Applications of these methods to vaccine development, stem cell
research and suppression assays
Poster Session
Abstracts
4. Analysis of Cell Cycle Progression and Division Data
The methods used to analyze cell cycle progression data obtained
by flow cytometry will be discussed and demonstrated along with
the methods used to quantitate cell division. To best understand
Speaker/Author
Index
ISAC 2013 Program and Abstracts
2. Cell Cycle Progression
This section will focus on the different methods for measuring cell
cycle progression by flow cytometry. Labeling mechanisms and
the information provided DNA binding dyes (“classic” cell cycle
analysis) will be compared and contrasted with alternative methods
including:
• Thymidine Analogs (BrdU, EdU)
• Proliferation-Related Antigens including cyclins and cyclin
dependent kinases
• FUCCI cell lines, vectors and mice
Commercial
Tutorials &
Exhibits
Tutorial Details or Outline:
1. Fundamentals of HT Flow
2. Assay Design for functional screening
3. Assays for Immunological screening
4. Automation and plate preparation
5. Running the assays
6. Data collection
7. Data analysis and processing
8.Reporting
9. Problems and solutions
1. Overview
The DNA cell cycle will be reviewed providing everyone with a
clear understanding of what needs to occur when in order for a
cell to enter into and progress through the cell cycle before finally
dividing. The differences between cell cycle progression and cell
division and how flow cytometry is used to measure each will be
explained.
Poster
Session
After participating in this tutorial, the participant will be more
aware of the advantages and possible disadvantages of having to
work in an automated, or semi-automated régime. They will have
a good knowledge of how to approach the preparation, and the
analysis of data. They will also learn some analytical tools that have
been designed particularly for high throughput and high content
analysis.
Tutorial Details or Outline:
Wednesday,
22 May
Tutorial Objectives: High throughput screening is now a real
opportunity with flow cytometry. It is now possible to collect
thousands of samples per day in a highly ordered way. However,
designing and preparing these assays does require some careful
attention to details and automated prep becomes an important
component. One advantage is that you only sample very small
volumes (like 1 uL) instead of 50 to 250 uL. This means instead
of needing far more cells to start, you may actually be able to run
far more samples than you might think. We will discuss several
different implementations of HT flow cytometry and the types
of assays that are usable in this format. This tutorial will also
discuss aspects very high content flow cytometry where you might
have a very large number of samples as well as a large number
of parameters such as might be found in CyTOF data where
parameters may exceed 20-30 per assay.
After participating in this tutorial attendees should have a clear
understanding of 1) events that must occur in order for a cell to
enter the cell cycle and to divide; 2) the difference between cell
cycle progression and cell division as measures of proliferation; and
3) advantages and limitations of specific flow cytometric methods
commonly used to quantify cell cycle progression and/or extent of
cell division.
Tuesday,
21 May
J. Paul Robinson1, Padmakumar Narayanan2, Rob Jepras3
1
BMS/BME, Purdue University, West Lafayette, IN, United
States, 2Amgen, Seattle, WA, United States, 3GSK, Middlesex,
United Kingdom
Examples of applications using the different methods will be used
to compare and contrast the information that is obtained using
different probes and techniques. We will end with an opportunity
for participants to discuss their real world problems and invite
you to send your challenging cases to one of the instructors for
discussion during the tutorial.
Monday,
20 May
5
Sunday,
19 May
Tutorial Details or Outline: Basic biosafety principles will be
covered and in particular how the principles of containment
and risk assessment apply to flow cytometry and cell sorting.
The development of an SOP is a consequence of a thorough risk
assessment and the steps required to formulate an instrument
specific SOP will be presented. Procedures, engineering controls,
disinfection, and personal protective equipment will be discussed
as part of the overall SOP development process. An overview of the
newly approved NIH Biosafety Policy for Cell Sorters will also be
presented.
Tutorial Objectives: This tutorial will consider flow cytometric
techniques for monitoring different aspects of cell proliferation.
After a brief review of cell cycle biology, we will discuss:
1) Principles, methods and analysis strategies for measuring
cell cycle progression using DNA binding dyes, thymidine
analogues, proliferation-related antigens and phase specific
expression vectors;
2) Principles, methods and analysis strategies for monitoring extent
of cell division using proliferation tracking dyes (CFSE, PKH26
and newer analogs of each).
Saturday,
18 May
Tutorial Objectives: This tutorial will provide the attendee with an
overview of biosafety principles, as they apply to flow cytometry
with emphasis on cell sorting. The process of risk assessment for
determination of biosafety containment levels and the development
of a Standard Operating Procedure (SOP) for flow cytometers will
be discussed.
Kylie Price1, Katharine A. Muirhead2, Paul K. Wallace3
1
Malaghan Institute of Medical Research, Wellington, New
Zealand, 2SciGro, Inc./MidWest Office, Madison, WI, United
States, 3Roswell Park Cancer Institute, Buffalo, NY, United
States
Special
Lectures
Kevin Holmes
Flow Cytometry Section, NIAID, NIH, Bethesda, MD, United
States
Congress
Overview
4
91
Congress
Overview
Sunday,
19 May
Saturday,
18 May
Special
Lectures
these we will examine the evolution over time of these methods
from the simple to the complex.
• Cell Cycle Analysis
Inside-out (S-Fit)
Outside-in
Non-linear least-squares analysis
• Cell Cycle Progression
Proliferative Fraction
Proliferative Index
Precursory Frequency
7
Tutorial Objectives:
Cell Sorting: Fundamentals, Applications and
Troubleshooting
This tutorial will cover two subjects:
Monday,
20 May
Tuesday,
21 May
Tutorial Details or Outline:
Wednesday,
22 May
The aim of this session is not to provide a "recipe book" type
approach to electrostatic cell sorting, but rather to stimulate the
participants to think about how cell sorting can be practically
applied to help solve scientific problems.
Poster
Session
The session will discuss how electrostatic cell sorting works and
why it has become widespread, and compare this technology
with other separation techniques. We will then cover the physical
constraints that affect cell sorters and the implications they have on
what goes into an instrument and what comes from an instrument.
We will discuss ways that the cell sorter can make a "bad" decision
and what to do about it.
Commercial
Tutorials &
Exhibits
8
Oral Session
Abstracts
It is hoped that opportunities will arise for active audience
discussion about cell sorting to occur.
Tutorial Objectives: Image-based experiments using cultured cells
have proven to be a powerful means of generating informationrich data for biological applications. This tutorial will instruct
biologists in the concepts and application of CellProfiler, an
open-source, freely-downloadable software package designed for
large-scale, automated phenotypic image analysis. We will work
through hands-on examples, including construction of an analysis
pipeline, optimization of module settings, automatic cellular feature
detection and measurement, and exporting results. We will also
briefly discuss the basic principles of supervised machine learning
in order to score phenotypes where phenotypic differences between
samples are subtle and/or complex. The tutorial participant should
gain a working knowledge of CellProfiler and how to process and
analyze their high- or low-throughput, high-content experiment.
Image Quantification and Analysis using CellProfiler
David Logan
Imaging Platform, Broad Institute of Harvard and MIT,
Cambridge, MA, United States
Poster Session
Abstracts
Analysis of Receptor Dynamics Using Flow Cytometry
Alexandre Chigaev1, Yang Wu2
Pathology Dept. MSC09 5025, University of New Mexico
Cancer Center, Albuquerque, NM, United States, 2Department
of Pathology and Center for Molecular Discovery, University
of New Mexico, Albuquerque, NM, United States
Tutorial Objectives:
1. Provide information on the fundamentals of electrostatic cell
sorting and an overview of the application of cell sorting in
practical situations.
2. Discuss some of the areas where things can and do go wrong,
and what steps can be taken to improve sorting experiments.
Speaker/Author
Index
9
5. Summary
The tutorial will conclude with an open discussion using real world
examples to illustrate what can and does go wrong and how best to
avoid these problems.
Geoffrey Osborne
Queensland Brain Institute / The Australian Institute for
Bioengineering and Nanotechnology, Univ of Queensland,
St Lucia, Brisbane, Australia
92
Tutorial Details or Outline:
1. Basic overview of CellProfiler
2. Hands-on example(s) of an image-based assay analysis pipeline
3. Discuss other capabilities of CellProfiler, including
interoperability with other tools
4. Example using a biologist-friendly machine learning tool,
CellProfiler Analyst
1
Part I Real-Time Flow Cytometry of Integrin Signaling: Lessons
Learned
Have you ever dreamed of performing signaling experiments on live
cells, in real-time, at natural receptor abundance, and without longterm pre-staining of cells using fluorescent dyes? We have been
doing these for more than a decade. A unique property of integrin
molecules is the ability to rapidly change ligand binding affinity, to
“stand-up” on the cell surface, and to respond to the ligation of the
binding pocket through the changes of the molecular conformation.
These changes generate a plethora of molecular conformations,
which, at the cellular level, are translated directly into different
modes of cell-adhesive behavior. G-protein coupled receptors,
tyrosine kinase receptors, and other signaling pathways (including
nitric oxide/cAMP pathway) rapidly regulate integrin conformation,
and modulate cell adhesion and mobilization, by triggering socalled “inside-out” signaling pathway. In the current presentation
we focus on the basic methods and novel unpublished results
including FRET-based measurement of molecular extension of LFA1 integrin, as well as rapid de-activation of VLA-4 integrin through
previously undescribed signaling pathways.
This work was supported by R01HL081062 and U54MH084960.
Authors declare no competing interests.
By the end of this section participants will not only learn about
real-time flow cytometry, experimental design, and interpretation of
the data, they will also understand the relationship between integrin
molecular conformation and immune cell adhesive behavior
(rolling, firm adhesion, or cell detachment). We will also discuss
practical questions related to probe design, and commercial sources
of existing probes.
Part II High Throughput Flow Cytometry in Drug Discovery
Now you’ve learned all the basics about monitoring surface protein
signaling in real time, it’s time to move on to the hot field of drug
discovery. In the second half of the tutorial, we will introduce
you to a newly developed approach that is not only suitable for
the real-time analysis of receptor trafficking, but also compatible
with high-throughput flow cytometry (HTFC). The later application
demonstrates that in addition to measuring individual samples from
test tubes, flow cytometry has the capability to collect data from
40 samples per minute, and thus contribute to the early stage of
drug discovery. In the sample approach, a newly available reporter
protein (FAP) tag was fused with target protein, and ligand induced
receptor trafficking was measured by flow cytometry in real-time.
We will share with you some basics of HTFC and the FAP reporter
system, experimental design of HTFC compatible assays, associated
data analysis, and work flow. Videos taken in our high-throughput
flow cytometry center at the University of New Mexico will also
be available to demonstrate the automated processes of the sample
plate preparation and screening.
ISAC 2013 Program and Abstracts
(Zucker an Fisher 2013, Protocols in Cytometry, Unit 1:28). This
paper provides useful information in evaluating flow cytometers
prior to purchase.
11
Seeing a More Colorful World: A Guide to
Polychromatic Flow Cytometry
Pratip Chattopadhyay
ImmunoTechnology Section, Vaccine Research Center, NIAID,
NIH, Bethesda, MD, United States
12
Analysis and Sorting of Rare Cell Populations
Robert Zucker1, Nancy Fisher2
1
Toxicology Assessment Division, U.S. Environmental
Protection Agency, Research Triangle Park, NC, United States,
2
Department of Microbiology and Immunology, University of
North Carolina at Chapel Hill, Chapel Hill, NC, United States
Tutorial Objectives: This tutorial will address how to find the
proverbial ‘needle in the haystack’ when needing to identify or
sort rare populations of cells. In the contemporary flow cytometry
laboratory, there is often the need to accurate identify rare cells,
such as circulating endothelial cell, endothelial progenitor cells,
tumor cells, or immune subpopulations such as plasmacytoid or
monocytoid dendritic cells.The student will gain an appreciation of
obstacles in the accurate identification of rare cells and of strategies
to overcome these obstacles and to assure better experimental data.
J. Philip McCoy
NHLBI and CHI, Bethesda, MD, United States
Poster
Session
Commercial
Tutorials &
Exhibits
Tutorial Details or Outline:
1. Definitions, and an overview of the need for rare cell
identification
2. Artifacts and sources of noise
3. Strategies for panel design and avoiding artifacts
4. Examples of rare event analyses
5. Verification of rare events, how do you know that you have
identified the cells of interest with fidelity?
6. Summary and final thoughts
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Wednesday,
22 May
Tutorial Details or Outline: We will discuss instrument
standardization, antibody conjugation and titration, panel
development, troubleshooting, and data analysis.
Evaluation and Purchase of an Analytical Flow
Cytometer: Some of the Numerous Factors to
Consider
Tutorial Details or Outline:
We discuss seven major issues to evaluate in the purchase of a new
flow cytometer:
• Applications,
• Hardware and specifications (applications) ,
Tuesday,
21 May
Tutorial Objectives: This tutorial will cover the latest tips and
tricks for successful polychromatic flow cytometry experiments.
Participants will leave the session with a practical, working
knowledge of how to develop or optimize the technology for their
laboratories.
10
Tutorial Objectives:
1) When purchasing a flow cytometer, the decision of which
brand, model, specifications, and accessories may be
challenging. The decisions should initially be guided by the
specific applications intended for the instrument. However,
many other factors need to be considered, which include
hardware, software, quality assurance, support, service, and
price and recommendations from colleagues. These issues are
discussed to help guide the purchasing process.
2) Student should obtain information that will be used to evaluate
different factors that are neccesary to evaluate in purchacing a
new flow cytometer.
Monday,
20 May
In the first part of the tutorial, we will focus on a new approach to
measure real-time receptor trafficking with high-throughput flow
cytometry. We will be discussing:
1. Background and overview of high throughput flow cytometry
and drug discovery
2. Methods for real time monitoring of receptor trafficking: design
and development of HTFC compatible assays using FAP as the
reporter protein tag.
3. De-convoluting the outcome from HTFC: primary screening,
dose response screen and counter screens
4. Conclusions: New approaches to discover new ligands
modulating receptor trafficking; Role of HTFC in drug discovery
Sunday,
19 May
Part II
Recommendations from colleagues.
The individuals evaluating these machines should have an
evaluation procedure in place to compare different units for their
research endpoints. If the wrong unit is chosen, it may result in
extra costs, lack of productivity, and less robust data that may not
fulfill the current or future objectives of the laboratory. Our attempt
is not to compare these machines or bead products used to test
these machines directly, but to provide some insight on what factors
to look for that may be overlooked by some scientists in their
evaluation process. Instruments should be tested with the samples
that are usually run in the laboratory. A series of quality assurance
(QA) bead tests should be performed so resolution, sensitivity, and
precision can be evaluated.
Saturday,
18 May
Part I
In the first part of the tutorial, we will present the use of small
ligand-mimicking fluorescent probes for the real-time analysis of
receptor occupancy, affinity and conformational changes. We will
discuss basics of real-time flow cytometry, as a novel approach that
allows performing signaling experiments on live cells, in real-time,
at natural receptor abundance. We will show how:
• Real-time flow cytometry can be used for the determination of
binding rate constants and dissociation constant
• Rapid changes in the ligand binding affinity are detected under
signaling through the cell receptors
• Molecular extension (unbending) can be detected using FRETbased methods
• Real-time binding of conformationally sensitive antibody is used
for the determination of the dynamics of intracellular signaling
• Software, ease of use, and power,
• Quality assurance testing,
• Service, support, and company
• Price and maintenance prices,
Special
Lectures
Tutorial Details or Outline:
Congress
Overview
By the end of the second half of this tutorial, we hope you will
get some idea about the role of high-throughput screening in the
field of drug discovery, gain some experience on setting up assays
suitable for high-throughput flow cytometry, be familiar with a new
type of biosensor, and bring home with some thoughts and ideas
about how FAP and HTFC can do for your research.
93
Congress
Overview
13
Quantitative FRET Microscopy
Special
Lectures
Gyorgy Vereb, Janos Szollosi
Department of Biophysics and Cell Biology Medical and
Health Science Center, University of Debrecen, Debrecen,
Hungary
Sunday,
19 May
Saturday,
18 May
Tutorial Objectives: The use of fluorescence (Förster) resonance
energy transfer (FRET) for assessing molecular interactions in
cellular systems is exponentially expanding. Several methods,
mostly for microscopy, have been proposed, but most of them that
made it to broad use owed to their simplicity suffer from being only
qualitative or even from being prone to misinterpretation of results.
The educational outcome of the tutorial is to provide the audience
with stable foundations for applying a simple, yet qualitative FRET
procedure that can be performed in any commonly available laser
scanning fluorescence microscope. Addition outcomes include
skills in interpretation of FRET data, and a broader knowledge on
the pros and cons of various FRET methods that allow an educated
choice of the approach most appropriate for the biological
question.
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Tutorial Details or Outline:
1. Introduction: What is FRET?
2. Various manifestations of FRET: donor quenching and acceptor
sensitization
3. The simplest generic approach to quantitate FRET from donor
quenching: implementation, interpretation, limitations
4. The simplest generic approach to quantitate FRET from
acceptor sensitization: implementation, interpretation, and
the very serious limitations that make this approach the least
recommendable (except a few specific cases)
5. Acceptor photobleaching (AccPb), as a self-controlled approach
in measuring microscopic FRET from donor quenching - the
method at its simplest
6. Artifacts that can arise during AccPb, and their correction
7. A simple ImageJ plugin for evaluating AccPb FRET
8. When time or space comes up as a 3rd dimension to be
considered: basic principles of intensity-based (ratiometric)
FRET measurements that are corrected for spectral bleedthrough
9. Summary, discussion, consultation for the participants about
their ongoing experiments or plans.
14
Commercial
Tutorials &
Exhibits
Growing a Cytometry Core Facility: Adding Calue
with Hardware and Education
Derek Davies1, Alfonso Blanco2
London Research Institute, Cancer Research UK, London,
United Kingdom, 2Conway Institute of Biomolecular and
Biomedical Research, University College Dublin, Dublin,
Ireland
Oral Session
Abstracts
1
Speaker/Author
Index
Poster Session
Abstracts
Tutorial Objectives: Core facilities are now common in all work
settings. Flow cytometry is a well-established technique but the core
faces particular challenges in the face of expanding technology.
In particular, cores need to bring added value to their users and
institutional setting. But how can core facility staff keep up with the
latest developments, how can they receive appropriate continuing
education and how can this be passed on to users of a facility? We
will discuss evaluation of technology and strategies for importing
this into a core and also how education on site, at relevant meetings
and by remote learning can benefit the facility. At the end of the
tutorial the delegate will be aware of the approaches that can be
taken to bring added value to the core, its staff and its users.
Tutorial Details or Outline: This tutorial is aimed at Core managers
or facility staff that work in established cores and who wish to
94
expand the repertoire of service provision in times of budgetary
restrictions and the ever-increasing time demands on a successful
core.
1. Overview of the structure of a core facility: this includes facility
planning, financial considerations, operational issues such
as scheduling, assessment of users needs and re-charge. (10
minutes)
2. How is a facility judged? We will present how to assess the
remit of the core and how to develop metrics that can be
used to assess success and act as leverage to move the facility
forward. (15 minutes)
3. How can the core grow? A successful facility needs to bring
extra capacity or capability but how can new or alternative
technologies be imported. What are the implications for the
facility and its staff? (15 minutes)
4. New approaches to education. New hardware brings new
challenges for education of facility staff. We will show how new
approaches to training are required whether this is by internal
training, external training, assisted sessions or remote learning.
(30 minutes)
5. Collaborations between core facilities and other labs at national
and international level to bring about standardisation. (15
minutes)
6. Summary and conclusions. (5 minutes)
15
CellOrganizer: Building Models of Cell Structure
from Microscope Images and Using Them for HighContent Screening and Cell Simulations
Robert Murphy1,2, Gregory Johnson1, Devin Sullivan1
1
Lane Center for Computational Biology, Carnegie Mellon
Univ, Pittsburgh, PA, United States, 2Department of Biological
Sciences, Biomedical Engineering, Machine Learning,
Carnegie Mellon University, Pittsburgh, PA, United States
Tutorial Objectives: CellOrganizer is an open source software
system that can learn models of the size, shape and spatial
distribution of cellular components directly from images. These
models are generative, which means that they can be used to
synthesize new images of cells that are statistically similar to the
ones they were trained on. Such images are useful for testing
image analysis algorithms, and can be used as the basis for
spatially-realistic cell simulations using systems such as Virtual
Cell and MCell. Perhaps most importantly, CellOrganizer models
represent a transportable means of representing the results of High
Content Screening (HCS) assays that is not dependent on a specific
instrument, assay or cell type. This tutorial will focus on how to use
CellOrganizer and how to interface it with other software.
The tutorial will begin with a brief overview of the conditional
structure of the models within CellOrganizer and the system
organization. The first part of the tutorial will focus on training
generative models. Students are strongly encouraged (but not
required) to bring a laptop. Attendees are also encouraged to
bring a fluorescent cellular image dataset of their own to use for
building a model, but datasets will be available at the tutorial for
attendees who do not have one. Ideally, images should be two or
three dimensional single cell images (i.e., already segmented) with
different fluorescence channels for a fluorescently labeled target
protein (ideally a protein showing a punctate or vescular pattern), a
cell membrane or cytosolic-labeled marker, and a DNA marker (but
these are not strict requirements). The second part will focus on
synthesizing cell images from the models and importing the images
or model parameters into other software systems. The last part will
focus on adding new capabilities to the open source system, such
as modules for building new types of components.
Students should leave this session with mastery of the principles
behind building probabilistic models from images and practical
experience with training and using them with CellOrganizer. They
ISAC 2013 Program and Abstracts
forces present in the acoustic standing wave field to enrich
bacteria in a continuous flow [7]. Further aspects on bacteria and
acoustophoresis will also be discussed.
Tutorial Details or Outline:
1. Overview of conditional structure within CellOrganizer
2. Explanation of system architecture
3. Training 2D and 3D models for
a. Nuclear shapes
b. Cell shapes
c. Vesicular or punctate proteins
4. Synthesizing in silico cells
5. Importing generated images into cell simulators
6. Using model parameters to analyze HCS experiments
7. Adding new functions
References
J. Dykes, A. Lenshof, I. Åstrand, T. Laurell, S. Scheding, PLOS One,
August 2011, 6, 8,
Brian Warner, Liping Yu, Marko Blom, Wilfred Buesink, Andreas
Lenshof, and Thomas Laurell, CYTO 2012.
Persson J., Augustsson P., Laurell T. and Ohlin M., FEBS J, 2008,
275, 5657-5666
B. Björn Hammarström, T. Laurell, and J. Nilsson, Lab Chip, 2012,
12, 4296-4304 DOI: 10.1039/C2LC40697G
Thomas Laurell
Measurement tech & Ind E E, Div. Nanobiotechnology, Lund
University, Lund, Sweden
18
The shape of cells is coupled to their environment by the actions of
signaling networks that dynamically regulate complex interactions
amongst components of the cytoskeleton, membrane, and cellsubstrate adhesions. But very little is understood as to how signal
transduction results in specific cellular forms. In my laboratory
we are trying to answer three broad questions: 1) How does
signaling network activity generate shapes that are important for
behaviors such as cell migration? 2) How do networks “sense”
cellular shapes? 3) When the systems that regulate cell shape fail,
or are re-engineered (e.g. cancer), what are the consequences? In
order to answer these questions we continue to assemble, using
high-throughput and high-content imaging, a compendium of
data describing the shape of millions of single cells from different
genetic backgrounds, across different growth conditions, and
following chemical perturbation or RNAi. We then implement novel
computational methods to analyze this “data cube”, and model
signaling networks that control cell shape.
Wednesday,
22 May
Poster
Session
Here I will discuss how we have gained new insights into
how signaling network activity underpins the morphological
heterogeneity of populations, and how the activity of key
transcription factors such as Nf-kappaB and YAP/TAZ activity
is influenced by this heterogeneity. Firstly, we have performed
genome-scale RNAi screens in Drosophila and found that wildtype Drosophila hemocytes and neuronal cells are remarkably
heterogeneous, and exist in a number of discrete stable states. We
show how signaling activity that drives transitions between stable
shapes, and that morphological heterogeneity is a structured,
tunable, and evolvable process. We also validate our findings made
in Drosophila using models of metastatic melanoma. Secondly,
through analysis of a chemical screen in breast cancer cells, we
quantify how Nf-kappaB and YAP/TAZ nuclear translocation is not
only dependent of the shape of individual cells, but also on the
shape of neighboring cells. We provide both systems-level and
mechanistic insights into how the shape of cells can modulate
transcriptional activity. These findings have consequences as to how
morphological heterogeneity can influence transcriptional programs
and ultimately disease progression.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Chris Bakal
Cancer Biology, Institute of Cancer Research, London, United
Kingdom
Tuesday,
21 May
A most challenging area for acoustophoresis is the processing
of samples with particles smaller than 1-2 micrometers since
the primary acoustic radiation force scales to the third power of
the radius. This also explains why there are only a few reports
on acoustophoresis for bacteria handling. Recent findings have
however opened up the possibility of using the secondary acoustic
Signaling Networks Regulating Cellular Growth and
Form
Monday,
20 May
Acoustophoresis intrinsically offers simple means for continuous
flow based sample preprocessing and clean-up prior to analysis.
Examples will be given where cell debris and dissolved labels are
removed prior to FACS redout using acoustophoresis [3]. On-line
sample preparation of dairy sample for somatic cell counting has
also been reported where interfering lipid particles are removed
prior to either Coulter Counter analysis of fluorescence readout
facilitating the endpoint analytical step [4]. Using affinity ligands
coupled to microbeads acoustophoresis further enables targeted
extraction of molecular and cellular species. Examples are given
where allergy antigen specific bacteriophages are extracted from a
phage display library [5] and molecular species are enrichment and
purifiedin an acoustophoretic sample pre-processing step before
mass spectrometry readout [6].
Sunday,
19 May
Translating Acoustophoretic Cell Handling to Clinical
Applications
FFA has also been developed to deplete thrombocytes in
acentrifugation-based peripheral blood progenitor cell (PBPC)
apheresis, intended for stem cell extraction inclinical therapy of
haematological disorders [2].
Saturday,
18 May
Carl Grenvall, Jacob Riis Folkenberg, Per Augustsson, Thomas
Laurell, Cytometry A, 2012, 81A, 12, 1076-1083
P. Augustsson, J. Persson, S. Ekström, M. Ohlin and T. Laurell;Lab
Chip, 2009, 9, 810–818
Free Flow Acoustophoresis, FFA, utilizes the fact that individual
cell types display cell different migration velocities in an acoustic
standing wave field, which enables continuous flow based
separation. More recently FFA has been developed to target clinical
use where purification of tumor cells (TC) from white blood cell
fractions have been accomplished [1]. A key question in this respect
is whether FFA, which is a labelfree separation, can provide TC
populations that are not detected (i.e. not expressing EpCAM) by
means of the current clinical standard method –Veridex.
Special
Lectures
Per Augustsson, Cecilia Magnusson, Maria Nordin, Hans Lilja,
Thomas Laurell, Anal Chem, 2012, 84, 7954-7962
17
Acoustophoresis in microfludic systems is gaining attention as
a viable technology platform for continuous flow based cell
manipulation, including cell separation, buffer exchange, valving,
concentrations, affinity extraction and cell interaction studies. We
have recently shown (submitted) that cells processed by means of
microchip acoustophoresis experience a low mechanical stress and
display no change in biological response when compared to control
samples. Thisnow opens the route to the development of a wide
range of clinical applications where several of the unitoperations in
standard cell assay protocols can be replaced by acoustophoretic
cell processing.
Congress
Overview
will be able to use them to compare results from different HCS
assays using the generative model parameters, and import synthetic
images into cell simulation systems.
95
Congress
Overview
19
Discovery of Small Molecules that Control Cell
Differentiation
Special
Lectures
Petr Bartunek
Institute of Molecular Genetics, Prague, Czech Republic
20
Saturday,
18 May
Expanding the Capabilities of Mass Cytometry
Scott Tanner, Alexander Loboda, Dmitry Bandura, Vladimir
Baranov, Olga Ornatsky
DVS Sciences Inc., Markham, ON, Canada
Sunday,
19 May
The Mass Cytometer is a specific, designed-for-purpose
implementation of an analytical atomic mass spectrometer. The
principal point of commonality is the Inductively Coupled Plasma
that provides atomization and ionization. This presentation will
focus on the unique adaptations that address the need for single cell
distinction, with the sensitivity and dynamic range that meet the
needs of biologic informatics.
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
The overall efficiency of a Mass Cytometer system in quantifying
antigens in real time single cell assays depends on several factors.
First, processes of aerosol generation, vaporization, atomization
and ionization of single cells define how quantitatively the singlecell induced ion cloud sampled through the plasma-vacuum
interface represents the composition of each single cell. Second,
ion transport through the interface, the ion optical path and the
time-of-flight analyzer define not only the absolute sensitivity
(effectively defining the minimum number of copies of antigen per
cell that can be detected given a specific cell-labeling efficiency),
but also how readily bright and dim labels can be distinguished
while retaining the specificity and high multiparameter capability
of the assay. Third, ion signal handling on the nano-second scale,
which includes ion detection, signal pre-amplification, digitization
and processing, defines the dynamic range of a single cell assay.
Integration of the ion signals over the mass-time windows,
combined with appropriate real-time Gaussian curve fitting of the
sequential spectra corresponding to microsecond scale transients,
provides additional gating information to resolve doublets and
debris from true single cell events.
Poster
Session
Using leukemia cell lines, we will demonstrate how the theoretical
fundamentals of these sequential processes can be translated into
hardware and software improvement of data fidelity.
21
Commercial
Tutorials &
Exhibits
Simultaneous Analysis of Multiple Fluorescent
Proteins and Fluorochromes by a Novel Spectral Flow
Cytometer
Oral Session
Abstracts
Michio Tomura1, Koji Futamura2, Nao Nitta2, Masaya Kakuta2,
Motohiro Furuki2
1
Center for Innovation in Immunoregulative Technology and
Therapeutics, Kyoto University Graduate School of Medicine,
Kyoto, Japan, 2Bio Science Business Department, Life Science
Business Division, Medical Business Unit, SONY Corporation,
Tokyo, Japan
Speaker/Author
Index
Poster Session
Abstracts
Introduction: We have already presented various biomedical
applications using spectral flow cytometry (FCM) at CYTO1-4. In
this presentation, we show the simultaneous analysis of multiple
fluorescent proteins (FPs) and fluorochromes with spectral unmixing
that improve signal resolution and reproducibility over traditional
filter based cytometers. FPs are a powerful reporter system in
biomedical research fields. The ability to detect multiple FPs and
fluorochromes simultaneously using FCM provides the opportunity
to differentiate among various cell populations, or to study gene
function and monitor protein-protein interactions in individual
96
cells. However, traditional flow cytometric analysis of multiple
FPs and fluorochromes has been difficult. We show several results
of simultaneous analysis including photoconvertible FPs such as
Kaede and KikGR by spectral FCM with a unique algorithm5.
Methods: We have further developed a novel spectral FCM as
previously reported3. Unlike traditional FCMs, our novel spectral
FCM uses a full spectrum 32 channel linear array PMT to detect the
entire fluorescence derived from all fluorescent probes over a range
from 500 to 800 nm. These spectra are analyzed to automatically
deconvolve the contribution of every fluorochrome, even in the
presence of significant spectral overlap, enabling more flexible
experimental design. One of the advantages of spectral FCM is
the recognition of the unique spectral emission profiles of each
fluorochrome, and is not limited to merely the peak wavelength. To
further exploit this strength, we analyzed various cells labeled with
adjacent FPs and fluorochromes such as GFP/FITC, KikGR-Green/
FITC, GFP/Venus (YFP variant), PE/KikGR-Red, and Fucci/KikGR,
where the emission peaks have almost identical wavelengths, but
the fluorochrome spectra differ. We have successfully demonstrated
eleven color analysis including KikGR-Green and -Red
simultaneously with bright and very useful fluorochromes such as
PE, PE-Cy5, and APC of which the combination is indistinguishable
on a traditional cytometer. We also analyzed and compared spectral
changes of KikGR during photoconversion by both spectral FCM
and confocal microscope.
Results: Using spectral FCM, we were able to demonstrate
successful deconvolution and identification of respective FP signals.
We also analyzed KikGR during photoconversion and detected
the increase of KikGR red signal together with the concomitant
decrease of KikGR-Green spectra in a time dependent manner.
There were good correlations between spectral changes by
spectral FCM and color images obtained by confocal microscopy.
Examining EGFP and EYFP labeled cells, the spectral FCM was able
to clearly distinguish EGFP+, Venus+ and EGFP+Venus+ populations.
These data show that spectral FCM has a reliable deconvolution
of multiple, spectrally overlapping fluorochromes enabling the
simultaneous and reliable detection of multiple FPs despite varying
spectral overlap or dramatic differences in relative intensities.
Conclusion: The improved spectral FCM provides the most reliable
and accurate analysis of multiple FPs and fluorochromes. Our
presentation will include updates on our spectral flow system and
application results at the conference in May 2013.
References:
Nobukazu Watanabe, CYTO2011
2
William Hyun, CYTO2011
3
Koji Futamura, CYTO2011
4
Nao Nitta, CYTO2012
5
Masashi Sekino, CYTO2012
1
22
Quantitative Real Time Single Cell Spectroscopy in
Flow
John Nolan1, Danilo Condello, Erika Duggan2
1
La Jolla Bioengineering Institute, La Jolla, CA, United States,
2
La Jolla Bioengineering Institute, San Diego, CA, United States
Introduction: The long standing interest in measuring the complete
emission spectra from individual cells in flow cytometry has
recently become a practical reality. Advances in optics, detectors
and electronics have enabled several approaches for single cell
spectroscopy in flow, opening the door to new instrument concepts,
labeling strategies, and biological applications. Key to continued
progress in this area is a systematic evaluation of calibration,
standardization, and data analysis approaches. We have developed
high resolution spectral flow cytometers capable of high speed
fluorescence and Raman scattering measurements. Here we
report on the sensitivity and dynamic range of these spectral flow
ISAC 2013 Program and Abstracts
Conclusions: This new measurement system preserves the efficiency
of traditional time-domain systems as well as throughput of
standard cytometry. It does not require frequency modulation that
traditional phase sensitive instruments require. In the future we plan
to evaluate the instrument for multiple-exponential fluorescence
decays, for exploring the fluorescence lifetime of various locations
across a cell, and for implementing new biological applications.
24
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Oral Session
Abstracts
Conclusion: We report remarkably durable Ag85A-specific CD4 T
cell responses up to 6 years after MVA85A vaccination. We propose
Commercial
Tutorials &
Exhibits
Methods: A flow cytometer was developed based on a traditional
design but incorporating a rapidly scanning cw laser beam
Poster
Session
Results: We successfully completed long-term follow-up in ~75%
of participants previously vaccinated with MVA85A. The maximum
follow-up time, in adults, was 6 years after MVA85A vaccination,
while the minimum follow-up time was 3 years, in infants.
Frequencies of Ag85A-specific CD4 T cells were highly durable
in all ages: response magnitudes significantly exceeded the prevaccination responses. Magnitudes of Ag85A-specific CD4 T cells
in MVA85A recipients were also significantly higher than those of
placebo recipients.These CD4 T cells almost exclusively expressed
Th1 cytokines and were predominantly polyfunctional (coexpressed IFN-g, TNF-a and IL2).These antigen-specific Th1 cells
displayed CCR7-CD45RA- effector memory or CCR7+CD45RAcentral memoryphenotypes, in roughly equal proportions.
Wednesday,
22 May
Background: Mycobacterium tuberculosis (Mtb) is one of the most
pervasive diseases today. The aim of prophylactic vaccination is to
induce long-lived immunity that provides a rapid recall response
upon pathogen encounter. Heterologous prime-boost regimens
may constitute the most promising vaccination strategy against
tuberculosis (TB). MVA85A is a new TB vaccine, which is designed
to boost immunity primed by the current TB vaccine, BCG.
However, whether such prime-boost strategies induce long-lived
antigen-specific memory responses is not known. The aim of this
study was to determine if specific T cell responses persist up to 6
years after vaccination with candidate heterologous boost vaccine,
MVA85A.
Background: Fluorescence decay measurements using a timedomain approach is a powerful and sensitive technology when
combined with epifluorescence microscopy. Time domain
measurement systems observe the fluorescence decay of molecules,
metabolites, or other fluorescent species inside of cells. When
images are acquired by fluorescence lifetime imaging microscopy
(FLIM) or similar relatives, highly resolved multi-pixel data can be
obtained. Lifetime values help indicate concentration independent
subcellular phenomena. Although FLIM systems retain high signal
to noise, they are still limited in efficiency and throughput. In this
contribution we present a new type of flow cytometer designed to
rapidly capture fluorescence decay profiles based on a time-domain
approach.
1
Tuesday,
21 May
Erica Smit1, Michele Tameris1, Elisabeth Jane Hughes1, Ashley
Veldsman1, Linda van der Merwe1, Hennie Geldenhuys1,
Mark Hatherill1, Helen McShane2, Willem Hanekom1, Hassan
Mahomed1, Thomas Scriba1
1
University of Cape Town, Cape Town, South Africa, 2University
of Oxford, Oxford, United Kingdom
Wenyan Li1, Giacomo Vacca2, Mark Naivar3, Jessica Houston4
New Mexico State University, Las Cruces, NM, United States,
2
Kinetic River Corp., San Jose, CA, United States, 3DarklingX,
LLC, Los Alamos, NM, United States, 4Chemical Engineering,
New Mexico State University, Las Cruces, NM, United States
Fluorescence Lifetime-Dependent Flow Cytometry in
the Time-Domain
Monday,
20 May
Remarkable Durability of Ag85a-Specific CD4 T
Cell Memory Responses up to 6 Years after Mva85a
Vaccination
Methods: We recalled adults, adolescents, children and infants,
who previously received a single intradermal MVA85A vaccination.
Peripheral blood mononuclear cells (PBMCs), were isolated and
frequencies of Ag85A-specific T cells measured by IFN-g ELISpot
assay. We characterized the T cell response in more detail with a
whole blood intracellular cytokine staining (ICS) assay. Intracellular
expression of IFN-g, TNF-a, IL-2 and IL-17 by CD4 and CD8 T
cells, as well as memory phenotypes of these cells (using CCR7 and
CD45RA), were measured by multiparameter flow cytometry, using
a nine-color antibody panel.
23
Sunday,
19 May
Conclusions: Spectral flow cytometry can offer the sensitivity and
dynamic range of conventional flow cytometry, with the added
benefits conferred by measurement of complete spectra from
individual cells. These benefits include: a straightforward approach
to report results quantitatively, improved resolution of overlapping
spectra, and the possibility, with high resolution systems, to
implement new types of labels such as nanoparticle SERS tags.
Supported by NIH EB003824.
Results: In our initial evaluation we measured fluorophores with
different lifetime values ranging approximately from 3 ns to 25
ns. Lifetime decay profiles from microspheres and cells showed
differently sloping fluorescence decays, in comparison to scatteronly pulses.
Saturday,
18 May
Results: Calibration of the spectral flow cytometers with
fluorescence intensity standards enabled us to characterize
detection efficiency (Q, photoelectrons per MESF). The sensitivity
and dynamic range of the spectral flow cytometers were at least as
good as our benchtop conventional flow cytometer. We measured
the binding capacity of antibody capture beads, and used these
calibrated capture beads to acquire reference spectra from
fluorescence- or SERS-labeled antibodies. These reference spectra
confer information about the spectra of individual antibodies,
as well as the brightness, enabling the spectral analysis to report
either the brightness of a cell (in units of photons or MESF) or the
abundance of a label in/on a cell (in units of antibodies per cell).
A comparison of real-time spectral unmixing with post-acquisition
spectral analysis off line gave equivalent results in terms of per cell
tag abundances, and paves the way for the development of spectral
cell sorters.
excitation source. The laser was focused to a tight spot size to yield
short interaction pulses when scanned over cells or microspheres
between 2 and 10 microns in diameter. The fluorescence decay was
deconvolved from the raw fluorescence and scattering cytometric
waveforms, which were collected using a high-speed data
acquisition system.
Special
Lectures
Methods: Our spectral flow cytometers employ imaging
spectrographs with holographic gratings to disperse the light
across high speed CCD detectors for high resolution spectral
measurements, and mirrors, filters, and PMTs/PDs for conventional
measurement of discrete spectral bands. The data acquisition system
provides full control of all detectors, as well as real time display and
spectral analysis of individual cells. Beads and cells were stained
with fluorescent antibodies and/or antibodies labeled with surface
enhanced Raman scattering (SERS) nanoparticle tags. The spectral
data were analyzed either by integrating the intensity across specific
spectral ranges, creating the equivalent of virtual bandpass filters,
or by classical least squares (CLS)-based spectral unmixing of the
spectra using the known spectra of labels, autofluorescence and
background signals as reference components. We used calibrated
intensity standards as well as reagent-capture beads to calibrate the
brightness and target abundance on unknown samples.
Congress
Overview
cytometers, and illustrate how spectral overlap and quantification of
both cell brightness and target abundance can be addressed using
spectral unmixing.
97
Congress
Overview
that suchlong-lived immunity represents a highly desirable attribute
of the T cell response induced by heterologous prime-boost
regimens against TB.
25
Special
Lectures
Functional Characterization of Lymphoid Subsets in
Chronic Myelogenous Leukemia by Mass Cytometry
Phospho-Flow Analysis
Saturday,
18 May
Jitakshi De1, Rosemary Fernandez 1, Neil Shah2, Holden
Maecker1
1
Human Immune Monitoring Center, Stanford University,
Stanford, CA, United States, 2UCSF Helen Diller Family
Comprehensive Cancer Center, San Francisco, CA, United
States
Monday,
20 May
Sunday,
19 May
Background: Mass cytometry (MC) is a novel approach to precisely
decipher cell-specific activity of cytokine-mediated signaling in
disease states, allowing simultaneous assessment of different celltypes in a complex biologic milieu. Despite enormous success of
tyrosine kinase inhibition (TKI) in chronic myelogenous leukemia
(CML) therapy, there is a 20-30% rate of relapse. TKI-refractory
CML stem/progenitor cells survive independent of BCR-ABL kinase
activity. Identification of residual CML cells and characterization
of mechanisms underlying therapy resistance and relapse are
necessary for cure. Here, we applied MC phospho-flow to study
potentiated oncogenic pathways in chronic phase and relapsed
CML.
Wednesday,
22 May
Tuesday,
21 May
Methods: Phospho-flow analysis was performed on fresh blood
samples from two patients with CML (one treatment-naive in
chronic phase, and the other with prior TKI therapy and a rising
BCR-ABL1 transcript) referred to UCSF Comprehensive Cancer
Center, in parallel with a healthy donor sample. Aliquots of each
were treated with IL3, IL6, or no stimulus, for 10 min at 37C, lysed
and fixed, and reacted with a series of rare earth metal isotopetagged antibodies including those towards lineage-determining,
activation, and maturation antigens; phosphorylated epitopes within
key regulatory proteins of JAK/STAT and MAPK pathways; and
IKB kinase. ICP-MS data acquired on CyTOFTM mass cytometer at
Stanford HIMC were analyzed on high-dimensional data analysis
algorithms.
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Results:
1. In chronic phase CML, subsets of CD19+ lymphoid cells had
differential STAT5 activation profile: IgD+/CD27-/CD33- naive
B cells had no baseline p-STAT5, but had marked (~29.2X) IL3induction; whereas IgD+/CD27+/CD33+/IL3R+ cells had high
baseline p-STAT5 and diminished (~2.5X) IL3-induction. IgD-/
CD33-/IL3R-/CD45dim progenitors had low baseline, and no
inducible STAT5 activity.
2. Compared to cellular counterparts from the healthy donor,
baseline p-STAT3 in CML cells was much lower with substantial
IL3 and IL6 induction. While the naive T cell compartment
was largely preserved in chronic phase, it was diminished in
relapsed CML.
3. In the relapsed case, rare IL7R+ subpopulations (CD19br/IgD-/
CD45dim progenitors and CD27+/CD45RA+ naive Th cells,
both comprising <1% of total cells) with median baseline
p-STAT5, p-38 MAPK, and IKB values markedly above that of
chronic phase CML cells were detected. In these rare IL7R+
cells, p-ERK1/2 inversely correlated with p-p38 MAPK, p-STAT5,
and IL7R expression.
Speaker/Author
Index
Conclusions: High-dimensional MC analysis offers novel insights
into cell-specific signaling deregulation in chronic phase and
relapsed CML. The data suggest differential cytokine responsiveness
correlates with IL-receptor expression and stages of maturation
in CML cells. In CD19+/IL3R+ cells, high baseline p-STAT5 is
consistent with constitutive activity, likely dependent on ABL
98
kinase activation and in vivo cytokine stimulation. Post-TKI therapy,
increasing STAT5 and p38 MAPK activity in IL7R+ lymphoid cells
(likely arising from residual CML stem/progenitor cells programmed
to cycle) could represent BCR-ABL activation and predict relapse.
Further studies are necessary to recognize cell-specific STAT5
signaling patterns associated with ABL kinase activity and disease
outcome. Compounds that alter the growth factor responsiveness of
TKI-refractory progenitors may be of clinical relevance.
26
Maternal BMI Affects Expression Pattern of Cord
Blood T- and NK-Cell-Subtypes
Attila Tárnok1, Jozsef Bocsi2, Christopher Blatt3, Anikó Szabó2,
Susanne Melzer2, Ingo Dähnert3
1
Department of Pediatric Cardiology, Leipzig, Germany, 2LIFE
Leipziger Forschungszentrum - Zivilisationserkrankungen,
Leipzig, Germany, 3Department of Pediatric Cardiology,
Universitat Leipzig, Leipzig, Germany
Acknowledgement: This publication is supported by LIFE - Leipzig
Research Center for Civilization Diseases, Universitat Leipzig.
LIFE is funded by means of the European Union, by the European
Regional Development Fund (ERDF) and by means of the Free
State of Saxony within the framework of the excellence initiative.
This publication is also supported by MaDaKos - a BMBF funded
project: with the project number 990101-088.
Background: Natural killer cells (NK-s) play an important role
in immune response and immune modulation, in autoimmune
diseases and tumour cell killing. Earlier studies demonstrated
immunological changes of NK function in adult obesity. In
newborns it was demonstrated that the composition of leukocyte
populations is affected by different biological and environmental
factors. Such parameters altering the lymphocytes are for example
the length of pregnancy, length of birth, method of delivery
(caesarean), nutrition of the mother or contamination with different
chemicals.
Aim: The aim of the study was to identify altered NK and NK-T
cell parameters in relation with maternal BMI (Body Mass Index).
Here we investigated the expression of NK and NK-T cell specific
receptors and antigens as FC gammaRIII (CD16), NCAM (CD56),
CD94, NKG2C (CD159c), NKG2D (CD314), CD3, CD4, CD8
by 10 colour flow cytometry (Gallios, Beckman-Coulter LTD,
Germany). FCS files were analysed by FlowJo (7.6.4. PC-version)
software.
Methods: 52 mothers were recruited in Heinrich-Braun-Klinikum
in Zwickau Saxony Germany and diveded into a high BMI Group
(I; n=17; BMI(28.1-37.8) median=32.1) and a low BMI Group (II
; n=35; BMI(18.0-25) median=22.1). EDTA anticoagulated cord
blood samples were collected and analysed within 24h postnatal.
T-and NK cells blood samples were characterized by two 10 colour
protocols.
Results and Conclusion: More than 2,000 parameters were
obtained from each analysis. Helper, cytotoxic and regulatory
T-subtypes and NK cell subpopulations expressing various
activation markers showed broad absolute cell count variations and
multiple antigen expression patterns. Both BMI group values were
overlapping. Cytomic data pattern classification by discriminant
analysis and cluster analysis is needed to reveal parameter
combinations that allow to discriminate between both groups and
to identify important immune modulation. Further investigation of
these parameters might help to understand the effects on T- and NK
cells of obesity in pregnancy.
ISAC 2013 Program and Abstracts
Methods: FlowDensity estimates thresholds for individual cell
subsets from the density distribution of each marker. It then chooses
the best threshold based on several automated and pre-defined
parameters, including the number of significant peaks identified,
the change in slope of the density distribution (especially useful for
detection of rare cell populations), percentile thresholds and the
number of standard deviations from the maximum peak. For rare
cell subsets, which are notoriously difficult to identify automatically,
flowDensity can overcome this problem using expert-provided
information. Once thresholds are set for a particular panel, they are
adjusted to each FCS file in an automated, data-dependent fashion.
This eliminates significant manual effort that would otherwise be
necessary to obtain similarly robust results.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Background: Standardization of immunological assays,
including flow cytometry, in terms of reagents, sample handling,
instrument setup, and data analysis, is an important barrier to
the successful cross-study and cross-center data analysis that is
required in order to characterize the biological variability in the
healthy human immune system1,2. This is one of the goals of the
“Human Immunology Project” and FOCIS (Federation of Clinical
Immunology Societies), who have developed five standardized
staining panels and reagents (termed Lyoplates), which aim to
standardized many of these experimental variables.
Poster Session
Abstracts
Background: Although various automated clustering algorithms
have recently been developed to identify cell populations in flow
cytometry data, they often fail to replicate a human expert’s gating
results, especially for rare cell populations. To address this problem,
we developed flowDensity, an automated gating algorithm that
emulates an expert’s sequential 2D gating strategy to automatically
Greg Finak1, John Ramey1, Jafar Taghiyar2, Rick Stanton3,
Aaron Brandes4, Peng Qui5, J Philip McCoy6, David Hafler7,
Holden Maecker8, Tim Mossman9, Richard Scheuermann3,
Ryan Brinkman2, Raphael Gottardo10
1
Fred Hutchinson Cancer Research Center, Seattle, WA, United
States, 2British Columbia Cancer Agency, 3J Craig Ventner
Institute, San Diego, CA, United States, 4Broad Institute,
Cambridge, MA, United States, 5University of Texas MD
Anderson Cancer Center, Houston, TX, United States, 6National
Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, MD, United States, 7Yale School of Medicine, New
Haven, CT, United States, 8Stanford University, 9University of
Rochester Medical Center, Rochester, NY, United States, 10Fred
Hutchinson Cancer Research Center, Seattle, WA, Canada
Oral Session
Abstracts
Jafar Taghiyar1, Radina Droumeva2, Mehrnoush Malekesmaeili2,
Greg Finak3, Raphael Gottardo4, Ryan Brinkman5
1
British Columbia Cancer Agency, Vancouver, BC, Canada,
2
British Columbia Cancer Agency, Vancoucer, BC, Canada,
3
Fred Hutchinson Cancer Research Center, Seattle, WA, United
States, 4Fred Hutchinson Cancer Research Center, Seattle, WA,
Canada, 5British Columbia Cancer Agency
FlowCAP: Comparison of Automated and Manual
Gating of Standardized Lyoplate Flow Cytometry
Data
Commercial
Tutorials &
Exhibits
Reproducing Manual Gating of Flow Cytometry Data
by Automating Cell Population Identification
29
Poster
Session
28
Wednesday,
22 May
This study provides evidence that the primary immune response
to the yellow fever vaccine virus is compromised in elderly, which
is caused by several age-related impairments at multiple layers of
immunity.
Conclusions: Automated flow cytometry methods developed to
date have focused on fully automated analysis and are especially
suited for discovery applications. However, such approaches do
not take expert knowledge into account, and as a result seldom
match manual results where this is desirable (e.g., for diagnosis). By
robustly emulating 2D sequential gating, flowDensity eliminates the
need for laborious manual analysis while successfully replicating
manual results. flowDensity will be available via BioConductor
under an open-source license.
Tuesday,
21 May
We observed a significantly delayed YFV-17D viremia peak in
the elderly, suggesting an impaired level of infection control and
viral clearance. Furthermore we detected a deferred onset of YFV17D-specific IgM and lower numbers of CD11c+ myeloid and
plasmacytoid DCs during the early phase of the response (day 4)
in the elderly group. Moreover higher numbers of acutely induced
plasmablasts at day 14 and a significantly weaker virus-specific
CD8+ T-cell response were characteristic for aged vaccinees.
Correlation analyses revealed that the magnitude of the virusspecific CD8+ and CD4+ T-cell responses is determined by the
size of the naïve CD8+ and CD4+ T-cell compartments prior to
vaccination, which is reduced in the elderly. Furthermore our data
indicated that low levels of plasmacytoid and CD11c+ myeloid DCs
in elderly translate into diminished subsequent T-cell responses.
Monday,
20 May
Immunosenescence describes age-associated deterioration of the
immune system and provides a key element in understanding the
increased incidence of infections, malignancies and autoimmunity
at advanced age. To assess age-related changes in the context of
an acute primary response to a viral challenge, we vaccinated 24
young (20-30 years) and elderly (55-70 years) individuals with the
live-attenuated yellow fever 17D (YFV-17D) vaccine and analyzed
their humoral and cellular response for the following month in
close intervals. Several multidimensional FACS panels and a
complex serology were applied at each of the ten study days to
comprehensively monitor the antiviral immune response.
Results: We applied flowDensity to two datasets to determine its
ability to gate rare cell populations. Firstly, we applied flowDensity
to the Flow Cytometry Critical Assessment of Population
Identification (FlowCAP) III challenge four to identify 26 target cell
populations in lyoplate data, for which it was the co-top performing
algorithm. Secondly, we used flowDensity to gate 13-colour flow
cytometry data from 66 different mice from the International Mouse
Phenotyping Consortium (IMPC), and compared it to the two
top algorithms from FlowCAP I, flowMeans and SamSPECTRAL.
FlowDensity correctly identified rare cell subsets overlapping larger
cell populations to within the variability of three human experts,
which the other algorithms failed to do.
Sunday,
19 May
Regina Stark1, Ronald Axel Schulz1, Julia Nora Maelzer1,
Cristina Domingo-Carrasco 2 , Tomas Jelinek 3 , Karsten
Juerchott4,5, Nina Babel6, Avidan U. Neumann5, Matthias
Niedrig2, Andreas Thiel1
1
Regenerative Immunology and Aging, Berlin-Brandenburg
Center for Regenerative Therapies, Charite, Berlin, Germany,
2
Center for Biological Security 1, Robert Koch-Institut, Berlin,
Germany, 3Berlin Centre for Travel and Tropical Medicine,
Berlin, Germany, 4Institute for Theoretical Biology, Humboldt
University of Berlin, Berlin, Germany, 5Systems Biology of
Infectious Disease, Charite, Berlin, Germany, 6Department
of Nephrology and Intensive Care, Charite, Berlin, Germany
Saturday,
18 May
Compromised Innate and Adaptive Immune
Responses during Yellow Fever Vaccination in
Elderly — A Multiparametric Longitudinal FACS
Study
Special
Lectures
provide results that match those of manual analysis.
Congress
Overview
27
Methods: In collaboration with the FlowCAP committee,
standardized samples (Cytotrol control cells) were distributed
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Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
to nine participating centers and analyzed by flow cytometry
using lyophilized reagents and SOP’s to minimize experimental
variability. Data from two of these panels (T-cell and B-cell) were
entered into the FlowCAP challenge, where challenge participants
analyzed the data using automated gating methods. The resulting
cell population statistics were compared against a consensus
manual gating scheme that defined standard major classes of T and
B-cells. To limit the subjective influence of inter-individual effects,
the data were gated centrally (i.e. by one individual) and the
automated gating results evaluated based on within and betweencenter variability, as well as bias relative to the manual gating
“ground truth”.
Results: The evaluation of the FlowCAP results showed that,
for both the T-cell and B-cell panels, several automated gating
algorithms could successfully recapitulate centralized manual
gating statistics for T-cell and all B-cell subpopulations with little
to no statistically significant bias, and within-center and betweencenter variability as low or lower than the centralized manual
gating approach.
Conclusions: These results show that automated gating algorithms
are ready for general use and can be applied today to standardized
flow cytometry assays. These approaches will be useful to future
endeavors to perform reproducible analyses and comparisons of
immunological data.
1. Maecker, H. T., McCoy, J. P. & Nussenblatt, R. Standardizing
immunophenotyping for the Human Immunology Project.
Nature reviews. Immunology12, 191–200 (2012).
2. Maecker, H. T. et al. Standardization of cytokine flow cytometry
assays. BMC Immunology6, 13 (2005).
30
A Computational Method for Creating and Using
Pre-defined Gating Sequence Templates to
Automatically Gate High-Dimensional Flow Data
Stephen Meehan1, Connor Meehan2, Wayne A. Moore3, David
R. Parks4, Leonore A. Herzenberg5
1
Genetics, Stanford University, Burnaby, BC, Canada,
2
Mathematics, University of Toronto, Toronto, ON, Canada,
3
Genetics, Stanford University, Stanford, CA, United States,
4
Stanford Univ, Stanford, CA, United States, 5Stanford
University
Background: Technological advances have enabled flow cytometry
experiments of ever increasing size and complexity. These
severely tax the investigator’s ability to analyze the resulting data.
Conventional gating analysis is widely criticized as subjective and
is not well reproduced between laboratories. Much attention has
been paid to developing more objective gating criteria and more
automated methods to accelerate the workflow, ranging from simple
gate repositioning tools to highly sophisticated parametric models.
However, except in the simplest cases, these have not achieved
widespread acceptance.
Methods: Density Based Merging (DBM) is a nonparametric
clustering algorithm that has proven useful in flow cytometry
where rigorous parametric models are unavailable. Unsupervised
clustering methods take a very pessimistic view of the data,
assuming that there is no a no a priori knowledge or expectation
of structures within the data. However, this is rarely true in real
experiments. DBM has been implemented to incorporate prior
knowledge in the form of a gating hierarchy that represents a known
strategy to reduce the data based on prior work.
Earth Movers Distance (EMD) is one of a class of metrics between
populations that arises in certain optimization problems. It takes its
colloquial name from the fact that if one treats two populations as
piles of earth (or cells), EMD represents the minimum work (mass
times distance moved) needed to transform one into the other,
i.e., the work/cost of moving one cell population to the position
100
occupied by another on the same or a comparable set of axes. The
population comparison with the lowest cost identifies the most
similar pair. We use EMD here to match/locate cell populations in
paired analyses of samples stained with comparable reagents.
Results: We have developed software (ClusterGenie) that allows a
knowledgable researcher to build a gating sequence template using
a representative set of selected training samples. For each step in
the gating hierarchy, the template contains the required biomarkers,
cluster identifying parameters and cluster aligning parameters.
Parameters entail information about the template clusters and
whether they need further analysis: e.g. bin sizes & thresholds,
etc.. At each stage, ClusterGenie analyzes samples for template
conformance by: using DBM to find clusters; using EMD to align
clusters with those in the training set in order to distinguish between
new, missing and recognizing clusters (in progress); reducing the
dataset to those clusters the template identifies for further analysis.
Conclusions: Conventional cluster analysis is essentially a single
sample operation. However, aligning the results from many
samples, commonly referred to as meta-clustering, is an important
part of many flow cytometry projects. Our method differs from
previous efforts in that we use fully nonparametric methods and
incorporate prior knowledge in the form of a hierarchical decision
tree embodied in the analysis template. Preliminary results indicate
that the use of DBM for cluster finding and EMD for cluster
matching will yield better results than prior approaches.
31
Simplicial Analysis in Flow Cytometry Data
Processing. Enabling Svm and Hdp Cell
Classification via Standardization of Cell-Signal
Simplices
Bartek Rajwa1, Ferit Akova2, Alex Pothen3, M. Murat Dundar2
Bindley Bioscience Center, Purdue University, West Lafayette,
IN, United States, 2Computer & Information Science, IUPUI,
Indianapolis, IN, United States, 3Computer Science, Purdue
University, West Lafayette, IN, United States
1
Compositional (or simplicial) data analysis deals with data
vectors in which the values are non-negative and sum to unity.
Apart from probabilities, such data also arise when the nonnegative measurements are scaled by the total of the components.
Geometrically, compositional data (CoDa) with N components are
represented in a sample space of the regular unit N-simplex (hence
the name simplicial analysis). Since this space is radically different
from the real Euclidean space associated with unconstrained data,
the traditional multivariate statistical approaches inevitably fail.
An appropriate methodology for simplicial analysis began
to emerge in the 1980’s with contributions from J. Aitchison,
who introduced transformations allowing for application of
standard multivariate tools such as principal component analysis.
Subsequently, the work has been expanded by J. J. Egozcue. Today
compositional analysis tools are used in a wide range of fields from
geology via econometrics and financial mathematics to biology and
clinical studies.
This presentation will demonstrate the use of simplicial methods in
a processing and automated (i.e., machine learning–driven) analysis
pipeline for flow cytometry data. The reported results reveal that
apart from the obvious application of these techniques in analysis of
composition of cell populations, simplicial procedures can also be
utilized as a crucial preprocessing step for intra- and interlaboratory
data standardization, normalization, and quality assurance. In
the latter role the proposed methodology takes advantage of the
fact that staining of single cells can be represented using CoDa
nomenclature, in which the signal associated with a particular
marker is part of the total fluorescence signal produced by a stained
cell. Treating the FC data vectors as N-tuples and separating the
total signal in subsequent analysis allows for innovative utilization
of the CoDa toolkit.
ISAC 2013 Program and Abstracts
Oral Session
Abstracts
Conclusions: The relevance of this method to study the role of NOX
enzymes in various pathologies, e.g. Multiple Sclerosis as well as
brain tumors or physiologic circumstances, e.g. in the intestine, is
demonstrated.
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Results: We probed in this way the modification in NAD(P)H
signals in different organs in health and disease. Thereby, we did not
focus on NAD(P)H metabolic modifications, i.e. the ratio between
free NAD(P)H and enzyme-bound NAD(P)H, which participates to
intracellular biochemical reactions, as mostly known from cancer
research. We exploit a unique property of enzyme-bound NAD(P)
H, i.e. its fluorescence lifetime depends on the enzyme to which
it is bound to. Thus, a specific fluorescence lifetime may function
as a finger-print of a certain biochemical reaction within the live
cell, catalyzed by a specific enzyme. In our case, we identify
the activation of the members of the NOX family (fluorescence
lifetime approx. 3650 ps, Niesner et al, J. Biophys., 2008), with
its prominent member NOX2 - the phagocytic NADPH oxidase,
against the pool of mithocondrial enzymes (fluorescence lifetime
between 1200 ps and 2500 ps).
Commercial
Tutorials &
Exhibits
Further, the sensitivity of the camera plus the high, data acquisition
rates afforded by two phases per image make it possible for us to
film dynamic lifetime events at a current rate of 10 frames/second.
Finally, we intend to present these results as well as results from the
latest version of the MEM-FLIM camera (v3) that is currently being
tested, which has 512 x 512 pixels with a square pixel size of 24
μm and an 80 MHz modulation clock.
Methods: We employed parallelized time-correlated single-photon
counting (TCSPC) to perform intravital marker-free FLIM.
Poster
Session
We have made lifetime measurements using the MEM-FLIM camera
for various samples, e.g. fluorescein solutions, fixed GFP-actin
stained HeLa cells, and live GFP-actin stained U2OS cells. As an
example, for the HeLa cells, the phase-based method for lifetime
estimation yields lifetimes of 2.59±0.40 ns for the MEM-FLIM
system and 2.66±0.49 ns for the reference system.
Wednesday,
22 May
Our Modulated Electron-Multiplied Fluorescence Lifetime Imaging
Microscope (MEM-FLIM) camera is modulated at 25 MHz and has
212 x 212 pixels with a square pixel size of 17 μm. In our current
MEM-FLIM camera system (v2), the spatial resolution, as measured
by the optical transfer function (OTF), is significantly better than
that of a conventional (i.e. reference) micro-channel plate (MCP)
based FLIM system. At both 500 cycles/mm and 1000 cycles/mm
the OTF is 2x better than that achieved with the reference system.
The (relative) sensitivity (ΔADU / Δintensity) of the MEM-FLIM
camera is 3x better than that of the reference camera when each
system is at its lowest gain setting. The dark current of the reference
system is 3.5x lower than that of the MEM-FLIM camera but, at the
short integration times used (≈100 ms), this is not a problem.
Tuesday,
21 May
Frequency-domain FLIM systems currently require an image
intensifier for low light levels applications, MHz modulation
frequencies, and very short exposure times. We have designed,
developed and tested a new generation of FLIM instrumentation
that uses an advanced design for 1) a modulated LED excitation
signal and 2) an application-specific CCD sensor that replaces
the image intensifier. The CCD device can be modulated at the
pixel level thereby permitting homodyne demodulation of the
fluorescence emission signal. All incoming light is captured by the
modulated pixels thereby recording two, widefield phase images at
once.
Background: In the last decades, intravital multi-photon laserscanning microscopy (MPLSM) proved to be the most versatile
tool to study cell migration and cell-cell interactions in health
and disease. Its power to enable investigations in genuine
environment - the living organism - in a dynamic way, with high
resolution constitutes its hallmark among high-performance
technologies used in cell biology and biomedicine. However, the
MPLSM technology is currently limited mostly to observations
of morphological changes within tissue over time, without
information about underlying molecular mechanisms. In order to
quantify intracellular molecular parameters and phenomena in
live organisms, complicated genetically encoded constructs must
be generated and expressed in corresponding transgenic mice,
e.g. calcium-binding proteins (such as CerTN L15), potassium
voltage indicators, apoptose indicators. This is a time-consuming
and artifact-prone undertaking. On the other hand, well-known
endogenous molecules can and have been used as probes of vital
phenomena in live cells. A prominent example are the ubiquitous
coenzymes nicotinamid adenosine dinucleotide (NADH) and
nicotinamid adenosin dinucleotide photsphate (NADPH), hereafter
NAD(P)H, the fluorescence of which have been used for decades
to quantify the metabolic state of cells. Especially, the power of
NAD(P)H fluorescence lifetime imaging (FLIM) has been used e.g.
in the context of cancer research. However, the current MPLSM
and FLIM technologies did not allow until now to use endogenous
chromophores as probes in vivo due to their low fluorescence
signals.
Monday,
20 May
Ian Young1, Qiaole Zhao2, Ben Schelen2, Raymond Schouten2,
Jan Bosiers3, Rene Leenen3, Inge Peters3, Kees Jalink4, Marcel
Raspe4, Sander de Jong5, Bert van Geest5
1
Bionanoscience, Delft University of Technology, Delft,
Netherlands, 2Imaging Science & Technology, Delft University
of Technology, Delft, Netherlands, 3 Teledyne DALSA,
Eindhoven, Netherlands, 4NKI - Dutch Cancer Institute,
Amsterdam, Netherlands, 5Lambert Instruments, Roden,
Netherlands
Raluca Niesner1, Agata Mossakowski2, Julian Pohlan2, Moritz
Radbruch3, Jannike Bayat-Sarmadi4, Friedemann Paul5, Anja
Erika Hauser2,4, Helena Radbruch2
1
Biophysical Analytics, Deutsches Rheuma Forschungszentrum,
Berlin, Berlin, Germany, 2Charité - University Hospital,
Berlin, Berlin, Germany, 3DRFZ, Berlin, Berlin, Germany,
4
Immundynamics, DRFZ, Berlin, Berlin, Germany, 5NCRC,
Charité - University Hospital, Berlin, Berlin, Germany
Sunday,
19 May
Next-Generation FLIM: Modulated All Solid-State
Camera System
Intravital Marker-Free Nad(P)H Fluorescence
Lifetime Imaging – in Vivo Selective Enzyme
Detection
Saturday,
18 May
32
33
Special
Lectures
The demonstrated examples will include applications of simplicial
processing for automated gating of longitudinal FC data (using
support vector machines - SVM), and use of simplicial techniques
for automated classification of rare event in data sets obtained
from multiple laboratories (utilizing SVM and hierarchical Dirichlet
processes - HDP).
Congress
Overview
The results presented show that owing to the properties of scale
invariance (vectors with proportional components represent the
same composition) and subcompositional coherence (analysis of
a subset of signals does not depend on the remaining signals), the
CoDa-type transformations may produce FC data representation
that allows for removal of many effects of experimental variability.
The standardization of FC simplices can pre-process and realign
data suffering from variability in staining efficiency, changes and
inconsistencies in detector gain settings, or improper compensation.
101
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
34
Ultra High Throughput Image Cytometry via
Continuous Scanning
Jeffrey Price1,2, Greg Gemmen1, Behrad Azimi1, Mirco Guigli2,
Jeffrey Price1,2
1
Vala Sciences Inc., San Diego, CA, United States, 2SanfordBurnham Medical Research Institute, La Jolla, CA, United States
High content screening (HCS) is a powerful tool used
predominately in academic research for large-scale cellular biology,
for cDNA and RNAi screens, and in pharmaceutical companies
for early drug discovery using large compound libraries. HCS
can be described as a multi-tiered approach to acquire and
analyze large volumes of biologically relevant data captured from
automated microscopes in various imaging modes (e.g., confocal,
epifluorescent, reflective, and transmitted). HCS combines modern
cell biology, automated microscopy, and robotic handling to
produce spatially and temporally resolved datasets that contain
important phenotypic and morphological parameters in the cellular
systems under investigation. However, large-scale screening of
tens of thousands to millions of compounds and potential clinical
diagnostic applications (e.g., rare cancer cell detection in blood)
is largely limited by image acquisition speed. Current imaging
instruments typically scan at peak speeds of up to 100,000 wells
per day, in contrast with μHTS plate readers capable of hundreds of
thousands of wells per day. This limitation is far more prohibitive in
the screening of assays where a small population of screened cells
is expected to be responsive. In these screens, a large number of
images would need to be collected for each condition in order to
obtain statistically relevant data. The time required to collect such
large amounts of data slows the screening down to impractical
limits which often disqualifies the assay from screening of large
libraries of compounds.
The dependency of the screening speed on the amount of data
collected is largely due to the fact that the acquisition is done
field-by-field, where the automated microscope moves to an area
of the sample, stops, collects images then moves to the next area
and repeats. Here, we propose a continues scanning method
where images are acquired simultaneously with the movement of
the sample at a given speed. By eliminating the need to stop and
start movement, scan times significantly improve. Furthermore,
since images are collected at all times during the movement,
the overhead of screening large number of cells per condition is
minimized.
Here we report significant progress in three color, epi-fluorescent,
continuous scanning HCS method that utilizes time delay
integration (TDI) imaging and reflective position focus control in
parallel, making screening rates of >250,000 full wells/day (4.3min
per 384- or 8.5min per 1536-well plate) a reality. Performing
autofocus in parallel with continuous motion imaging allows for a
much larger percentage of the overall scanning time to be devoted
to light collection, thereby removing many of the inefficiencies
inherent in currently available instruments and making image
brightness and signal-to-noise a function of light source and assay
brightness.
35
Automated Intracellular Fret Measurements using
Hyperspectral Microscopy and Feature Extraction
Silas Leavesley1,2, Andrea Britain3, Thomas Rich2,3
Chemical and Biomolecular Engineering, University of
South Alabama, Mobile, AL, United States, 2Center for Lung
Biology, University of South Alabama, Mobile, AL, United
States, 3Pharmacology, University of South Alabama, Mobile,
AL, United States
1
Background: Förster resonance energy transfer (FRET) is a
fluorescence microscopy tool that has been invaluable in
102
understanding spatially-dependent phenomena in living cells.
Many techniques have been implemented to quantify the level (or
efficiency) of energy transfer. These range from simple estimates
based upon the intensity of one or two detection bands to complex
experimental approaches and equipment configurations, such
as fluorescence lifetime or acceptor photobleaching. These
techniques have advantages and disadvantages, which have been
widely documented and debated. For example, fluorescence
lifetime calculations can be made insensitive to changes in total
fluorophore concentration, which is advantageous when samples
have varying fluorophore concentrations. However, identification
of many fluorescent signals, in addition to the FRET pair, is very
difficult to perform using lifetime and photobleaching techniques.
Hyperspectral microscopy techniques may also be used to measure
FRET efficiency.In theory, hyperspectral microscopy and spectral
flow cytometryapproachesallow measurement of FRET efficiencies
in experiments with many fluorescent labels and high tissue
autofluorescence.
Methods: In this work, we have combined hyperspectral
microscopy with automated image analysis and feature extraction
to measure subcellular FRET efficiencies in time-lapse confocal
microscopy studies. HEK-293 and pulmonary aortic endothelial
cells were grown on 25 mm round coverslips and transfected with
either a cytosolic or plasma membrane-localized CFP-EPAC-YFP
probe. EPAC changes conformation upon binding cAMP; the CFPEPAC-YFP probe displays approximately 45% FRET efficiency at
basal cAMP levels and 37% FRET efficiency at saturating cAMP.
Two days post-transfection, cells were labeled with Hoechst 33342
and imaged on a Nikon A1R spectral confocal microscope (405 nm
excitation, 432-606 nm emission in 6 nm increments).
Results: Non-negatively constrained linear unmixing was used
to calculate the abundance of Hoechst, CFP, and YFP. Image
processing and feature extraction using Cell Profiler (The Broad
Institute) software was then performed. Time-lapse single-cell FRET
efficiencies (whole cell and cytosolic) were then calculated. Initial
tests with forskolin and rolipram displayed a characteristic increase
in cytosolic cAMP with an average standard error-of-the-mean of
0.9% (over the time-course, n=8 trials).
Conclusions: These results indicate that hyperspectral imaging
or flow cytometryapproaches can be used to measure FRET
efficiencies in the presence of additional fluorescent labels – and
potentially autofluorescence – with high accuracy and precision.
In image cytometry experiments, additional labels would yield
valuable information for feature extraction, such as nucleus
identification, as shown here. Future work will focus on localizing
FRET signals to specific subcellular domains and performing studies
in whole-vessel pulmonary vascular preparations.
36
Amplifying DNA Beads Assay in Flow Cytometry: A
Platform Technology Based on Exonuclease III-Aided
Target Recycling
Jie Lu, Dayong Jin
Macquarie University, Sydney, Australia
Introduction: Exonuclease III(Exo III) has been recently
discovered to “recycle” target molecules, thus resulting in the
PCR-like sensitivity1,2. Here we reportapplication of Exo III
amplification technique to flow cytometry, yielding a highlysensitive, reproducible, separation-free, and high-throughput DNA
quantification platform.
Method: The schematic diagram was shown in Fig. 1. TheExo III
can catalyse the stepwise removal of mononucleotides in a 3’ to 5’
direction from probe’s 3’ blunt end, removing fluorescence reporter
and ultimately releasing the target. The released target can then
recycle to hybridize withsecondarysurface DNAprobes in sequence,
thereby improving sensitivity. Owing to the high-density coverage
of carboxyl groups on polystyrene beads, different ratios of Cy5
ISAC 2013 Program and Abstracts
Methods: We are developing an innovative approach
(GlycoSenseTM) capable of real-time glycoprofiling based on
combining multiplex suspension array technology with glycanspecific reagents. In this method, flow cytometry is used to detect
binding between glycans and glycan-specific reagents that are
conjugated to spectrally-unique microspheres. By combining the
individual reagents into a multiplex suspension array, the entire
analysis can be obtained in less than a minute on a basic cytometer.
Saturday,
18 May
Conclusion: This work shows the great potential to apply Exo
III recycling technique to flow cytometry, leading to both high
sensitivity and analysis speed.
lead times also make corrective action during commercial cell
cultivation impossible, with the only recourse to specification
failures being lot rejection, at a cost of $5-$50M. The availability
of robust, rapid, and simple analytical methods for glycoprofiling
during biologics production would fundamentally alter
biopharmaceutical R&D and commercial manufacturing.
Special
Lectures
Results and Discussions: The beads carrying high-density probe
DNA achieved the lowest limit of detection (3.2 pM), which was
10 times lower than medium-density ones (37.1 pM). Moreover,
the formerresulted in a sigmoid working curve with larger dynamic
concentration range (38 pM ~ 625 nM) compared to the later (from
610 pM∼39 nM). These results showExoIIIaided recycling technique
can significantly improve the sensitivity by a factor of56.8 to
68.4 compared to conventional nucleic acids bead assay (direct
hybridization).3
Congress
Overview
modified DNA probes were conjugated onto 15 µm beads via EDCactivated conjugation. The surface probe DNA loading after target
DNA recycling were quantified by flow cytometry method (Cytek
DxP6; 25 mW 639 nm lasers).
Sunday,
19 May
Results: The GlycoSenseTM method is not intended to replace
full-scale characterization of biologics but makes in process
glycoprotein sampling and monitoring possible. In preliminary
experiments, this method was used to analyze the terminal
glycosylation patterns of standard glycans and glycoproteins. The
results were comparable to that of other glycan analysis techniques.
Monday,
20 May
Conclusions: The GlycoSenseTM approach is a rapid, simple
glycoprofiling method that requires no specialized equipment or
training. It complements the current methods of glycan characterization
while addressing the unmet need for real-time glycoprofiling tools.
38
Multiplexed Microsphere Protease Assays and High
Throughput Flow Cytometry for Drug Discovery
Figure 1. The Schematics of Exo III technique and its aided flow
cytometry DNA beads assay
Development of a Glycoprofiling Method Using
Multiplex Microspheres
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Results: We have demonstrated multiplex microsphere based
protease assays and high throughput flow cytometry based
screening that discovered several interesting inhibitors to the
proteases. The average Z’ value for each substrate was greater than
0.7 in our screenings and hundreds of compounds were found with
nominally active (hits). We will present confirmatory assays and
dose response curves for the identified compounds. We will also
present the key kinetic parameters (Km, kcat and kcat/Km) of a protease
as measured in microsphere-based and solution phase assays.
Additionally, we have performed detailed inhibition mechanim
studies of these compounds by full-length and peptide FRET assays
to determine whether these compounds work with the distal sites or
cleavage sites, further to determine whether they are competitive,
noncompetitive and uncompetitive inhibitors.
Poster Session
Abstracts
Background: Glycans (complex carbohydrates) covalently
attached to the surface of a protein play a significant role in
the bioactivity of molecules such as therapeutic antibodies or
erythropoietin. Consequently, improper glycosylation has a direct
impact on the safety and efficacy of glycoproteins. The US Food
and Drug Administration (FDA) requires that the glycoprofiles
of all therapeutic glycoproteins (biologics) fall within certain
specifications. However, the heterogeneity of glycans and their
structural isomerism make their characterization a time consuming
task. Currently, glycoprofiling requires several weeks for completion
by highly trained personnel using specialized instrumentation
and is therefore not suitable for in process monitoring. These long
Oral Session
Abstracts
Loretta Yang1, Christine Leoff2,3, Robert Woods2,3
Glycosensors and Diagnostics, San Diego, CA, United States,
2
CCRC, University of Georgia, Athens, GA, United States,
3
Glycosensors and Diagnostics, Athens, GA, United States
1
Methods: Here we have developed a multiplex substrate set
containing four substrates (SNAP-25 and VAMP-2 fusion proteins,
a fusion protein bearing LF cleavage site, and a negative control
substrate) for Botulinum neurotoxin type A & F light chains and
Bacillus anthracis lethal factor respectively. The HTS platform uses
streptavidin coated fluorescent suspension microsphere arrays
where each microsphere population bears a unique fluorescent
protease substrate. To measure protease activity, we add three
proteases simultaneously, where each cleave their substrate to
cause loss of fluorescence on specific microsphere populations.
The HTS has been implemented in 1536-well plates containing the
>350,000 compounds of the library.
Commercial
Tutorials &
Exhibits
37
Poster
Session
References:
(1) Zuo, X.; Xia, F.; Xiao, Y.; Plaxco, K. W. J Am Chem Soc2010,
132, 1816.
(2) Luo, M.; Xiang, X.; Xiang, D.; Yang, S.; Ji, X.; He, Z. Chem
Commun (Camb)2012, 48, 7416.
(3) Spiro, A.; Lowe, M.; Brown, D. Appl Environ Microbiol2000,
66, 4258.
Background: Due to their toxicity, it is of great importance to
identify potential inhibitors to Lethal Toxin of B. anthracis and the
Botulinumneurotoxins. We have developed a simple and efficient
protease assay that enables the use of full-length protease substrates
and multiplexed assays, provides high-throughput screening (HTS)
for drug discovery. HTS by flow cytometry is an efficient method
to discriminate from a large number of compounds and identify
the potent protease inhibitors, not only because it requires small
sample volumes, it is high throughput and robust, but it is also
sensitive, reproducible, and accurate.
Wednesday,
22 May
Tuesday,
21 May
Jingshu Zhu1, Larry Sklar2, Bruce Edwards2, Steven W. Graves
1
University of New Mexico, Albuquerque, NM, United States,
2
Univ of New Mexico
103
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
Conclusions: Our multiplexed microsphere protease assay
combined with high throughput flow cytometry will be applicable
to a broad range of proteases due to its ability to rapidly discover
inhibitors from large chemical libraries. Moreover, we have
demonstrated that it can also provide critical kinetic parameters that
are helpful in the study of the inhibition mechanisms. Finally, the
use of full length substrate in our study is a marked improvement
compared to peptide-based assays, because full-length substrates
more accurately mimic the complex interactions of many proteases
with their natural substrates. These interactions often include
binding at distal sites and potential conformational changes of both
the substrate and the protease.
Additionally, there are indications that the improved bead recovery
leads to higher assay sensitivity for detections of low affinity targets,
which will have enormous benefits to the field of diagnostics. The
acoustic trapping protocol enables automated assay preparation,
generic to anybead-based flow cytometry assays.
References:
[1] M.B. Meza, DrugDisc.Tod., 5 (2000) 38-41
[2] M. Evander, J. Nilsson, LabChip 12 (2012) 4667-4676
[3] B. Hammarström, M. Evander, H. Barbeau, M. Bruzelius, J.
Larsson, T. Laurell, J. Nilsson, Lab Chip10 (2010) 2251-2257
39
Significantly Improve Bead Recovery of Flow
Cytometry Assay by Utilising a New Technology
Platform for Sample Incubation
Maria Tenje 1 , Neil LeBlanc 2 , Mikael Evander 1 , Björn
Hammarström1, Hongyan Xia3, Axel Tojo1, Sándor Belák2,3,
Thomas Laurell1,4
1
Department of Measurement Technology and Industrial
Electrical Engineering, Lund University, Lund, Sweden,
2
Department of Virology, Immunology and Parasitology,
National Veterinary Institute, Uppsala, Sweden, 3Department
of Biomedical Sciences and Veterinary Public Health, Swedish
University of Agricultural Sciences, Uppsala, Sweden,
4
Department of Biomedical Engineering, Dongguk University,
Seoul, Korea, Republic of (South)
We present a novel technology platform that increasesthe
performance ofbead-based flow cytometry assays. Acoustic trapping
in a microfluidicformat was used to perform the micro bead
processing steps prior to Luminex(Luminex Corp.) analysis. The
proposed method significantly improved bead recovery, a critical
parameter for assay optimisation. The microfluidic format also
significantly reduced the assay time
Bead based assayshas become a standard within diagnostics [1].
However, with the automated wash station protocol for magnetic
beads (Tecan Hydroflex) a significant bead loss is noted during
the assay steps. This increases assay cost since increased amount
ofmicro beads are requested to ensure assay reliability. We have
therefore developed a microscaled non-contactacoustic trapping
method where the beads can be retained in a flow and sequentially
be incubated with the sample andassay reagents, resulting in a
significantly reduced bead loss.
Acoustic trapping is a technique to position and retain beads in
a non-contact mode at distinct places in a microfluidic platform
[2]. The beads are drawn into a rectangular glass capillary (crosssection: 2mmx200 µm)that is docked to an800 µm wide piezo
electric transducer (PZT) (Fig. 1(a)) that extends across the capillary
[3].The reaction site is specified by the acoustic pressure field
gradient locally generated by the PZT.The PZT drives a l/2 acoustic
standing wave with a pressure minimum in the centre of the
channel above the transducerwhere the beads will be trapped, (Fig
1(b)). Sample and reagents were incubated with the beads in a slow
flow past the bead cluster.For the first time, we now demonstrate
how acoustic trapping can be utilised as a reaction site for beadbased flow cytometry assays.
The performance of the acoustic trapping assay was compared to
conventional protocols using micro titre plates and an automated
wash station, runningthe 6-plex Flock Monitor assay (BioVet) on
vaccinated poultry sample. The beads were analysed in a Luminex
flow cytometer. Bothprotocols displayed the same assay results
as compared to thecontrol assay. The bead recovery for the Tecan
wash station protocol was found to be~30% and the acoustic trap
showed a bead recovery of > 70%, (Fig. 2).Most importantly, the
acoustic trapping platform also enabled a reduction in the assay
time from 2.5 hrs to only 45 min with maintained assay sensitivity.
104
40
Navigating the Labyrinth of Regulated Flow
Cytometry in Drug Development
Virginia Litwin1, Jennifer J. Stewart2, Jennifer Olsen1, Joel
Puchalski1, Cherie Green3, Christopher Wiwi4
1
Covance, Inc., Chantilly, VA, United States, 2Flow Contract
Site Laboratory, Kirkland, WA, United States, 3Amgen, Inc.,
Washington, DC, United States, 4Celgene Corp., Summit, NJ,
United States
As a potential new drug entity progresses from the drug discovery
phase to commercial launch, supporting analytical data are
generated in laboratories adhering to different regulations such as
Good Laboratory Practices (GLP), Clinical Laboratory Improvement
Act (CLIA) regulations, or Good Manufacturing Practices (GMP).
Designing, validating, and implementing flow cytometry based
assays in regulated environments presents unique challenges not
encountered with other analytical technologies commonly used
in drug development (JIM, 363:104-119, 2011. JIM, 363:120-134,
2011).
This workshop will begin with brief presentations describing the
various regulated environments encountered throughout the life
cycle of a new drug entity. A presentation on GLP will be followed
by a discussion of CLIA regulations. This presentation will focus on
how the intended use of the data influences the validation process-exploratory biomarkers and patient stratification markers will be
ISAC 2013 Program and Abstracts
Procedures (SOPs) and 3) procedures for safe operation and
validation of aerosol containment systems. The impact of this
Policy has been widespread, with adoption by many laboratories
and Institutions outside of the NIH. The ISAC biosafety committee
is drafting a revision of the 2007 ISAC Biosafety Standard to
incorporate much of the NIH Policy into the International Standard.
This workshop will outline the NIH Policy and also present sample
questions/scenarios of real-life situations encountered by cell sorter
operators and guidelines to determine the appropriate biosafety
procedures and practices.
Quantitative Cytometry - Calibration and
Standardization
John Nolan1, Bartek Rajwa2, Rachel Errington3, Gyorgy Vereb4,
Stephen J. Lockett5, Anil Parwani6, Gustavo K. Rohde7
1
La Jolla Bioengineering Institute, La Jolla, CA, United
States, 2Purdue University, 3Cardiff University, 4University
of Debrecen, 5Optical Microscopy and Analysis Laboratory,
SAIC, Frederick National Laboratory, Frederick, MD, United
States, 6University of Pittsburgh, Pittsburgh, PA, United States,
7
Carnegie Mellon Univ, Pittsburgh, PA, United States
Lili Wang1, Robert Hoffman2
1
NIST, Gaithersburg, MD, United States, 2BD Biosciences, San
Jose, CA, United States
45
Withdrawn.
46
Use of Kinetic Imaging Cytometry to Develop
Pharmacological Approaches to Cardiac
Regeneration and Preservation of Contractile
Function
Mark Mercola
Bioengineering, Sanford-Burnham Med Res Inst. and Univ.
Calif., San Diego, La jolla, CA, United States
Heart failure has relatively few treatment options and remains a
major cause of death in the developed world. Consequently, there
has been tremendous interest in developing novel therapeutic
approaches that regenerate myocardium and/or preserve function.
We have developed human pluripotent stem cell-based assays
for cardiomyocyte regeneration and preservation of physiological
function. A new instrument, the Kinetic Imaging Cytometer (KIC)
developed by Vala Sciences (San Diego), served a critical function
our screening by providing high throughput, cell-by-cell analysis
of calcium transients. Calcium handling is central to the contractile
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Early prediction of mitochondrial perturbation (MP) and impairment
of hepatocellular bile acid transport (HBAT) by novel drug
candidates is becoming a critical feature in drug development.
In this workshop we will describe approaches for screening and
diagnosing drug candidates for their potential to cause MP and
HBAT utilizing various fluorescence based platforms. We discuss
the strength and limitations of various screens and provide
recommendations of where to position these assays during the drug
commercialization process.
Oral Session
Abstracts
Biosafety standards specific for flow cytometry and in particular,
cell sorting are important for ensuring the safe operation of these
instruments. Recently, the National Institutes of Health (USA)
has adopted an NIH Biosafety Policy for Cell Sorters for the NIH
Intramural laboratories. This Policy provides direction in the
following key areas: 1) design of cell sorting laboratories. 2) the
creation of laboratory or instrument specific Standard Operating
George Babcock1, Padma Narayanan2
1
University of Cincinnati, Cincinnati, OH, United States,
2
Amgen, Seattle, WA, United States
Commercial
Tutorials &
Exhibits
Since the first ISAC guidelines published in 1997, the ISAC has
provided the research community with valuable guidance to the
biosafety of cell sorting. In 2007, these guidelines were updated
and upgraded to standards to best reflect the importance of these
procedures and policies in laboratories around the world.
Functional Analysis of Mitochondria and Transporters
Poster
Session
Kevin Holmes1, Stephen P. Perfetto2
Flow Cytometry Section, NIAID, NIH, Bethesda, MD, United
States, 2Vaccine Research Center NIH, Bethesda, MD, United
States
1
44
Wednesday,
22 May
Biosafety: Biosafety Policy Meets Real Life Scenarios
Tuesday,
21 May
42
Long term and cross laboratory collaborative studies require
appropriate calibration and standardization protocols. This
workshop will concentrate on standards and reference materials for
both instrument calibration and assay calibration/standardization.
The workshop will summarize current state of standards and
reference materials, as well the gaps to be filled and needs
remaining for cytometry standardization. The workshop will have
a series of presentations followed by discussion, with the aim of
defining goals for a collaborative study with which assay calibration
and standardization can be demonstrated through the use of
standards, reference controls, and a validated assay procedure.
Monday,
20 May
At the workshop we will present and describe the goals of the ISAC/
CYTO University program. We will specifically detail aspects for
the current plans for image-based cytometry online education. Thus
the workshop includes an overview of current microscopic imaging
modalities and techniques, a review of current training programs
in microscopy (live and online), as well as a review of other online
resources, amongst other topics. The workshop will provide an
opportunity for all interested parties to participate in discussions to
shape the curriculum and methods for image cytometry education.
Sunday,
19 May
43
The Delivery of an Image-based Cytometry Education
Resource at the ISAC University
Saturday,
18 May
41
ISAC’s CYTO University is an exciting new endeavor where the
emphasis is to develop the GOTO online cytometry education
portal, supported and contributed by experts and superb educators
at the society. This workshop is open to everyone who wants to
contribute to this endeavor. The aim at ISAC/CYTO U is to develop
a comprehensive online education portfolio, one area of which
is focused on image cytometry where the goal is to provide a
learning environment to teach the fundamental concepts and tools
for undertaking quantitative cell/tissue based analysis using image
derived data. In short, our aim is to provide education on ‘how
to convert images into parameters’ that describe the underlying
biology. While there is a plethora of educational materials
available regarding microscopy with linked applications in cell
biology currently available online, it is clear that an integrated peer
reviewed, online resource centered around image-based cytometry
education is currently lacking.
Special
Lectures
Attendees at this workshop will learn how flow cytometry is used
in regulated environments and have the opportunity to share their
experiences and challenges in this arena. In addition, participants
will learn how adhering to the regulations can increase the quality
of their data.
Congress
Overview
used as examples. Finally, the application of GMP for cellular
therapeutics will be addressed. This presentation will include
the unique challenges of implementing flow cytometric methods
suitable for GMP lot release and introduce the concept of the
Quality by Design (QbD) approach to method characterization.
105
Congress
Overview
Special
Lectures
Saturday,
18 May
performance of cardiomyocytes, and KIC was used in our screens for
the automated acquisition of kinetic parameters of cardiomyocyte
function. Illustrative of molecules from our studies include the first
selective small molecule inhibitor of TGFbeta signaling. Mechanistic
studies revealed a direct link between TGFbeta signaling and control
of chromatin modifying machinery that is responsible for committing
multipotent progenitors to cardiomyocyte lineage. A second
example is a potential RNA therapeutic that preserves cardiac
function in preclinical mouse model through targeting a microRNA
that suppresses cardiomyocyte contractility.
47
Non-Genetic Cell Population Heterogeneity:
Implications for Cell Differentiation and Cancer
Progression
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Sui Huang
Institute for Systems Biology, Seattle, WA, United States
Background: Even a clonal (isogenic) population of cells exhibits
drastic cell-cell variability with respect to phenotype, including
transcriptome. This non-genetic heterogeneity has a characteristic
dynamics indicating that the apparently stochastic phenotype
fluctuations of individual cells is not simply due to “gene expression
noise” alone but instead, often involves unrecognized metastable
sub-types of cells. Considering such heterogeneity changes the
traditional paradigms of “molecular pathways” that is largely
based on analysis of population averages, opening new vistas of
cell behavior previously not appreciated. In particular, the new
individual cell resolution perspective has profound implications on
our understanding of cancer progression.
Methods: Using a combination of transcriptomics in FACS sorted
subpopulations, quantitative flow cytometry and single-cell
qPCR of clonal cultures of tumor cells undergoing defined state
transitions, combined with mathematical modeling, we dissect
the dynamics of cell population heterogeneity and show how the
“phenotypic plasticity” of cells with the same genome can affect
clinically relevant processes, such as development of resistance to
chemotherapy.
Results: We present a series of results obtained by the integration
of single-cell resolution measurements of gene expression and
mathematical modeling that offer an explanation for the fractional
and incomplete nature of induced state transitions (such as
differentiation). We also find that the rapid development of cancer
drug resistance – at least in the initial phase – is not due to selection
of preexisting genetic mutants but can be induced by the drug itself,
thus defying the orthodoxy of a Darwinian evolution that drives
cancer progression.
Conclusions: In this overview we present, using concrete examples
of experimental findings, the importance of single-cell resolution
measurement in the assessment of molecular changes during cell
phenotype switches. The often neglected aspects of non-genetic
heterogeneity coupled with high-dimensionality in cell fate changes
must be considered when analyzing the response of cancer cells to
therapeutics.
48
Live Cell Analysis of Ncam Polysialylation in Microcommunities Using the Novel Combination of an
Antibody-Mimetic EGFP-Endosialidase and the
Viability Dye DRAQ7
Paul Smith1, Marie Wiltshire1, Sally Chappell1, Laurence
Patterson 2, Steven Shnyder 2, Robert Falconer 2, Rachel
Errington1
1
School of Medicine, Cardiff University, Cardiff, United
Kingdom, 2Institute of Cancer Therapeutics, University of
Bradford, Bradford, United Kingdom
106
Background: Modification of neural cell adhesion molecule
(NCAM) by polysialic acid (polySia) is thought to impart antiadhesive/migratory properties to cells of neuroendocrine tumors
including small cell lung cancer (SCLC), offering a target to limit
spread (Falconer et al., Curr Cancer Drug Targets. 2012 12(8):925).
The impact of polySia-NCAM on SCLC cell proliferation is unclear
given the anchorage-independent growth of cells as clusters in
vitro and propensity to undergo transition to adherent forms. We
hypothesized that polySia-NCAM expression enhances in vitro/in
vivo proliferative capacity independent of cell adherence. To test
this we developed a model SCLC system tractable for 2D/3D cell
culture, functional adherence and live cell polySia-NCAM analysis.
Methods: Our novel approach to tracking polySia-NCAM
expression in live cells exploited a combination of EndoN-GFP (a
polySia antibody-mimetic eGFP-tagged endosialidase) and the nontoxic viability dye DRAQ7. Adherent variants were successively
enriched from NCI-H69 cell clusters and subjected to non-toxic
removal of polySia and switching from 2D (attached) to a 3D
(cluster) growth format on hydrophobic surfaces. Cells were profiled
for both selective adherence to matrix components using multiple
substrate arrays and polySia-NCAM heterogeneity using live and
fixed cell flow cytometry and imaging. Proliferation was assessed
by cell counting and Q-tracker® 705 flow cytometry. In vivo growth
was determined by volume expansion of subcutaneous NCI-H69
xenografts in Balb/c immunodeficient nude mice.
Results: Adherence to extracellular matrix substrates, with laminin
preference, was independent of polysialylation. PolySia-negativity
was not predictive for adherence. Selection for adherence resolved
polySia+ and polySia- sub-populations for NCI-H69, and high
polySia+ sub-populations for two other SCLC cell lines (SHP-77
& COR-L279). Highly polySia+ NCI-H69 cells showed enhanced
growth rate in 2D and 3D cultures and in vivo. Growth rate of
subpopulations correlated positively with polySia expression but
not adherence. Surprisingly, co-culture of polySia+ and polySiacells resulted in an overall increase in the relative frequency of
polySia+ cells, over several days, not attributable to cell death or
differential proliferation.
Conclusions: Live cell analysis of polysialylation shows modified
expression profiles in NCI-H69 mixed micro-communities,
revealing homeostatic influences not realised in isolated variant
subpopulations. We suggest a new model of enhanced polySia
expression upon SCLC cell detachment and spread with a
concomitant gain of proliferative advantage. We conclude that
polySia expression presents a dynamic target, independent
of adherence potential, for controlling spread and tumor cell
proliferation. [Acknowledgements: Grant support, EPSRC (UK) &
Yorkshire Cancer Research; support studies, Dr E Furon (Cardiff) &
P Cooper (Bradford); EndoN-GFP (Prof J Finne and Dr A Jokilammi,
Univ of Helsinki)].
49
Cytometric Assessment of Dna Damage- and mtorSignaling, the Factors Contributing to Aging and
Senescence
Zbigniew Darzynkiewicz, Hong Zhao, Dorota Halicka
New York Med Col, Valhalla, NY, United States
Background: Two conceptual models are being promoted to
explain the aging phenomenon. Cumulative DNA damage caused
by reactive oxygen species (ROS), by-products of oxidative
phosphorylation, is one of them (ROS concept). Constitutive
stimulation of the mitogen- and nutrient-sensing mTOR/S6 signaling
is the second mechanism (TOR concept).
Methods: The flow- and laser scanning- cytometric methods
were developed to measure the level of the constitutive DNA
damage/ROS- as well as of mTOR/S6- signaling in individual
cells. Specifically, persistent activation of ATM and expression
of γH2AX in untreated cells detected with phospho-specific Abs
ISAC 2013 Program and Abstracts
Methods: We created surrogate disease samples using an acute
myeloid leukemia (AML) cell line,
HL-60; labeled with CFSE. By spiking fluorescently labeled
tumor cells into healthy donor blood (HD), we could work in an
environment of certainty that leukemic cells were (1) present and
(2) at known concentrations. Initial development experiments
demonstrated limited stability (<6 hours)of pHH3 signal in blood
collected in EDTA when held at ambient temperature.However,
after assay optimization, we found thatpHH3 signal was stabilized
by collecting blood in EDTA plus a preservative (Cyto-Chex® BCT),
followed by red blood cell lysis and fixation. Additional stability
was gained when lysed/fixed cells were stored in 90% methanol
at -70°C. This is a key result, as it enables preservation of samples
at clinical sites, with subsequent transport and batch analysis in
a central laboratory.Qualification experiments were designed to
measure specificity of the pHH3 reagent, sample stability, and
discrimination of signal to noise for pHH3. Lower limit of detection
and intra- (triplicates), and inter-assay (repeat draws) precision was
also characterized.Blood from 9 HD, collected in Cyto-Chex® BCT
then spiked with untreated or nocodozole-treated HL60 cells at 3
different concentrations of pHH3+ cells (Low: ~50; Med: ~500;
Hi: ~5,000) was used in qualification experiments. Percent and #
of pHH3+ cells in >G2M were measured. A range of ±3 SD for %
pHH3+ was pre-defined during development experiments.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Background: Aurora Kinases play an essential role in cellular
division by controlling chromatid segregation. This family of
proteins is often over-expressed in many human tumors and is being
evaluated as anti-cancer targets with pan and selective-Aurora
Kinase inhibitors.One substrate for Aurora Kinase A/B is Histone
H3 at Serine10. Phosphorylation of Histone H3 (pHH3) has
been associated with chromosome condensation and segregation
during mitosis and meiosis, and therefore serves as a reasonable
marker of the G2M position in cell cycle. To better understand
the pharmacologic effects of Aurora Kinase inhibitors in vivo, we
developed a sensitive flow cytometric assay to measure cell cycle
and detect rarepHH3+ tumor cells in blood, using intracellular
staining with anti-pHH3 antibody with DAPI (DNA content).
Poster
Session
Jurkat cells were treated with 20µM caspase inhibitor(ZVAD.FMK)
for 1 hour. To induce apoptosis, the cells were treated with 2µg/
ml camptothecin, 0.5µg/ml anti-CD95 antibody (anti-CD95) or
25µg/ml cycloheximide. The cells were labelled with Annexin
V-Pacific Blue to detect phosphatidylserine (PS) exposure; 7-amino
actinomycin D to detect the loss of plasma membrane integrity;
cytochrome c-FITC to detect the release of cytochrome c from
the mitochondria; tetramethylrhodamine ethyl ester (TMRE) to
detect the loss of mitochondrial membrane potential; or LC3BAlexa Fluor 488 to detect autophagosome formation. Following
immunostaining, single cell data acquisition was performed using
the BD FACSCantoTM II.
Cherie Green1, Connie Ma2, John Ferbas2, Gloria Juan3
Clinical Immunology, Amgen, Ventura, CA, United States,
2
Clinical Immunology, Amgen, Thousand Oaks, CA, United
States, 3Molecular Sciences, Oncology, Amgen, Thousand
Oaks, CA, United States
1
Wednesday,
22 May
Apoptosis is a type of programmed cell death (PCD) that is crucial
for all multi-cellular organisms. The cellular milieu triggers the
initiation of this process, and once the cells have succumbed to the
death cues they are degraded and discarded. The fact that apoptosis
is a self-killing process is now well accepted. However, the
question that has intrigued many researchers is how a cell commits
to death and what factors instigate this decision-making process.
In theory, it is believed that cells can be rescued if the apoptotic
stimuli are removed and survival mechanisms are restored prior
to the point-of-no-return. Empirically, however, a single event that
defines the point-of-no-return in apoptosis remains to be exacted.
Many would even argue that considering the complex signalling
network of apoptosis, and its importance in cellular activity, it is
unlikely that a single event would be the control for this process.
Here, we investigate the effect of caspase inhibition on apoptosis of
a leukemic T cell line.
Development and Qualification of a Whole Blood
Assay to Monitor pHH3 and Cell Cyclein Patients
with Acute Myeloid Leukemia (AML) Treated with
Aurora Kinase Inhibitors
Tuesday,
21 May
Rubina Pal, Yaw Ohene-Abuakwa, Tim Rutherford, Frances
Gibson
St George's Univ of London, London, United Kingdom
51
Monday,
20 May
Dissecting the Intricate Network of the Cell Death
Machinery: Simultaneous Detection of Apoptosis and
Autophagy Using Flow Cytometry
Sunday,
19 May
50
Saturday,
18 May
Conclusions: Although the primary target of each on these agents
may be different the data are consistent with the downstream
mechanism in which the reduction of translation rate through
mTOR/S6K signaling is coupled with a decrease in the energy
production through oxidative phosphorylation and leads to a
decline in the level of ROS, mitochondrial potential and oxidative
DNA damage. The decreased rate of translation induced by
these agents may slow down cells hypertrophy and alleviate
other features of cell aging/senescence. The reduced oxidative
DNA damage is expected to lower predisposition to neoplastic
transformation which may result from defective DNA repair at
the sites coding for oncogenes or tumor suppressor genes. The
data suggest that combined assessment of constitutive γH2AX
expression, mitochondrial activity (ROS, ΔΨm) and mTOR
signaling provides an adequate gamut of cell responses to evaluate
effectiveness of suspected gero-preventive agents.
Caspase inhibition completely attenuated anti-CD95 induced
apoptosis. 4, 24 and 48 hours post-treatment with ZVAD.FMK
and anti-CD95, the cells revealed no PS exposure, release of
cytochrome c, or loss of mitochondrial membrane potential.
However, ZVAD.FMK merely delayed the process in camptothecin
or cycloheximide treated cells as the above events were detectable
after 24 or 48 hours. The initial inference of this observation is
that caspase activation is an upstream event in the death receptor
pathway, and hence blocking caspase activation restores cell
viability. However, further experimentation revealed that Jurkat cells
might be protected from apoptosis by the cell survival mechanism,
autophagy. Autophagy detection using LC3B-Alexa Fluor 488
revealed that the cells that remained viable, after treatment with
any one of the apoptosis inducers in the presence of ZVAD.
FMK, were LC3B positive. The effect of simultaneous inhibition of
caspase activation and autophagy is currently under investigation. A
discussion of the methods of immunostaining and these results will
be the focus of this presentation.
Special
Lectures
Results: The reported gero-preventive agents: rapamycin,
metformin, 2-deoxyglucose, berberine, resveratrol, vitamin D3
and aspirin, all decreased the level of constitutive DNA damage
signaling as seen by the reduced expression of γH2AX.They also
decreased the level of intracellular ROS and mitochondrial transmembrane potential ΔΨm, the marker of mitochondrial energizing.
All these agents also reduced phosphorylation level of mTOR,
RP-S6 and 4EBP1 in A549, TK6, WI-38 cells and in mitogenically
stimulated human lymphocytes. The most effective was rapamycin.
Congress
Overview
reports constitutive DNA damage induced by endogenous ROS.The
phosphorylation of Ser235/236-ribosomal protein (RP), of Ser2448mTOR and of Ser65-4EBP1 informs on constitutive signaling along
the mTOR/S6 pathway.
107
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Conclusion: We have developed a robust biomarker assay to
monitorpHH3 and cell cycle in blood. This assay provides a
valuable analytical tool to understand pharmacodynamic effects
of Aurora Kinase inhibitortreatment in patients with hematologic
malignancies.
52
Understanding Subcellular Filament Networks from
Fluorescence Microscopy Images
Gustavo Rohde1, Saurav Basu2, Jieyue Li2, Kris Dahl2, Robert
Murphy2
1
Carnegie Mellon Univ, Pittsburgh, PA, United States, 2Carnegie
Mellon University, Pittsburgh, PA, United States
Background: Network-like filament distributions play important
roles in eukaryotic cells. These include cell motility, wound healing,
cancer (including metastasis), intra cellular transport, and many
other phenomena.Microtubules (tubulin), for example, are often
the target of cancer treatment drugs (taxol) given their crucial role
in cell reproduction. Detailed study of subcellular distributions has
often ben hindered by the lack of appropriate imaging technologies.
High-resolution alternatives (e.g. electron microscopy) are capable
of extracting information pertaining to relatively small subcellular
regions. However, they are poor options for high content, high
throughput quantitative studies. Fluorescence microscopy (e.g.
confocal) are good alternatives for high throughput studies.
However, their resolution is often not high enough to obtain
detailed measurements pertaining to filament distributions.
Method: Here we describe a suite of techniques based on
computational analysis of fluorescence microscope images that
enables one to study in detail quantitative properties (e.g. number
of filaments, curvature profiles, length distributions, subcellular
distribution, etc.) of filament networks in 2D and 3D confocal
fluorescence images of live and fixed cells. The software takes
as input raw images, and based on linear filament modeling
techniques, output the desired quantities. More specifically, the
idea is to consider all possible line (tube)-like structures within
the geometry of the image being used, and from these determine
which is most likely to have been present given the measured
image data available. In instances when filaments are sparsely
distributed relative to the image resolution, direct estimation is
possible. In instances where image resolution is too limited for
direct estimation, indirect estimation of many important parameters
is still possible.
Results: We present results showing that parameter (number
of filaments, lengths, etc.) extraction pertaining to both actin
and microtubules is possible in both live and fixed cells. More
specifically, we use the system to understand effects of the presence
of carbon nanotubes in the actin distribution of cultured cells. We
also present results demonstrating the application of our method to
understanding differences in microtubule distributions of different
cell types from 2D fluorescence microscopy images. Finally, given a
set of many images where both vesicular proteins and microtubules
are visible for the same cell, we also show that our modeling
approach can also be used to study the relationship of arbitrary
vesicular proteins in terms of association to filament subcellular
filament networks.Amongst other applications, we utilize data from
the Human Protein Atlas to show that the framework can be used to
identify proteins that could be potential cancer biomarkers.
Speaker/Author
Index
Poster Session
Abstracts
Results: All acceptance criteria were met for this qualification.
Experiments with unconjugated anti-pHH3, isotype control, and
blocking peptide demonstrated specificity. Samples that were held
at -70°C in 90% methanol were stable for up to 4 months.Intra- and
inter-assay precision for samples with greater than 25 pHH3+ cells
was less than 25% CV across triplicates and repeat draws.
108
53
New Approaches to Study Neuronal Connectivity in
A High-Content Format
Natalie Prigozhina, Jordan Seldeen, Fabio Cerignoli, Ranor
Basa, Jeffrey Price, Patrick McDonough
Vala Sciences, Inc., San Diego, CA, United States
Proper neuronal function is critical for overall physiological
function, health, and cognitive abilities. Disorders involving
altered neurotransmission and neuro-circuitry are debilitating to
individuals, costly to society and include inherited mutations (e.g.,
fragile-X, Huntington’s disease, multiple sclerosis, and Parkinson’s
disease), psychological disorders (e.g., autism, schizophrenia,
bipolar disease) and dementia (e. g., Alzheimer’s and old age).
Furthermore, damage to the brain or spinal cord due to accidents
or strokes results in impaired neurotransmission and deterioration
of neural networks. Additionally, a phenomenon termed “chemobrain’ reported since the late 1980s, involves a decline of learning
and cognitive function, likely related to altered neurotransmission,
which occurs in patients receiving anti-cancer drugs. The potential
mechanisms that cause this disruption remain largely unknown, but
may involve the disruption of adult neurogenesis and damage to
existing normal mature neurons in the brain.
Unfortunately, there are few model systems for use in developing
therapeutic medications or treatment strategies for these afflictions.
They primarily rely upon performance of electrophysiological
measurements, done on an individual cell basis at low throughput
and at high expense. The goal of our research is to develop highcontent, high-throughput methodology for testing the effects of
compounds and genomic constructs on neuronal function synaptic
transmission, establishment of neuronal networks, neuronal survival
and the differentiation of neuronal precursor cells.
Here we would like to present our in vitro assay system to quantify
the effect of test compounds, both long term and acute) on
neuronal transmission. We are developing an in vitro neuronal
system, where the neurons spontaneously form functional synapses
and exhibit spontaneous or induced synchronized activity. We
then assay the activity of the neurons using fluorescent calcium
and/or voltage indicators in the presence or absence of known
pharmacological agents in order to identify positive and negative
controls. The 10 to 20 second time-lapse movies are recorded at
video rate using Kinetic Image Cytometer (KIC) and analyzed by
automated image analysis software CyteSeer (both developed by
Vala Sciences).
We successfully cultured, imaged and analyzed primary rat
hippocampal and human iPSC-derived neurons in a high
throughput format (384 well). Our preliminary results demonstrate
that neurons form interconnected neuronal networks and exhibit
occasional spontaneous synchronized flashes meaning they form
functional synapses with each other. We also showed that the
activity of the neurons in our system can be modulated (inhibited
or activated) by long or short term treatments with pharmacological
drugs. Finally, we have correlated the calcium based activity
assessment with expression and colocalization of pre- and
postsynaptic markers using immunolabeling technique.
54
Addressing Uncertainty in the Automated Evaluation
of Stem Cell Colony Quality
Michael Halter1, Ya-Shian Li-Baboud1, Adele Peskin1, Peter
Bajcsy1, Daniel Hoeppner2, Anne L. Plant3
1
NIST, Gaithersburg, MD, United States, 2Lieber Institute for
Brain Development, Baltimore, MD, United States, 3NIST
Laboratories that culture pluripotent stem cells must make
subjective decisions about which colonies to passage and which
ISAC 2013 Program and Abstracts
Sample pre-enrichment is a common process strategy in cell sorting
applicationswhich is used to reduce the complexity of the sample
and reduce overall sorting time.However, it can still be a timeconsuming procedureand may compromise biological function.1
Here we demonstrate an in-line sample pre-enrichment modular
tool on a modified BD Influx™ cell sorter combining magnetic cell
separation and acoustic cell concentration technologies2 to preprocess the sample immediately before injection into the flow cell
(Figure 1).This new pre-enrichment process can speed sorts 3–4
timesoverall and specifically eliminates the time that cells sit after
pre-enrichment which greatly reduces cell-cell aggregation and
subsequent additional losses due to coincidence and doublets.
Treg cells were directly sorted from a PBMC sample and from the
same sample which had been pre-enriched for CD4 T lymphocytes
using the BD IMagTM CD4 T Lymphocyte Enrichment Kit using the
in-line process. Without pre-enrichment, the rateat which the
sorted cells were collected was observed to be 244 cells/sec (total
over-threshold event rate of 4,000 events/sec) while with preenrichment the sort rate was increased to 940 cells/sec(total overthreshold event rate of 10,000 events/sec). This higher rate allowed
1.2x106 Treg cells to be sorted in about 24 minutes or about 3.5
times faster than the un-enriched sort procedure. Waste fluid from
the acoustic concentrator was subsequently analyzed and found to
contain about 7% of the total cells coming from the magnet. Typical
cell loss during the concentration step varies depending on sample
concentration and flow rate through the concentrator and ranges
typically from 1%–10%.
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
The magnetic depletion step removes sample components prone
to aggregation, such as monocytes, and improves the spatial
distribution of cells immediately prior to sorting. Aggregate
removal is shown in Figure 2 using a BD Influx™ Sort Analysis
Tool software application which displays the position of all cells
in the sample stream, calculates the proximity of the nearest
neighbors, andadditionally expresses a measure of dispersion
as the Entrainment Factor (EF = observed frequency / expected
frequency). When EF = 1, the sample distribution can be described
by normal Poisson distribution, >1 indicates aggregation and<1
indicates ordering. We have observed repeatedly that the preenrichment process reduces EF to <1, and we are able to measure
small increases in both electronic detection and sort efficiency
of 1%–5%. When EF>>1, loss of processed events occurs due to
more frequent electronic aborts and reduced sort efficiencies on the
processed events due to coincidence within drop packets.
Oral Session
Abstracts
Poster Session
Abstracts
We have demonstrated that the in-line enrichment modular tool on
the BD Influx™cell sorternot onlycuts down the time to sort large
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Brian Warner1, Liping Yu1, Joe Trotter1, Maria Jaimes1, Marko
Blom2, Wilfred Buesink2, Andreas Lenshof3, Thomas Laurell3
1
BD Biosciences, San Jose, CA, United States, 2Micronit
Microfluidics, Enschede, Netherlands, 3Lund University, Lund,
Sweden
Tuesday,
21 May
Determining the responses of a biological system to various
perturbagens is a key paradigm for understanding the basic
processes that underlie that system.Automated, high content/high
throughput microscopy methods offer an opportunity to efficiently
perform such experiments by surveying a large population of cells
at low cost. Here we describe a non-parametric, cross-validation
method to determine the pairwise similarity between populations
from different conditions while simultaneously considering the
relationship between experimental replicates as a baseline for
evaluation. Using images of Arabidopsisprotoplasts under various
perturbagen conditions, we segment regions of interestto form
a distribution of responses for each experimental replicate. We
next calculate features to describe the subcellular pattern in each
regionand construct a confidence interval describing the ability
of each feature to discriminate between the distributions of samecondition experimental replicates. Using different subsets of the
replicates and iteratively discarding the features with the largest
Accelerated Cell Sorting Using In-Line Sample PreEnrichment
Monday,
20 May
Gregory Johnson1, Joshua Kangas1, Alexander Dovzhenko2,
Karsten Voigt2, Santosh Bhavani1, Klaus Palme2, Robert
Murphy1,2
1
Carnegie Mellon University, Pittsburgh, PA, United States,
2
Albert-Ludwigs-Universität Freiburg, Freiburg, Germany
56
Sunday,
19 May
A Non-Parametric Method for the Automated
Discovery of Perturbagen Responses in High-Content
Analysis
Saturday,
18 May
55
same-condition discriminative ability, we evaluate the error of a
nearest neighbor classifier between different-condition distributions.
We determine there to be a difference in control versus condition
when the error between different experimental conditions is
statistically greater than the error between same-condition
replicates. Using the same method, we evaluate hierarchical
relationships between experiments to discover phenotypes present
in the data. We demonstrate the robustness of this method by
using images from automated widefield microscopy images to
identify an initial set of chemical compounds that affect subcellular
distributions of various proteins, and confirm the results with the
same method by confocal microscopy.
Special
Lectures
A quantitative description of stem cell colony quality was generated
by first using a reference set of colony images that were assigned
a quality score by expert biologists. In this case, we found that
colony scoring by different expert biologists did not always
provide a consistent score. Using the inconsistent scoring data, we
determined the uncertainty in the colony scoring decision. Using
our set of image analysis features and we examined approaches to
training a classifier using the reference set of scored colony images.
Interestingly, some image analysis features were more correlated
with each expert biologist, suggesting that the set of image analysis
features we used capture at least some of the information used
when making a manual assessment of stem cell quality. Each class
definition includes an uncertainty derived from the uncertainty in
the expert scoring. We compare two approaches to classification:
Random Forest, which is a sequential (decision tree) method, and
a linear combination approach in which a set of features is applied
simultaneously. We find that the linear combination method
appears to be somewhat more successful at correct classification.
However, the decision tree method allows us to identify features
that are less important to correct classification. This work provides
a quantitative and unambiguous definition of stem cell colony
quality and illustrates how uncertainty can be incorporated into this
definition.
Congress
Overview
to discard based on their visible microscopic characteristics. To
the expert in culturing stem cells, these characteristics indicate
desirable properties such as pluripotency and viability and
undesirable properties such as cell death and spontaneous
differentiation. Because stem cell colony quality assessment
is subjective, culturing these cells can be inconsistent between
laboratories and even from passage to passage within the
same laboratory. If an automated approach to stem cell quality
assessment could be developed, consistency and reproducibility
would be improved, and automation of expansion and passaging
of stem cells would be made possible. In this study, we have used
descriptive explanations of visual cues from stem cell experts
to provide insight into the image features that are critical in
identification of desirable colonies. Using this insight, we have
designed image analysis algorithms to quantify characteristics of the
colonies thought to be associated with quality (i.e. pluripotency) of
the stem cell colony.
109
Congress
Overview
Special
Lectures
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
samples, but alsoincreases detection and sorting efficiency. The
mitigating effects on cell-to-cell aggregation are transitory—a preenriched sample that is left to sit for some period of time (1–2hr)
begins to degrade and aggregationrecurs. This suggests that time to
sort after pre-enrichment is of great importance which provides a
compelling argument for the advantages of the in-line procedure.
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Methods: Microflow1’score technology is a fiber-optic flow-cell
with sheathless fluidics. The fiberized design ensures fixed optimal
positioning of optical components. The laser and the detection
sub-systems are also fiberized. It is a simple to use, self-contained
FC with: low mass (10kg), small size (34cm W x 19cm D x 20cm
H), safe fluid management, low power consumption (11W) with
battery operation (10 D batteries), minimal sample and waste
volumes (less than 3mL) and Velcro patches to fastenFC. Prepared
biological samples are delivered into the flow cytometer via a
6-chamber cartridge that locks to the FC.In preparation for the
ISSdemonstration, a cartridge was loaded with stress biomarkers: a
customized Th1/Th2 assay (CBA multiplexed kit by BD) to support
monitoring secreted cytokines, SupT1 cell line and human blood
cells prepared for CD4 and CD45 surface marker detection.
Thecartridge also includes calibrationmicrofluorospheresand
flushing solutionsto validate performance.Samples were also
characterizedwith BD’s FACSArray, our reference method. To further
reduce sample volume to 500µl, analytes canbe injected with a luer
slip syringe.Two Flight Models (FM) have been tested on ground
using syringes and cartridges.
Results: Results indicate consistency between both syringe and
cartridge injection. Multiplexed cytokines assays were within
5% of the reference method. Leukocyte sub-populations were
discriminated but hadsome counting discrepancies when
comparing to the reference instrument.Overall, there was
acceptable correlation between the FM and EQM units.
Speaker/Author
Index
which isincompatible with microgravity (2). In the past, NASA has
been successfully using a modified Guava FC (3) during parabolic
flights. Recently, a miniature sheathless FC, Microflow1Engineering
Qualification Model (EQM), also performed well during simulated
microgravity (4).Microflow1, developedby INO for the Canadian
Space Agency, is scheduled for a technology demonstration onboard the ISS early in 2013.
58
57
Microflow1 a Portable Flow Cytometer for Space
Travel: In Preparation for the International Space
Station Demonstration
Christophe RIVIERE , Ozzy MERMUT , Genevieve DUBEAULARAMEE2, Luchino COHEN2
1
Biophotonics, INO, Quebec, QC, Canada, 2Canadian Space
Agency, Saint-Hubert, QC, Canada
1
1
Background: Flights in space have demonstrated that weightlessness
is responsible for physiological changes includingaltered immune
system (1). A flow cytometer (FC) will help to monitor physiological
adaptation onboard the International Space Station (ISS).Most
commercial flow cytometers depend on hydrodynamic focusing,
110
Conclusions: Microflow1 EQM unitis compatible with simulated
microgravity and the FM units have acceptable on-ground
performance. Microflow1 or its next generationmay well
be selected as apermanent installationon the ISS for online
biodiagnostic/monitoring of astronauts during extended space
missions.
1. Williams D, Kuipers A, Mukai C, Thirsk R. Acclimation
during space flight: effects on human physiology. CMAJ
2009;180:1317-23.
2. Crucian BE, Norman J, Brentz, J, Pietrzyk R, Sams CF:
Laboratory outreach: student assessment of flow cytometer
fluidics in zero-gravity. Laboratory Medicine 2000, 31:569-572.
3. Sams CF, Crucian BE, Clift VL, Meinelt EM. Development of a
whole blood staining device for use during space shuttle flights.
Cytometry, 1999;37:74–80.
4. Christophe Rivière, Sébastien Leclair, Ozzy Mermut, Geneviève
Dubeau-Laramée, Daniel Provençal, Luchino Y. Cohen,
Cyto2012, Leipzig, Germany, poster #461 and manuscript in
preparation.
An Extremely Parallel Flow Cytometer for Rapid
Cellular Analysis
Steven Graves, Pearlson P. Austin Suthanthiraraj, Menake E.
Piyeasena, Andrew Shreve
Center for Biomedical Engineering, University of New Mexico,
Albuquerque, NM, United States
Introduction: Flow cytometry is an invaluable tool for cellular
analyses such as the determination of DNA content, the detection
of pathogens in blood, and immunophenotyping of cells.Current
flow cytometers achieve particle analysis rates as high as 70,000
events per second, while delivering sample at roughly 100 µl/
minute. To increase throughput, higher analysis rates have been
achieved by running four flow cytometer analysis heads in parallel.
ISAC 2013 Program and Abstracts
Congress
Overview
However, new application areas requiring detection of very rare
events (e.g. circulating tumor cells, hematopoietic stem cells, fetal
cellsmaternal blood) require dramatically higher throughput, both
in terms of volumetric delivery and particle analysis rates.
Unbound kG-1a cells focused in the acoustic node whereaskG-1a
cells focused in the pressure antinode when bound to elastomeric
colloids.
Poster
Session
Conclusion: We demonstrate a new form of cell separation
technology which uses discriminatory acoustic forces on
elastomeric colloids to isolate cells of interest as a new generation
technology for ultrasensitive detections and separations.
1. Herzenberg, L, et al. Sci Am. 1976. 234(3): 108-117
2. Miltenyi, S, et al. Cytom. 1990. 11(2): 231-238
3. Laurell, T, et al. Chem Soc Rev. 2007. 36: 492-506
4. Piyansena, M, et al. Anal Chem. 2012. 84: 1831-1839
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Introduction: Fluorescence activated cell sorting (FACS) is the gold
standard for rapidly detecting and separating cells withhigh fidelity.
However, conventional FACSrequires copious sheath fluid and
serial (cell-by-cell) analysis. Alternatively, magnetic activated cell
sorting (MACS) provides batchwise separation, yet typically with
less purity and limited capabilities for multi-marker selections.1,2
Results: By modulating monomer ratios and reaction conditions,
elastomeric particles exhibited uniform and tunable size, density,
and compressibility, allowing for precise acoustic control. As
apreliminary surrogate demonstration for AACS, elastomeric
particles werebound to positive acoustic contrast polystyrene beads,
where the bound complexdisplaced the pressure antinode.
Wednesday,
22 May
Charles Shields IV1,2, Leah Johnson1, Lu Gao3, Gabriel Lopez1,2
1
Biomedical Engineering, Duke University, Durham, NC,
United States, 2Triangle MRSEC, Durham, NC, United States,
3
Mechanical Engineering and Materials Science, Duke
University, Durham, NC, United States
Elastomeric particles were synthesized from nucleation and growth
of hydrolyzed multi-functional siloxanemonomers. Particles were
adsorbed with streptavidin, which bound to biotinylated anti-CD34
antibodies on kG-1a leukemia cells. Only cells expressing CD34
were labeled, thus allowing discriminatory separation.
Tuesday,
21 May
Acoustofluidic Cell Sorting via Negative Acoustic
Contrast Capture Colloids
Methods: A PZT excitesthe microfluidic deviceat resonance
whereupon ultrasonic standing waves form across the flow cavity.
Here, channel walls correspond to pressure antinodes where
particles with low density and low modulusrelative to the media
(i.e., elastomeric particles)focus; the centerline corresponds to the
pressurenode where particles withhigh density and high modulus
relative to the media (i.e., cells) focus. When cells are labeled, the
complex displaces to the antinode when the primary radiation force
on the elastomeric particles is greater than that on the cell. After
separation, a downstream channel trifurcation allows for isolated
cell collection.
Monday,
20 May
59
Sunday,
19 May
Conclusions: In this work, we will present a compact affordable
parallel flow cytometer prototype that retains the optical properties
of conventional flow cytometry yet has a realistic path to analysis
rates >1x106 events/s and sampling rates of 50 mL/min. This system
represents an important technological advance in parallel flow
cytometry that will be an important for the analysis of extremely
rare event applications in flow cytometry.
Saturday,
18 May
Results: We have developed parallel microfluidic flow cellsby using
deep reactive ion etching to introduce many acoustic focusing
channels into a single silicon wafer. We have made these flow cells
with up to 100 parallel channels that each supported 3 well-defined
acoustically focused streams of cells, for a total of 300 streams.
Furthermore, using an EMCCD camera for image analysis, we have
demonstrated well-focused streams at flow rates as high as 15 mL/
min. For coupling to our optical detection system, we will present
the development of a multinode acoustic flow cell that supports 16
to 32 focused sample streams. We will also present the coupling of
this flow cell to our line focused laser interrogation system and our
array detection system. Finally, we will present our work towards
testing the integrated flow cytometer using calibration microspheres
and cell samples.
Special
Lectures
Methods: In short, the laws of physics preclude simply speeding
up the sample delivery rate to further increase the analysis rate
of flow cytometers. Therefore, we are pursuing the development
of parallel flow cells coupled with highly parallel detection
approaches to create a compact affordable flow cytometer that can
analyze up to 1x106cells/s and samples at 50 mL/min. We used
multinode acoustic standing waves to create many parallel flow
streams in a single flow channel or several flow streams in multiple
microfabricated flow channels. These channels are the basis of
our parallel flow cells that we have coupled to a newly developed
optical detection platform. This platform uses a line-focused laser
in combination with an array detector (e.g. multianode PMT or
EMCCD) to detect scatter and of fluorescencefrom up to a few
hundred flow streams simultaneously.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Poster Session
Abstracts
We propose a system as aprototype for a new generation
of acoustically activated cell sorting (AACS) that isa viable,
and potentially superior, alternative to FACS and MACS.
Acoustophoresis provides cell separation capabilities based
on acoustic properties of cells and fluid.3Using elastomeric
particleswith antibody affinity labels(Fig. 1), AACS can separate
rare cells (e.g., circulating tumor cells)from native fluidsby acoustic
radiation forces. These forces are independent of flow, allowing
continuous focusing at switchable flow rates for ultrasensitive
detection when coupled with fluorescence based flow cytometry.4
111
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
60
61
Complex Changes in Invariant Natural Killer T (iNKT)
Cells in Patients with Different Clinical Forms and
Treatments of Multiple Sclerosis
Immune Monitoring of Human Kidney Allografts with
Biopsy Multicolor Flow Cytometry and Cytokines
SARA DE BIASI1, Milena Nasi1, Anna Maria Simone2, Diana
Ferraro2, Francesca Vitetta2, Lara Gibellini1, Marcello Pinti3,
Cinzia Del Giovane4, Patrizia Sola2, Andrea Cossarizza1
1
Department of Surgery, Medicine, Dentistry and Morphological
Sciences, University of Modena and Reggio Emilia, Modena,
Italy, 2MS Center, Neurology Clinic, Nuovo Ospedale Civile
Sant’Agostino Estense (NOCSAE), Modena, Italy, 3Department
of Life Sciences, University of Modena and Reggio Emilia,
Modena, Italy, 4Department of Diagnostic and Clinical
Medicine and Public Health, University of Modena and Reggio
Emilia, Modena, Italy
Introduction: The pathogenesis of Multiple Sclerosis (MS) is
still poorly understood. Associations between MS and defects
in invariant natural killer T cells (iNKT) have been reported, but
contrasting data exist, due to methodological limitations. iNKT cells
are potent cytokine producers, have immunoregulatory potential,
and can be divided into functionally distinct subsets. A more
detailed characterization of iNKT cell subsets is needed to better
clarify their role in MS, particularly in different forms of the disease
and its treatment. Among T cells, a recently described population
exists that has strong proinflammatory capacity (CD8+CD161+ T
cells), and is altered in MS. Aim of our study was to evaluate the
amount of these cells, along with that of iNKT, in different forms of
MS.
Methods: We studied 66 patients followed by the MS Center
(NOCSAE, Modena): 52 with a Relapsing Remitting (RR; 46 taking
therapy, 6 without treatment), 9 with a Primary Progressive (PP)
and 5 with a Secondary Progressive (SP) MS. 16 age- and sexmatched subjects were healthy controls (CTR). iNKT and CD8+,
CD161+ cells were analyzed on fresh PBMC. CD3+ T cells were
volumetrically counted using a CyFlow Counter (Partec, Germany).
Then, isolated PBMC were stained with different mAbs for the
analysis on a 6-color high speed acoustic focusing flow cytometer
(Attune, Life Technologies, USA), using different mAbs, i.e. antiVa24Ja18 TCR, -CD4, -CD8, -CD161, -CD3, -CD19 and -CD14. A
minimum of 5 million cells were acquired, and data analyzed by
FlowJo 9.6 and Stata 11.0 softwares using Ranksum and Anova test.
Results: In comparison with CTR, MS patients with a RR form
displayed a statistically significant lower number (but not
percentage) of iNKT cells. Patients with other MS forms (whose
number was lower) showed a similar trend. Among iNKT cells, the
CD4+ subset did not vary in the different forms or in comparison
to CTR, while the CD8+ subset was always diminished. The CD4CD8- subset was significantly lower in the RR form compared
to CTR. Among iNKT cells, the population of CD8+CD161+
cells was lower in all MS patients. When compared to CTR, PP
and SP patients had a lower number and percentage of CD8+
T cells; CD8+, CD161+ T cells (both dim and bright subset)
were lower in all SP patients vs. CTR. Concerning the role of MS
treatment, RR patients taking Fingolimod (a drug that blocks the
sphingosine-1-phosphate receptor S1PR1 and traps lymphocytes in
the lymphnode) had a lower number of CD3+ T cells and a lower
percentage of iNKT expressing CD4. Patients taking natalizumab
(blocking alpha 4-integrin), interferon or glatiramer acetate
displayed no gross changes in iNKT cells in comparison with CTR.
Conclusion. The changes in iNKT cells and their subpopulations we
observed in RR patients, along with the trends present in the other
patients, suggest a role for these cells in the pathogenesis of MS.
Functional studies are ongoing to better understand the activity of
such cells.
112
Kimberly Muczynski, Nicolae Leca, Susan Anderson
Medicine/Nephrology, Univ of Washington, Seattle, WA,
United States
Background: Biopsies, the standard for assessing kidney transplants,
lack information about immune processes and are often insufficient
for guiding therapy. Different injuries can appear histologically
identical, as “nonpecific inflammation,” creating a dilemma for
treatment. For example, rejection and BK nephropathy both
have infiltrating leukocytes in kidney tubules but have opposite
treatments. Leukocytes in fibrotic areas can represent either
resolving inflammation or active injury. Also, leukocytes circulating
in the local microvasculature, which may be inciting inflammation,
are lost in histology processing but retained when a biopsy is
processed for cytometry.
Methods: To better assess allograft inflammation, I devised a
collagenase digestion protocol for reducing kidney biopsies to cell
suspensions for multicolor flow cytometry which preserves epitopes
of interest and used a high sensitivity Luminex platform assay to
measure secreted cytokines within the tissue. Allograft leukocytes,
antibody-bound endothelial cells and cytokines were assessed.
Results: The following observations have been made: 1) Rejection
is associated with reduction in allograft granulocytes, increased
CD3 cells and a predominance of CD8 over CD4 T cells. The ratio
of CD8:CD4 cells identify rejection early, before it is detectable by
histopathology. 2) BK nephropathy is associated with increase in
CD56+ (NK) cells and a low CD8:CD4 ratio. 3) Increased antibody
on endothelial cells is associated with transplant glomerulopathy
and can be used to follow clinic course. 4) IL-6 and -8 are
associated with renal ischemia. High levels of IL-4 predominate
in rejecting renal allografts with donor specific antibodies and
complement activation.
Conclusion: Cytometry, to define cells and cytokine levels within
kidney transplants, reliably distinguishes harmful nonspecific
inflammation from immune processes that do not require
adjustments of immunosuppression. It should be used as an
adjunct to traditional histopathology. Results may also define
targets for more specific immunosuppressants.
62
Metastatic Breast Cancer Stem/Progenitor
Populations Survive Consolidation Chemotherapy,
Circulate and Disseminate to Bone Marrow
Albert Donnenberg 1 , Vera S. Donnenberg 2 , Rodney
Landreneau2, Adam Brufsky1
1
Medicine, University of Pittsburgh School of Medicine,
Pittsburgh, PA, United States, 2Cardiothoracic Surgery, Univ
of Pittsburgh, Pittsburgh, PA, United States
Despite the prognostic value of circulating tumor cells in late stages
of breast and prostate cancers, the “stem/progenitor phenotype”
and tumorigenicity of these cells have never been demonstrated.
To determine immunophenotypic signatures of metastatic breast
cancer cells we examined coexpression of epithelial markers and
stem/progenitor markers in malignant pleural effusions from 9
breast cancer patients. Nonhematopoietic pleural effusion cells
expressed cytokeratin and EpCAM, but the proportion varied widely.
CD90 was the most prevalent stem/progenitor marker studied (14.5
± 4.1%, mean ± SEM), followed by CD133 (10.7 ± 6.24%) and
CD117 (3.4 ± 1.0%). These populations were largely mutually
exclusive and each had significant proportions of cells with >2N
DNA. Aneuploidy was limited to cells bearing epithelial markers.
ISAC 2013 Program and Abstracts
Background: Knowledge of the contextual information cells derive
from their physical, chemical and cellular microenvironment within
tissues is essential for the understanding and effective treatment of
many diseases, especially cancer. Producing uniform, controllable
in vitro 3D models of tissues and tumors is a critical requirement
to advance the nascent field of tissue cytometry. Improved methods
are also needed for generating 3D models containing multiple cell
types, as well as for in situ assay of cells and microenvironments.
Introduction: Our approach to addressing these limitations
involves the generation and assay of spherical cellular aggregates
(spheroids). We have engineered a novel apparatus that utilizes
fluid flow and ultrasonic vibrations to produce uniform populations
of calcium alginate microspheres (CAMs) containing viable cells.
Proliferation of cells in suspension-cultured CAMs results in the
generation of completely cellular spheroids of uniform size and
cellular composition.
Monday,
20 May
Methods: Our apparatus consists of a pressure chamber
producing stable flow of a sodium alginate pre-gel solution
containingfluorescently labeled human tumor cells through
a precisely fabricated glass micronozzle. The micronozzle is
coupled to a piezoelectric transducer that produces vibrations
that result in the formation of cell-containing microdroplets. The
microdropletscrosslink upon interaction with calcium ions in
a receiving solution, producing CAMs. Droplets are visualized
through a 10x objective coupled to a camera and an LED array
that strobes atthe vibration frequency. For initial interrogation of
the microenvironment, the pre-gel solution is functionalized with
pH-sensitive fluorescein. CAMs and the resultant spheroids are
assayed both in situ by confocal microscopy and by flow cytometry
following dissociation to single-cell suspensions.
Tuesday,
21 May
Wednesday,
22 May
Results and Conclusions: Varying the flow rate and PZT modulation
frequency controls the size of CAM populations. We can produce
uniformly sized CAMs from 100-250 µm diameter at frequencies
of 10-30 kHz, with initial cell concentrations up to ~25% of
tissue cellularity. Assay of the spatial distribution of cellswithin
CAMs during subsequent culture demonstrated proliferation and
the development of cell-cell and cell-matrix interactions. CAMs
and spheroids were dissociated to single cells for flow cytometry
analysis of proliferation, viability and quantification of ratios of
different cell types in co-culture. Functionalized, pH-sensitive
CAMs containing cells will be used to monitor pH gradients created
by cellular metabolism. This technology impacts the field of tissue
cytometry by providing a high-throughput method for creating
uniform 3D tissue models. Adding other integratedfluorescence
sensors (glucose, lactate, oxygen) will create a complex but
quantifiable microenvironment that will greatly improve tissue
cytometry applications andour understanding of tissue and tumor
biology.
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Recent studies provide strong evidence for a role for B cells in
the pathology of RRMS. In order to explore the effect of IFNβ-1b
treatment on B cell phenotype and function in RRMS patients,
blood was drawn from 10 RRMS patients before treatment and
again 2 and 6 months after injections every other day of IFNβ-1b.
Cryopreserved PBMCs were thawed and stained with 10-color
panels of antibodies against B cell surface antigens. At baseline,
RRMS patients have increased frequencies of naïve CD19+CD20+
B cells and decreased frequencies of memory B cells when
compared with age-matched healthy controls. Treatment alters the
composition of circulating B cell subsets, leading to an increase
after six months in circulating naïve B cells and a decrease in both
memory and B1 cells, both cell types of which are potentially
pathogenic in RRMS.In addition, B1 cells from RRMS patients
showed a higher level of CD5 expression, which may indicate a
higher level of regulatory B cells in this population. Furthermore,
several phenotypic differences were seen between RRMS patients
and healthy controls, including significant differences in IgM,
CD95, SIGLEC-10, CD24, CD307d, PD-1 and CD305 expression.
The balance of these phenotypic differences indicates a B cell
population in RRMS patients that is more inflammatory and less
able to be regulated. Although the number of subjects in this
study was limited, these findings suggest that alterations in B cell
phenotype and function may be a mechanism by which IFNβ-1b
reduces disease activity.
Jacqueline De Lora, Daniel Kalb, Alice Martinic, Antoinette
Trujillo, Travis Woods, Andrew Shreve, James Freyer
Center for Biomedical Engineering, The University of New
Mexico, Albuquerque, NM, United States
Sunday,
19 May
Daniel Mielcarz1, John DeLong1, Alan Bergeron2, Kathleen
Smith1, Alexandra Heyn1, Lloyd Kasper1, Jacqueline Channon1
1
Microbiology/Immunology, Geisel School of Medicine at
Dartmouth, Lebanon, NH, United States, 2Medicine, Geisel
School of Medicine at Dartmouth, Lebanon, NH, United States
A High-Throughput Method for Creating Uniform 3D
Tissue Models
Saturday,
18 May
Relapsing-Remitting MS Patients Have Significant
Differences in Circulating B Cells Subset and
Phenotype Compared with Healthy Controls, and
IFNβ-1b Treatment Can Alter the Composition and
Phenotype of These Subsets
64
Special
Lectures
63
Congress
Overview
We then attempted to detect cells coexpressing stem/progenitor
and epithelial markers as rare therapy resistant populations in
the bone marrow and blood of breast cancer patients. We made
use of unique cryopreserved hematopoietic stem cell backup
products (bone marrow n=3, leukapheresis n=4, 3 of which were
used for in vivo tumorigenicity experiments) collected after high
dose cyclophosphamide/etoposide stem cell mobilization. Flow
cytometry was performed before and after immunomagnetic
selection for EpCAM+ cells. An average of 1.3 x 109 cells was
separated. Patient samples were compared to normal bone marrow
(n=10) and normal leukapheresis products (n=4). Two of three
patient bone marrow samples had detectable stem cell marker+
populations (CD90+, CD133+, CD117+) that were tumor specific
because they were: 1) absent in normal samples; 2) cytokeratin+; 3)
enriched by EpCAM separation. By the same criteria, 3 of 4 patient
leukapheresis samples had CD133+ tumor cells. The data indicate
that stem cell marker+ cytokeratin+ tumor cells survive high dose
chemotherapy, circulate and disseminate. In order to determine
the tumorigenicity of circulating tumor-stem/progenitor cells, 3
leukapheresis products were immunomagnetically separated for
EpCAM+ and/or CD90+ cells. Recovered cells were admixed with
adipose-derived stromal cells and injected into mammary fat pads
on NSG mice (20 injections/specimen). Xenografts were monitored
for 6 months, when animals were sacrificed and tested for tumor
formation. Despite the presence of CD90+ EpCAM+ cells in all
specimens tested, only 3 sites out of 60 injected generated tumors,
all from the same specimen. Detailed genomic and histological
analyses are in progress.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
113
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
65
Analysis and Sorting of Antigen-Specific Antibody
Secreting Cells in Mixed Cultures Using the Affinity
Matrix Technology
Adriano Taddeo1, Hyun-Dong Chang1, Velia Gerl2, Bimba
Hoyer2, Andreas Radbruch1, Falk Hiepe2
1
Deutsches Rheuma-Forschungszentrum (DRFZ), Berlin,
Germany, 2Medizinische Klinik III m. S. Rheumatologie &
klinische Immunologie, Charité - Universitätsmedizin Berlin,
Campus Mitte, Berlin, Germany
One of the most time and labor intensive procedure in the
production of monoclonal antibodies (mAbs) by hybridoma cell
lines is the identification of antigen-specific antibody-producing
clones within a heterogeneous cell population. Moreover, some
hybridomas can lose the ability to produce mAbs by continuous
culture and recloning is therefore recommended if hybridomas
have been in culture for a long period of time and monoclonality or
stability of the hybridoma needs to be confirmed. Indeed, despite
their biological and practical significance, only a few methods
are presently available for the analysis of secreted antibody on the
single cell level by antibody secreting cells (ASCs). These methods
may offer a valid alternative for selecting the antigen-specific cells
allowing a faster and more accurate selection of the clones of
interest. Here we describe a new concept for cytometry and sorting
of live ASCs based on the specificity of the produced antibody.
Basically, the secreted antibody is retained on the cell surface of the
secreting cell, making it accessible to the powerful technologies for
detection of surface markers allowing FACS analysis and live-cell
sorting of antigen specific ASCs.
ASCs were incubated with an affinity matrix composed of 1) an
antibody binding specifically to ASCs coupled to 2) the antigen
specific to the antibodies secreted by the ASCs. For this proof-of
concept experiment an anti-CD138-OVA-construct (affinity matrix)
was generated and anti-OVA secreting hybridoma clones were
used as model of ASCs. Anti-OVA IgG1 secreting hybridoma cells
were mixed at different ratio (from 2:1 to 1:10000) with aspecific
cells (X63-Ag6.653 myeloma cells or anti-OVA IgG2a secreting
cells). After a short incubation for allowing the antibody secretion,
the anti-OVA antibodies secreted by ASC binds to the antigen
brought to the surface of the ASC by our matrix. The OVA-specific
ASCs were then stained with a fluorochrome labeled anti-IgG1
antibody and visualized by FACS. The affinity matrix technology
allowed us to clearly show a specific labeling of cells producing
antibodies with the desired specificity and the desired IgG subclass
independently of the ratio of specific/aspecific cells.
These results show that using the affinity matrix technology it
is possible to select ASCs in an antigen-specific way. Therefore,
this technology represents a useful tool for reducing the time and
manipulations necessary for hybridoma selection and cloning and
provides an innovative method for the analysis of secreted antibody
on the single cell level by antibody secreting cells (i.e. hybridoma
cells and plasma cells).
has proposed that the potency of cells used in therapies be
characterized by relevant molecular properties. Yet, there have
been no measures of potency developed for cellular transplantation.
Hematopoietic reconstituting cells (HRC) are transplanted in the
treatment of a variety of diseases. The number of CD34+ cells
transferred is clearly associated with engraftment but it does not
provide an indication of the potency at the level of the individual
cell. There are 2 sources of HRC that are known to be superior
to bone marrow HRC in terms of potency on a cell-by-cell basis:
G-CSF-mobilized peripheral blood HRC and umbilical cord blood
HRC.
We have used an enzymatic amplification system for enhancing the
sensitivity of flow cytometric analysis to assess HRC. In a previous
study we have demonstrated that our analytical paradigm could
detect transcription factor expression levels that correlated with
granulocyte-macrophage colony forming units and erythrocytic
burst-forming units (Cytometry Part A 83A:127-133, 2013). In
the current investigation we studied HRCfrom bone marrow,
G-CSF-mobilized peripheral blood, and umbilical cord blood
in order to assess cellular potency. We chose 16 molecules for
analysis including pathway molecules (such as phospho-Akt),
transcription factors (such as HoxB4), a transcriptional repressor
(Bmi-1), a translational regulator (Musashi-2), an anti-apoptotic
molecule (Mcl-1), an ubiquitin-conjugating enzyme (UBC13), and
an autophagy protein (Atg7). All of the 16 molecules have been
previously associated with HRC function based on the results of
experimental manipulations.
Our results provide us with a measure of cellular potency based
both on the expression levels of the 16 molecules and on the
correlations of the molecules with each other. Additionally, we
have uncovered a linear sequence of molecular associations that is
related to the potency of the cells.
By analyzing molecules known to be important in function, we
have developed acogent measure of cellular potency. We speculate
that this technology can be useful in developing measures that
may be useful in assessing the transplantation of other cells for
therapeutic purposes.
67
Sorting of Specific Lymphocyte Populations from
Peripheral Blood Progenitor Cell Products Using a
Novel Micro-Chip Based Acoustophoretic Platform
Anke Urbansky1, Andreas Lenshof2, Arshad Jamal3, Josefina
Dykes3, Ingbritt Åstrand-Grundström3, Thomas Laurell4,
Stefan Scheding3
1
Stem Cell Center, Lund University, Lund, Sweden, 2Lund
University, 3Lund University, Lund, Sweden, 4Department of
Measurement Technology and Industrial Electrical Engineering,
Lund University, Lund, Sweden
David Kaplan1, Hillard M. Lazarus2, Nicholas M. Kaye1
1
Pathfinder Biotech, Cleveland, OH, United States, 2Medicine,
Case Western Reserve University, Cleveland, OH, United States
Background: Processing of peripheral blood progenitor cells
(PBPC) for clinical transplantation or research applications aims
to effectively select or deplete specific cell populations. Usually,
fluorescence- or magnetically-based sorting techniques are used for
PBPC processing. Here, we investigated the performance of a novel
microchip-based ´acoustophoresis´ technique, utilizing ultrasound
for the separation of lymphocyte subsets from PBPC. By applying
an acoustic standing wave field on to a continuously flowing cell
suspension in a micro channel, cells can be separated depending
on their physical properties, such as size and density.
Cellular transplantation is a relatively new therapeutic modality.
Various stem cells and specific immune cells have been used
successfully in treating a variety of clinical conditions. It is
important to develop potency measures for the cells to be
transplanted in order to ascertain that the cells will perform as
expected. In fact, the Federal Drug Administration, which has
jurisdiction over cell transplantation for therapeutic purposes,
Study design and Methods: PBPC samples were obtained from
patients (n= 16) and healthy donors (n=6). Following density gradient
centrifugation, cells were labelled with either anti-CD4 or anti-CD8
microbeads (Dynal) and sorted on an acoustophoresis-microchip.
In parallel, control magnetic sorting was performed. PBPC samples,
target and waste fractions were analysed for separation efficiency,
recovery, T-cell function and progenitor cell content.
66
Developing Measures of Cellular Potency for
Therapeutic Transplantation
114
ISAC 2013 Program and Abstracts
In recent years, flow cytometry has benefitted greatly from the
recognized MIFlowCyt standards that cover key areas such as
quality control, instrument performance, experimental set up and
data reporting. With the emergence of image cytometry platforms
that preserve the high event throughput of flow cytometry while
delivering high content, quantitative imaging, this is an opportune
time to open a cross-platform multi-discipline dialogue as to how
MIFlowCyt and Imaging standards could be applied to image
cytometry. While there are many aspects to this topic that deserve
in-depth discussion and both the time and dedication outside the
time constraints of this workshop, we will aim to initiate discussion
and dialogue pertaining to a need to standardize data acquisition,
analysis and reporting for IC in a manner akin to MIFlowCyt.
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Hans Minderman1, Andrew Filby2, Anne Plant3, Michael
Halter3, Padma Narayanan4, Amanda White5
1
Rosewell Park Cancer Institute, Buffalo, NY, United States,
2
Cancer Research UK, London, United Kingdom, 3National
Institute of Standards and Technology, Gaithersburg, MD,
United States, 4Amgen, Washington, DC, United States,
5
Cincinnati Children's Hospital, Cincinnati, OH, United States
Oral Session
Abstracts
Over the past decade, new imaging modalities have revolutionized
the field of immunology at the level of systems, cellular, and
molecular immunology. I will discuss our work using two-photon
microscopy and calcium indicators imaged by TIRF microscopy to
investigate T cell activation. The lymph node can be understood
at the systems level as a filter that promotes cellular motility
and responses to antigen. Under basal steady-state conditions, T
lymphocytes home from the blood into the diffuse cortex of the
lymph node and migrate rapidly in a random walk that optimizes
the detection of antigen. T cells sample the surface of resident and
tissue-derived dendritic cells, and if the specific peptide MHC is
Integrating Standards at the Interface of Flow and
Image Cytometry
Commercial
Tutorials &
Exhibits
Michael Cahalan
University of California, Irvine, UCI, CA, United States
71
Poster
Session
Single-Cell Approaches to Investigate the Immune
Response
The cerebrospinal fluid (CSF) supports buoyancy of the cortex, and
buffers it from injury. It is also the conduit in which lymphocytes
traffic through the parenchyma of the cortex. Thus, CSF immune
profiling can provide a useful tool to track inflammatory processes
occurring in the central nervous system (CNS). For the last decade,
our laboratory has gained expertise in characterizing lymphocyte
dynamics in the CSF of patients with Multiple Sclerosis (MS), an
autoimmune disease of the CNS. The goal for this presentation is
to review our understanding of lymphocyte profiles in the CSF of
MS patients and compare them to lymphocyte profiles in the CSF of
patients at risk to develop MS and patients with other neurological
diseases of the CNS. The presentation will also include a discussion
on the limitations of characterizing lymphocyte dynamics in CSF,
and provide examples of how lymphocyte profiling in the CSF may
be used as a tool to guide novel therapeutic strategies.
Wednesday,
22 May
69
Nancy Monson
UT Southwestern Medical Center, Dallas, TX, United States
Tuesday,
21 May
Leukocyte adhesion under flow is mainly mediated by selectins
and their ligands. To study the contact area (footprint) of rolling
neutrophils at high resolution, we developed quantitative dynamic
footprinting (qDF), a TIRF-based technique that provides nanometer
resolution in the vertical z axis. Neutrophils roll over a selectin
substrate in a microfluidic device. This method shows the z distance
(height) of the plasma membrane or a cytosolic marker. The cell
morphology can be represented as hills and valleys. Using this
technique, we confirmed the existence of long tethers (up to 20 μm)
and discovered slings, cell-autonomous adhesive structures. We
adapted this method for multicolor use, thus enabling simultaneous
visualization of surface topography and a molecule, visualized as a
fusion protein or by fluorescently labeled antibody. Slings contain
patches of PSGL-1, the main ligand for P-selectin. When slings
peel from the substrate, each PSGL-1 patch provides resistance
to peeling and slows the cell down. PSHL-1 patch spacing is
consistent with the membrane content of microvilli, suggesting
that multiple microvilli are pulled into the same sling. Slings wrap
around the rolling cells and interact with the cell body by LFA-1
binding to ICAM-2.
Immune Profiles in the Central Nervous System:
What We Know, What We Need To Know, and What
It Means
Monday,
20 May
Klaus Ley
Inflammation Biology, La Jolla Institute for Allergy &
Immunology, La Jolla, CA, United States
70
Sunday,
19 May
Image Cytometry of Cell Adhesion
Saturday,
18 May
68
encountered, a proximal signaling cascade generates IP3 within
the T cell, in turn depleting the endoplasmic reticulum Ca2+ store.
Depletion triggers oligomerization of STIM1 proteins in the ER,
which then physically translocate, while still in the ER membrane,
to the plasma membrane. STIM1 forms µm-scale puncta and
triggers adjacent Orai1 channels to open, enabling a local Ca2+
influx and downstream gene expression responses. Genetically
encoded Ca2+ indicators fused to the cytosolic tails of Orai1 can
be used to detect local Ca2+ entry. Under TIRF microscopy, the
signals are sufficiently large to detect the opening of single- Orai1
channels. We are also using the dominant-negative E106A Orai1
mutant to inhibit Ca2+ influx in T cells to further evaluate functional
roles in homing to lymphoid organs, chemotaxis and integrin
signaling, gene expression, motility, and cell death pathways. The
presentation will include highlights of these studies.
Special
Lectures
Conclusion: The acoustophoresis technique can be utilized
to efficiently sort bead-marked lymphocyte populations from
PBPC samples with high purity and recovery without impairing
lymphocyte function. Acoustophoresis is, thus, an interesting
technology for PBPC processing, which has furthermore the
potential to offer a single platform technique for multi-parameter
cell separation.
Congress
Overview
Results: The mean separation efficiency of CD4+ lymphocytes to
the target fraction was 87 ± 7% with acoustophoresis compared to
95 ± 8% for control magnetic sorting (n=19). Viability of sorted cells
was 95 ± 4% (acoustic) and 97 ± 3% (magnetic). Following further
technical improvement of the chip, purities obtained with acoustic
sorting of CD8+ lymphocytes were 96 ± 3% compared to 93 ± 4%
for magnetic sorting (n=3). The median recovery of acoustically
selected lymphocytes was 57%. Leukocyte subpopulation analysis
performed after CD4 selection showed a relative increase of CD8,
CD19, CD34, CD56, CD14 cells in the non-target fractions. These
changes were due to the removal of CD4 cells and found to be
within the expected range. Functional testing of sorted CD4+ cells
showed unimpaired mitogen-induced proliferation capacity as well
as cytokine production. Results from hematopoietic progenitor cell
assays indicated a preserved colony-forming ability post-sorting of
non-target cells.
115
Congress
Overview
Special
Lectures
Attila Tarnok1, Ryan Brinkman2, Vera Donnenberg3, Henning
Ulrich4, Susan Vice5
1
University of Leipzig, Leipzig, Germany, 2British Columbia
Cancer Agency, Vancouver, BC, Canada, 3Univ of Pittsburgh,
Hillman Cancer Center, 4Instituto de Química, Univ of Sao
Paulo, 5Wiley, Hoboken, NJ, United States
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
72
Writing, Publishing and Reviewing: Advice and Tips
from Cytometry A
Scientific journals require certain quality standards from
manuscripts to be acceptable for further reviewing and publication.
There are some very common reasons why a paper gets reviewed
and accepted or rejected. This workshop aims to highlight all major
aspects of manuscript writing, submission and communication with
the reviewers, points out what can (and very often does) go wrong
and how to do it right in order to improve your chances to get
your paper published. Special emphasis will be taken to focus on
the needs for a biomedical and technical oriented journal such as
Cytometry Part A including MIFlowCyt, FlowRepository and OMIP.
Experts from the Cytometry Part A editorial board and from the
publisher Wiley-Blackwell will contribute. Winner of the best paper
award of Cytometry Part A will briefly present the winning work.
Authors may bring their manuscript drafts for discussion with the
editors after the workshop.
73
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
High Throughput Flow Cytometry in Pharma
Bruce Edwards1, Rob Jepras2
HSC and Cancer Center, University of New Mexico,
Albuquerque, NM, United States, 2 GlaxoSmithKline,
Middlesex, United Kingdom
1
Background: Flow cytometry has long been acknowledged as a
technology unmatched in the ability to combine high-content
measurements and analytical throughput of single cells and
particles in suspension. However, only recently with the advent of
high throughput sampling technology has flow cytometry’s multiexperiment throughput begun to approach the point of practicality
for efficiently analyzing hundreds-of-thousands of samples. This
combination of both high content and high throughput has led to
the renaming of the technology as high capacity flow cytometry.
Objectives: In this workshop we are soliciting input from industrial
and academic laboratories that have experience with high
capacity flow cytometry on topics such as: assay development
and validation, techniques and tricks for efficient sample and
plate preparation, perspectives on the role of high capacity
flow cytometry in high content screening and systems biology,
recent developments in hardware and software, favorite auxiliary
equipment excluding the flow cytometer/sampling platform, and
future needs and trends.
Conclusions: We hope that attendees who do not use high
throughput/capacity flow cytometry will leave inspired to try it
and that all participants will gain new insight and ideas for both
theoretical and practical approaches. Presentations will be limited
to several slides to allow for extended discussion.
74
Mesenchymal Stem Cell Identification and
Characterization
Vera Donnenberg1, Keith Wonnacott2, Albert D. Donnenberg3,
Henning Ulrich4, Anne Plant5
1
Univ of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA,
United States, 2National Institutes of Health, Bethesda, MD,
United States, 3Univ of Pittsburgh, Hillman Cancer Center,
4
Instituto de Química, Univ of Sao Paulo, 5National Institute
of Standards and Technology, Gaithersburg, MD, United States
116
Adult mesenchymal stromal cells also known as mesenchymal
stem cells (MSC) are known for their multipotency and
immunomodulatory potential. Both native and culture-expanded
MSC are characterized by the expression of antigens such as CD44,
CD73, CD90, CD105, and vimentin, and can be isolated from
bone marrow, adipose tissue, aortic and vascular tissues. Isolated
adipose derived- as well as in vitro expanded bone marrow derivedMSC are able to differentiate into a wide variety of lineages, making
autologous MSC an attractive candidatefor a broad spectrum
of regenerative applications. Further, their immunosuppressive
potential allows the use of allogeneic as well as autologous MSC for
immune-modulation.
Despite the various clinical applications, we lack clear
definitions and standardized procedures for the identification and
quantification of MSC and evaluation of their function in order
to determine the dose, purity, sterility, safety, and the potency of
the implanted cells. Multiparameter flow and imaging cytometry
represent methodologies uniquely suited for the detection and
the monitoring of cellular products utilizing MSC. The cytometry
community is poised to develop a series of validated protocols for
MSC preparation, detection and monitoring using a cytometry panel
design, as well as protocols for inter-laboratory comparison.
Special emphasis will be taken to focus on the needs for the
development and availability of validated, robust, automatable,
GMP-compatible methods for the identification and isolation of
human MSC. Experts from the Food and Drug Administration
Cellular Therapies Branch were invited to participate, the National
Institute of Standards and Technology the cytometry community
and cellular products manufacturing and testing laboratories will be
available for a panel discussion.
The goal of this workshop is to outline a set of procedures central
to assay qualification, validation and quality assessment, as well as
training and qualification of personnel.
75
Malaria Cytometry
Howard Shapiro1, Grace Chojnowski2
1
Howard M. Shapiro, M.D., P.C., West Newton, MA, United
States, 2Queensland Institute of Medical Research, Brisbane
QLD, Australia
Malaria kills ~ 800,000 people each year; most are children under
5 and many of the rest pregnant women. Most deaths occur in
Africa and Asia and are due to Plasmodium falciparum; other
parasite species (P. vivax, P. malariae, P. ovale[2 species], and
P. knowlesi) are less deadly. Precise numbers of infections and
deaths are uncertain because diagnoses are often made without
confirming the presence of parasites in patients’ blood. Although
prompt diagnosis allows cure in a few days with inexpensive drugs,
emergence of resistant strains is a major concern. Insecticide
spraying and distribution of bed nets help control the Anopheles
mosquito vectors, and vaccines and new drugs are under
development.
Although high-power microscopy of well-prepared blood smears,
using Giemsa’scentury-old stain, remains the most reliable method
of detecting and counting malaria parasites, its success depends on
well-trained observers being able to detect subtle morphological
features of parasitized red blood cells, often a problem because
there may be only a few such cells present on a slide, making it
unlikely the microscopist will encounter even a single parasite in
the time available for observation.
Multiparameter flow cytometry, used for decades to detect other
rare cell types in blood, e.g., stem cells and circulating tumor
cells, has been viewed as too complex and costly for use in
malaria diagnosis, but has recently begun to be used for research
in malaria biology and pharmacology andin a few field studies
of parasites at low densities.Although current flow and imaging
cytometry technology promise to make apparatus substantially
ISAC 2013 Program and Abstracts
ISAC 2013 Program and Abstracts
Background: In many cancers, tumor-infiltrating lymphocytes (TILs)
indicate levels of tumor immunogenicity and are a strong predictor
of survival. In particular, increased levels of regulatory T cells
(Tregs) are associated with poorer prognosis in some cancers. An
understanding of the phenotype and spatial distribution of TILs in
situ within tumor regions would be advantageous. However, visual
TIL assessment cannot easily determine the type of lymphocyte in
situ and multimarker quantitation is difficult with standard methods.
Here we present a multi-marker, computer-aided event-counting
method for determining the phenotypes of lymphocytes in follicular
lymphoma sections using a multispectral imaging (MSI) and
automated tissue segmentation and counting approach.
Speaker/Author
Index
Introduction: Multiplexingin image cytometry remains a
challenging task to implement, although the value of multiplexed
biomarker analysis is increasingly recognized in tissue pathology,
regenerative medicine and drug screening. Conventional image
cytometers are dominantly fluorescence based systems that have
limited multiplexing capability (< 4 simultaneous measurements)
due to the relatively broad fluorescence emission bands. In
James Mansfield1, Chris van der Loos2, LS Nelson3, C Rose3, HE
Sandison3, S Usher3, JA Radford3, KM Linton3, Richard Byers3
1
PerkinElmer, Hopkinton, MA, United States, 2Amsterdam
Medical Center, Amsterdam, Netherlands, 3Pathology,
University of Manchester, Manchester, United Kingdom
Poster Session
Abstracts
Er Liu, Erika Duggan, John Nolan
La Jolla Bioengineering Institute, San Diego, CA, United States
Phenotyping Tils in Situ: Tissue Cytometric
Enumeration of Intra- and Extra-follicular Foxp3+
Regulatory T Cells in Follicular Lymphoma
Oral Session
Abstracts
Multiparameter Image Cytometry Using Surface
Enhanced Raman Scattering Tags and Spectral Imaging
78
Commercial
Tutorials &
Exhibits
77
Poster
Session
Results from studies conducted to assess the value of SCNP
in probing the immune system in different clinical situations,
including prediction of response to cancer immunotherapy (such
as ipilimumab) in patients with metastatic melanoma will be
discussed.
Conclusions: Nanoparticle SERS tags can be used for image
cytometry in either an indirect or direct immunostaining format,
in a manner that is compatible with concurrent fluorescence
staining. We have developed a set of more than a dozen spectrally
distinct SERS tags that are suitable for either spectral flow or image
cytometry, allowing for highly multiparameter data to be acquired
with a single excitation wavelength. The SERS tags are sufficiently
bright to allow high speed data aquistion (50 msec/spectra), and
imaging speed in the current system is limited by the mechanical
movement of the stage.
Wednesday,
22 May
Single Cell Network Profiling (SCNP) is an approach for analyzing
and interpreting post-translational protein modifications (e.g.
phosphorylation, acetylation etc.) at the single cell level. This
technology allows for simultaneous characterization of a range
of critical cellular signaling networks in different cell subsets in
complex tissues andthe effects of therapies on signaling networks.
Using viable cells, measurements are made on endogenous
proteins before and after exposure to extracellular modulators such
as growth factors, cytokines or drugs which are chosen to evoke
cellular responses that echo how the signaling system is normally,
or abnormally, patterned. The proteomic readout in the presence
or absence of a specific modulator is termed a “signaling node”.
Signaling nodes are evaluated within cells from samples that have
associated relevant clinical information, for instance regarding
response to the therapy of interest. Multivariate analysis can then
be performed to create predictive models that can be validated in
subsequent independent studies.
Tuesday,
21 May
Alessandra Cesano
Nodality, Inc., South San Francisco, CA, United States
Results: Indirect immunoSERS of cells labeled with primary
antibodies against individual cell surface makers showed excellent
concordance with indirect immunofluorescence (Pearson
correlation coefficient 0.88). Spectral imaging (spot scanning,
50 msec/spot) of SERS tags on antibody capture beads produced
spectral images that confirmed our ability to resolved five different
SERS tags from a single spectral image (with p<<0.05 when
comparing positively stained single SERS samples over negative
samples on the same SERS channel). Finally, we show that
multiplexed staining of cells using fluorescence markers for image
segmentation and direct SERS tag antibody conjugates enables
multiparameter image cytometry of breast cancer cell lines.
Monday,
20 May
Insights into the Immune System via Single Cell
Network Profiling: Towards Improved Disease
Classification and Therapeutic Selection
Sunday,
19 May
76
Methods: Four breast cancer cell lines (MDA-MB231, MDA-MB435,
BT474, MCF-7) were cultured, fixed and stained with monoclonal
antibodies against five cell surface markers (HER2, CD24, CD29,
CD44, and CD61) respectively, for indirect immunostaining with
DyLight 647- or SERS-goat anti-mouse antibodies, or in multiplex
with direct SERS conjugates using five spectrally distinct SERS tags.
Cell cytoskeleton and nuclei were also stained with FITC-phalloidin
and DAPI respectively. A customized spectral imaging system was
used to collectfluorescence/SERS images for blue (DAPI), green
(FITC) and far red (DyLight647 or SERS) emission using band pass
filters and high resolution spectral image data for red (633 nm)
excited SERS. Classical least squares spectral unmixing (Matlab)
was used to reconstruct images of individual SERS tag staining
from the spectral image data, using reference spectra obtained
from antibody-funtionalized SERS tags bound to antibody capture
beads. Image segmentation (Cell Profiler) using the nuclear (DAPI)
and cytoskeletal (FITC-phalloidin) markers was used to identify
individual cells and the median pixel intensity per cell for the
immunofluorescence, immunoSERS, and unmixed spectral images
was calculated (FCS Express Image).
Saturday,
18 May
At the 2013 workshop, we will present results of collaborative
attempts to develop protocols suitable for most widely used types
of flow cytometers; the finalized protocols will appear in Current
Protocols. The workshop will discuss parameters and probes for
malaria cytometry, notably DNA content and base composition and
RNA content and content of the malaria pigment hemozoin, and
how they can be measured and used in malaria detection, species
identification, and determination of developmental stages and
drug effects. Instrumental modifications necessary for hemozoin
detection will be discussed, as will the possible configurations of
simple multiparameter flow and image cytometers suitable for work
in the field.
the present work, we used surface enhanced Raman scattering
(SERS) tags and multispectral Raman imagingformultiplexed
immunolabeling for image cytometry.
Special
Lectures
The inaugural Malaria Cytometry Workshop at CYTO 2012
represented the first formal collaboration among several laboratories
active in the field, providing core managers, researchers,
and clinicians with an overview of the principal cytometric
characteristics of malaria parasites and the problems that need to
be solved to allow cytometry to play a role in the fight to eradicate
the disease. The 2012 Workshop organizers have produced an
Overview chapter, now in press, for Current Protocols in Cytometry.
Congress
Overview
more affordable and accessible, in order for cytometry to become
useful in malaria research and diagnosis, it will be necessary to
bring malaria researchers and clinicians unfamiliar with cytometry
together with cytometrists unfamiliar with malaria.
117
Congress
Overview
Special
Lectures
Saturday,
18 May
Conclusion: Understanding the number and location (intra- and
extra-follicular) of Tregs is an assay with potentially important
clinical prognostic implications. Thus study shows that an
automated method for counting Tregs can be developed for
follicular lymphoma. This multimarker phenotyping and counting
approach shows the potential for broad applicability in the
enumeration of a wide range of specifically phenotyped TILs in situ
in many solid tumors.
Monday,
20 May
Results: Results indicate that machine-learning software can be
trained to accurately recognize follicular and non-follicular regions
within each core. MSI enabled the accurate quantitation of two
immunostains in the sample without crosstalk. The number of Tregs
were determined for each core and ranged from 0 to 453 per core
(median 58, stddev 97.5).
Sunday,
19 May
Methods: A single section of a tissue microarray containing 70
follicular lymphoma cores from [42 male, 28 female, 17 alive
and 53 dead at the end of followup, survival range 2-171 months
(median 55 months)] was stained for CD3, Foxp3 and hematoxylin.
Each core was imaged using MSI and the individual staining of each
marker separated from each other using spectral unmixing. The
images were analyzed using software which had been trained to
recognize the follicular areas based on the tissue morphology. Then
the Foxp3 status of each CD3+ TIL was then determined and the
number of each Treg (Foxp3+ T cell) counted for both the intra- and
extra-follicular tissue compartments.
79
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Biophotonic Nanoswitches – Light-Activation of the
BH3 Pathway in Cellular Systems
Rachel Errington , Robert Mart , Sally Chappell , Catherine
Watkins1, Marie Wiltshire1, Arwyn Jones3, Paul Smith1, Rudolf
Allemann2
1
School of Medicine, Cardiff University, Cardiff, United
Kingdom, 2School of Chemistry, Cardiff University, Cardiff,
United Kingdom, 3 Cardiff School of Pharmacy and
Pharmaceutical Sciences, Cardiff University, Cardiff, United
Kingdom
1
2
1
Background: The intrinsic apoptosis pathway is regulated by
the interaction of pro- and anti-apoptotic members of the Bcl-2
family of proteins. These interactions are mediated through highly
conserved alpha-helices and corresponding grooves. The design,
synthesis and characterization of biophotonic nanoswitches
derived from Bcl-2 helices have been achieved. The conformation
of these peptides and hence their activity in vitro and in vivo can
be controlled with external light pulses. Peptide behavior was
explored in a highly accessible lymphoma cell system compatible
with conventional probes for monitoring mitochondrial health and
cell cycle status. We demonstrate that fine temporal control can
be achieved with such peptide-based biophotonic nanoswitches
over early events in the commitment to cell death. The irreversible
nature of the apoptosis pathway suited a ‘switch-on’ peptide
system where apoptosis is induced by photoswitching rather than
a ‘switch-off’ approach, where apoptosis onset is merely delayed,
and once committed cannot be retrieved, this gives a convolution
that is difficult to decipher and quantify against a biophotonic
manipulation.
Methods: Switch-on - Peptides (eg Ac-BakI81Fi,i+7-XL) were
synthesized by standard solid phase Fmoc chemistry followed by
intermolecular azobenzene cross-linking. InCell measurements We have studied human tumor cells with different p53 signaling
capacity (SU-DHL-4; DOHH2 cells) that thus show different
primed classification and sensitivity for the triggering of the
intrinsic pathway for apoptosis by DNA damage. We used JC-1
and DRAQ7™ to dissect the early apoptotic events. The time
dependent cytochrome C release was monitored by single cell
analysis (image and flow). Permeabilized cells in a respiration buffer
were used to test the switches in vivo thus removing the burden of
118
peptide cargo delivery. UV/VIS spectra of peptide at 360nm were
obtained using Nanodrop 2000.
Results: To calibrate the relationship between the prevailing
apoptotic trigger and the time-dependent restraint on proliferation
we have used human lymphoma cell lines, assessed for their BCL-2
family/p53 preset for rapid mitochondrial membrane collapse using
the BH3-mimetic ABT-737. We then profiled the BH3 trigger in
permeabilized tumor cell systems using the fully-stapled or photoresponsive peptides designed to impose time-limited targeting of
critical Bcl-2 family components. Our photoswitcheable peptides,
derived from the pro-apoptotic Bak and Bid were used to target the
anti-apoptotic Bcl-xL in vitro where a 20-fold increase in binding
affinity to ~40 nM was observed for the light-activated state, which
reverted to the dark-adapted state with a half-life of 20 minutes
at 37 oC. We show the switches in action causing mitochondrial
membrane potential collapse and cytochrome C release at the
single cell level, the threshold for which is more sensitive in
DOHH2 cells, with unique patterns of mitochondrial recruitment
and breakdown. Photoswitched Ac-BakI81Fi,i+7-XL showed consistent
cell cycle specific patterns of activity with different apoptotic
triggering activity in cells in G2 phase relative to G1 with
implications for the resistance to cell death triggering induced by
cell cycle specific cytotoxic agents.
Conclusion: Peptides provide real benefits over small molecules,
in that it is possible to control their activity. It should allow us to
shape the delivery of active molecules in a spatio-temporal context.
80
Quantitative Co-imaging of Activated Signaling
Proteins and Downstream Individual Transcripts in
Breast Cancer Single Cells
Sunjong Kwon1, Michel Nederlof2, Koei Chin1, Joe Gray1
Biomedical Engineering, Oregon Health & Science University,
Portland, OR, United States, 2Quantitative Imaging Systems,
Inc., Pittsburgh, PA, United States
1
Backgrounds: The Akt and ERK pathways are important signaling
cascades in cancer biology and their aberrant signaling are involved
in changes of cell survival, proliferation, and differentiation in
various malignancies. Simultaneous visualization of these pathways
activation and subsequent gene expression in single cells is needed
to understand sensitivities and responses to drug treatment in
cancer cells with cell-to-cell variability. The expression level of Fra1(Fos-related antigen 1, FOSL1) is highly related with proliferation
and invasiveness of breast cancer cells. Akt or ERK signaling has
been reported involved in Fra-1 expression, but it has not been
determined in breast cancer cells.
Methods: To address if Fra-1 expression is regulated by Akt or ERK
signaling activation in breast cancer cells in situ, we simultaneously
visualized and quantified both pAkt/pERK levels and individual
Fra-1 mRNA particles in MCF7 breast cancer cells, combining
immunocytochemistry (ICC) and single-molecule fluorescent in situ
hybridization (smFISH), called as “immuno-smFISH”.
Results: Intensity of pAkt was clearly promoted with insulin
treatment and Fra-1 mRNA particles increased peaking with 2 hr
treatment, suggesting that Fra-1 is an Akt-inducible gene in breast
cancer cells. However, inhibition of PI3K or MEK signaling by
specific drug treatment abrogated increased Fra-1 mRNA level by
insulin or EGF treatment, indicating that upstream of not only Akt
but ERK signaling regulate mRNA level of Fra-1. Unexpectedly,
treatment of Akt inhibitor facilitated membrane localization of pAkt
and did not affect significantly upregulated Fra-1 expression by
insulin or EGF treatment, confirming that Fra-1 expression is not
solely dependent on Akt signaling.
Conclusions: Our immuno-smFISH could be applied to pinpoint
target cancer cells of aberrant signaling and subsequent end-point
gene expression in human tumor biopsy samples and xenograft
tissues.
ISAC 2013 Program and Abstracts
82
Standardisation of Whole Blood Immune Phenotype
Monitoring for Clinical Trials: Panels and Methods
from “The One Study”
The Cytokine Secretion Assay Reveals an Analog to
Digital Switch in Cytokine Expression
Conclusions: Our results indicate that NFAT is the decisive
molecular switch which transforms the analog TCR signal into a
digital, all-or-non decision for IL-4 expression in Th2 memory cells.
Wednesday,
22 May
83
Determination of Antigen Localization and Trafficking
when Targeted to C-Type Lectin Receptors using
Human Antibody-Antigen Fusion Protein Constructs
Harboring Specificity to CD205 or CD206
Poster
Session
Joseph Tario, Jr. 1, Tibor Keler2, Paul Wallace1
Flow and Image Cytometry, Roswell Park Cancer Institute,
Buffalo, NY, United States, 2Celldex Therapeutics, Needham,
MA, United States
1
Commercial
Tutorials &
Exhibits
The use of antibodies to target tumor antigen directly to antigen
presenting cells is a promising immunotherapeutic intervention
that has been employed with increasing frequency. Recently, two
antibody-based antigen targeting vehicles (clones B11 and 3G9)
have been developed, which harbor specificity for the mannose
receptor (MR; CD206) and DEC-205 (CD205), respectively. Both
targets are Type 1 C-type lectin receptors which are expressed on
monocyte-derived dendritic cells (DCs). Several investigations
have demonstrated that when antigen is genetically coupled to
B11 or 3G9, the resultant fusion proteins (FPs) can be bound and
internalized by DCs, which in turn, process the targeted antigen
and present it to reactive T cells; eliciting both Class I and Class
II mediated responses using in vitro and in vivo models. It was
recently demonstrated that similar levels of cross presentation
resulted from the targeting of the tumor-associated antigen
NY-ESO-1 to DCs via either FP clone. This immunologicallyadvantageous result suggests that both FPs might be similarly
processed by DCs; however this hypothesis is not supported by
the published literature, which reports that internalized CD205
and CD206 traffic to the lysosome and early endosome (EE),
respectively.
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Results: A transcription factor complex composed of RNA
polymerase II, NFAT, p65, c-Maf, p300, Brg1, STAT6 and GATA-3
assembles at the Il4 promoter in dependence of T cell receptor
(TCR) activation, but only in Th2 cells expressing IL-4 and not in
Th2 cells not expressing IL-4. Pharmacological inhibition of the TCR
signaling transducer NFAT inhibited transcription factor complex
assembly at the Il4 promoter. Grading of NFAT inhibition resulted
in the modulation of the number of IL-4 producing Th cells, but did
not alter the amount of IL-4 produced per cell.
Tuesday,
21 May
Conclusions: Local performance and central analysis of our ONE
Study flow cytometry panels in an international multi-centre cell
therapy trial yields acceptable variability for statistical comparisons.
These panels and procedures with WB (vs. frozen PBMCs) allow
unmanipulated analysis of changes in absolute cell numbers
of subsets, that could generally be useful for all laboratories
performing immune monitoring for either multi-centre or local
clinical trials.
Monday,
20 May
Results: WB age testing revealed that staining must be performed
within 4 hours of sample collection to keep variability low. In
particular, the variability of monocyte counts and their subset
composition exceeds 20% if samples are stained at later time
points. Inter-assay analyses revealed a variability of 0.05 to 20%,
depending on the frequency of the particular subset, with smaller
subsets showing higher variability. The inter-assay variability
performance defined cut-offs for each recorded subset, which is
the basis for assessing statistically significant differences achieved
by the different cell therapies. Inter-lab comparisons between all
participating centres, upon target file transfer and WB shipment,
revealed good precision, with a variability of 0.05 to 30%.
Methods: We have used the IL-4 cytokine secretion assay, which
allows the identification and isolation of viable IL-4 secreting cells
to segregate Th2 cells which express or do not express IL-4. In order
to analyze the molecular requirements for the reexpression of IL-4
in Th2 cells we have characterized the transcription factors bound
to the Il-4 promoter by chromatin immunoprecipitation (ChIP)
assay.
Sunday,
19 May
Methods: We designed six 7-to 9 -colour leukocyte profiling
panels capturing the following characteristics: 1) normal immune
phenotyping, 2) T cells subsets (αβ/γδ), 3) T cell activation, 4)
T effector/memory and Tregs, 5) B cells and 6) dendritic cells.
Standardisation was assured by applying the following strategy: age
of blood (0 vs 4 and 24 hours), age of stain (0 vs 4 and 24 hours),
intra-assay and inter-assay variability (WB staining: three healthy
individuals on three consecutive days), PMTs were defined using
Flow- set Pro beads and fluorescent target files were transferred to
participating centres, all equipped with a Navios flow cytometer
(Beckman Coulter).
Background: Naive T helper (Th) lymphocytes develop a memory
for interleukin-4 (IL-4) expression when activated in the presence of
IL-4, i.e. in later reactivations of these Th cells, re-expression of IL-4
occurs independently of instructive costimuli. They have become T
helper type 2 (Th2) cells. However, not all Th2 cells with a memory
for IL-4 reexpress IL-4 in a given reactivation. Here, we have analyzed
the molecular requirements for the reexpression of IL-4 in Th2 cells.
Saturday,
18 May
Objective: Immune monitoring via flow cytometry is critical
for studying effects of novel therapeutics aimed at e.g. reducing
transplant rejection or autoimmune disease. We initiated an
international trial through a consortium called The ONE Study
(www.onestudy.org), aimed at using different cell therapies to
promote tolerance to renal allografts. To compare the effectiveness
of different cell therapies, our consortium developed a robust
immune monitoring panel and procedures for whole blood (WB)
leukocyte profiling.
Hyun-Dong Chang, Juliana Koeck, Farahnaz Hatam, Markus
Bardua, Stephan Kreher, Hanna Bendfeldt, Ria Baumgrass,
Andreas Radbruch
Deutsches Rheuma-Forschungszentrum Berlin, Berlin,
Germany
Special
Lectures
Mathias Streitz1, Tewfik Miloud2, Michael Kapinsy3, Michael
Reed4, Edward K. Geissler5, James Hutchinson5, Katrin Vogt1,
Stephan Schlickeiser1, Anders H. Kverneland1, Christian
Meisel1, Hans-Dieter Volk1,6, Birgit Sawitzki1,6
1
Medical Immunology, Charite - Universitaetsmedizin Berlin,
Berlin, Germany, 2Immunotech S.A.S., Marseille, France,
3
Beckman Coulter GmbH, Krefeld, Germany, 4Beckman Coulter
Inc, Miami, FL, United States, 5University Hospital Regensburg,
Regensburg, Germany, 6Berlin Brandenburg Center for
Regenerative Therapies, Charite - Universitaetsmedizin Berlin,
Berlin, Germany
Congress
Overview
81
119
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
To explain this discrepancy, we predicted that the binding of clone
3G9 to a unique epitope on CD205 modulates its constitutive
trafficking pattern after internalization. We have employed confocal
microscopy to measure the localization and trafficking of each
internalized FP in monocyte-derived DCs, to determine whether
their trafficking pathways intersect. Our hypothesis is supported by
experiments which measured the prolonged antigen persistence of
both FP in pulsed immature and matured DCs.
Our measurements indicate that both internalized FPs traffic
to Class I-containing, EEA1-positive early endosomes where
they persist for up to 48 hours in viable DCs. The observed
colocalization of B11-FP and 3G9-FP was unexpected, and
provides evidence that the binding of 3G9-FP to CD205 modifies
the canonical trafficking of internalized DEC-205 receptor,
committing it to EE instead of to the lysosome for antigen processing
and presentation. In concordance with our prior findings, and
with antecedent reports from the literature; we conclude that
both FPs traffic to the EE, which serves as a reservoir for antigen
storage that promotes the cross presentation of targeted antigen.
Additionally, we conclude that consistencies in the trafficking of
both FPs explains the similar levels of cross presentation that have
been observed in models where the same antigen is targeted to DCs
using either clone B11 or 3G9.
84
Dissection of Anti-CTLA4-Induced Cytotoxic T Cell
Responses in Melanoma
Pia Kvistborg1, Daisy Philips1, Sander Kelderman1, Bianca
Hemskerk1, Christian Ottensmeier2, Daniel Speiser3, Christian
Blank1, John Haanen1, Ton Schumacher1
1
Department of immunology, Netherlands cancer institute,
Amsterdam, Netherlands, 2Southampton University Hospital,
Southsampton, United Kingdom, 3Ludwig cancer center,
Lausanne, Switzerland
There is strong evidence that melanoma-reactive T cell responses
induced by immunotherapeutic interventions such as anti-CTLA4
(Ipilimumab) treatment can exert clinically meaningful effects.
However, there is very little information on how these therapies
influence tumor-specific T cell responses. Furthermore, as the
number of potential melanoma-associated antigens to which
these responses can be directed is very high, classical strategies to
map cytotoxic T cell reactivity do not suffice. Knowledge of such
reactivities would be useful to design targeted strategies, selectively
aiming to induce immune reactivity against these antigens.
We have addressed these issues by designing MHC class I
molecules occupied with UV-sensitive ‘conditional’ peptide ligands,
thereby allowing the production of very large collections of pMHC
complexes for T cell detection. Secondly, we have developed a
‘combinatorial coding’ strategy that allows the parallel detection of
dozens of different T cell populations within a single sample. The
combined use of MHC ligand exchange and combinatorial coding
allows the high-throughput dissection of disease- and therapyinduced CTL immunity. We have used this platform to monitor
immune reactivity against a panel of 145 melanoma-associated
epitopes in patients receiving Ipilimumab treatment.
Comparison of PBMC samples from 32 melanoma patients preand post-therapy indicated a significant increase in the number of
detectable melanoma-associated CD8 T cell responses (p=0.004).
Furthermore, kinetic data on T cell responses during Ipilimumab
therapy suggests that this broadening generally occurs within
weeks after start of therapy. The pattern of reactivities detected
is highly patient specific, and this is most pronounced for
reactivities directed against cancer/testis antigens. The magnitude of
melanoma-specific T cell responses that was detectable prior to start
of therapy was not significantly altered (p=0.8).
These results establish the pattern of melanoma-specific T-cell
reactivity induced by anti-CTLA4 treatment and form a benchmark
120
for evaluation of other immunotherapeutic interventions, like antiPD1 treatment, that are currently undergoing clinical evaluation.
Furthermore, our data suggests that the clinical activity of
Ipilimumab may be mostly due to epitope spreading, rather than
through enhancement of pre-existing immune activity.
85
Flow Cytometric Analysis of Single Lipid Membrane
Vesicles
Samuel Stoner, John Nolan
La Jolla Bioengineering Institute, San Diego, CA, United States
Background: Cell-derived lipid membrane vesicles (ectosomes,
exosomes, and apoptotic bodies) have been implicated in a
wide varietyof important biological roles. These includecell-cell
signaling, antigen presentation, and transfer of genetic material.
It has been proposed that these microvesicles may serve as useful
biomarkers for a number of conditions including cardiovascular
disease, autoimmune disease, and cancer. These vesicles, which are
thought to range in size from 50-500nm, are present in a number of
biological fluids and are commonly analyzed via conventional flow
cytometry. However, their small size and low refractive index makes
detection and analysis of single vesicles extremely difficult and
prone to misinterpretation of results, especially when using light
scatter as a trigger parameter. The lack of appropriate standards for
analysis of these particles only adds to this difficulty.
Methods: Here we have developed a nanoparticle flow cytometer
(NPFC) optimized for high-sensitivity fluorescence-based detection
of single lipid membrane vesicles. The instrument performance was
characterized in terms of Q, B, and resolution limit. Liposomes
of defined composition were prepared by extrusion through
membrane filters ranging in size from 50-400nm to use as controls
for flow cytometry microvesicle experiments. Multiple lipophilic
dyes (PKH67, DiOC6(3), and di-8-ANEPPS) were characterized
for their ability to stain these liposomes, and a protocol for
subsequent detection in the NPFC was developed and extensively
characterized. Cell-derived microvesicles were generated from
cultured THP-1 monocytes via stimulation with ionophore A23187,
and single particles were stained and analyzed in the NPFC.
These vesicles were also assessed for a variety of surface markers
including phosphatidylserine (PS, using annexin V), CD14, and
tissue factor (TF).
Results: Characterization of the NPFC found a 5-10 fold
improvement in fluorescence detection efficiency (Q) compared
to a commercial instrument, which resulted in resolution limits
(separation index = 1) of 230 MESF FITC and 71 MESF PE.
Detection of individual liposomes was successfully triggered based
on fluorescence signal from lipophilic dyes, and analysis of different
liposome size populations showed fluorescence intensity to be a
good marker of vesicle surface area.Serial dilution of the liposomes
resulted in decreased event rates, but no change in median
fluorescence intensity, indicating that their measurement was not
compromised by coincidence. Characterizationof liposomes over
time revealed them to be stable for greater than 4 months at 4°C,
and thus potentially useful reference standards.Assessment of PS
composition of THP-1 derived vesicles via annexin-V staining
revealed two distinct populations of PS+ and PS- particles.
Interestingly, assessment of othersurface markers revealed the
presence of both TF and CD14 on single vesicles to fall below the
detection limit of even our high sensitivity instrument.
Discussion: Microvesicles present in biological fluids are likely to
be heterogeneous with respect to cell of origin and composition,
making the analysis of single microvesicles essential.Here we
demonstrate the usefulness of optimized instrumentation and
fluorescence-based triggering for the detection of individual small
lipid vesicles. We believe this approach is a first step towards
both more rigorous characterization of vesicle populations and
standardization of vesicle measurements via flow cytometry.
ISAC 2013 Program and Abstracts
Novel Approach for Characterizing Circulating
Microparticles Using Imaging Flow Cytometry
Extracellular Vesicles from Plasma of Healthy
Donors, Characterized by Cryo-Electron Microscopy,
Receptor-Specific Gold Labeling and Flow Cytometry
Alain Brisson, Nicolas Arraud, Romain Linares, Sisareuth Tan,
Céline Gounou
CBMN, University of Bordeaux, Bordeaux, France
Poster Session
Abstracts
Extracellular vesicles (EVs) derived from activated or apoptotic
cells are found in plasma and other body fluids in physiological
conditions and are present at elevated levels in various diseases,
including cardiovascular diseases and cancer. EVs present surface
receptors originating from their parental cells, which has promoted
the concept of using EVs as biomarkers of diseases. However,
research on EVs is rendered difficult by their small size and the
limitations of analytical methods currently in use.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
88
Oral Session
Abstracts
Background: Microparticles (MP) are small vesicles of 0.1-1µm
released by cells of almost all origins in response to activation
or apoptosis. Apart from normal blood cells, tumor cells are
also (or even more) prone to generate MP. These may be found
in circulating blood/plasma and behave both as biovectors of
Commercial
Tutorials &
Exhibits
Philippe Poncelet1, Jérémie Bez1, Tarik Bouriche1, Wolfram
Ruf2
1
R&T, BioCytex, Marseille, France, 2Immunology and Microbial
Science, The Scripps Research Institute, La Jolla, CA, United
States
Poster
Session
Study of Potential Markers for Tumor-Derived Mp in
an in Vitro Model of Spiked Whole Blood
Results: All three major subsets of MP were clearly discriminated
among AnnV+ MP with a majority of (CD41+) PMP which intensely
co-stained for CD9 and CD61+, as expected. CD15+ Leu-MP
mostly co-stained with CD11b and CD66abe. In the highest
spiking level the major fraction of Epcam+ (CD326) and EGFr+
were found mostly out of all major MP subsets, thus suggesting the
presence of tumor-MP that can be defined as AnnV+/ Epcam+/ EGFr+
(CD15neg/CD41neg/CD235aneg) MP. This multi-color approach
also delineated a fraction of CD9+ but CD41neg, CD66abe+ but
CD15neg and CD61+ but CD41neg that may also be part of the
tumor-MP subset. Although part of the faint albeit detectable TF
(CD142) staining came along with PMP, a significant part was also
associated with the lineage neg tumor-MP subset, in agreement
with the major TF-dependent FXa generation activity measured in
the highest level of spiking. Conclusion: Tumor-derived MP can be
generated in whole blood in vitro following incubation of tumor
cells for a few hours at 37°C. These Tumor-MP mainly display a
specific phenotype with absence of the hemopoietic lineage-related
markers CD15, CD41 and CD235a and expression of some cancerassociated markers including EGFr, Epcam and TF.
Wednesday,
22 May
87
Tuesday,
21 May
Conclusions: By combining flow cytometry with imaging we were
able to demonstrate improved detection, phenotyping and absolute
counting of MPs, while also providing morphological confirmation
and the ability to distinguish true single events from cell debris
or particle aggregation. Evaluating MPs below 200nm and sizing
remain a challenge as some MPs remain below the detection
threshold of the ImagestreamX imaging system and standardized
biological calibrators remain to be developed.
Monday,
20 May
Results: Although both standard and imaging flow cytometry could
resolve all latex bead populations, only the ISX could resolve the
two smallest bead population above the background noise of the
instrument using scatter parameters. The more biologically relevant
0.2u liposomes had approximately 10 fold less scatter intensity
than the smallest latex bead (0.22u) by imaging flow cytometry and
were not easily resolvable above background using scatter alone.
However, in contrast to standard flow cytometry, could be easily
resolved when combined with either brightfield or fluorescent
parameters. Labeled MP preparations were easily identified using
scatter intensity profiles by both methods, however there were
major differences in numbers and phenotypical profiles. The ISX
provided the possibility of identifying MP aggregates as false double
positive stains and cellular debris from true MPs, whereas standard
flow cytometry was unable to distinguish these types of events.
Sunday,
19 May
Materials & Methods: Flow cytometry is the most commonly used
technique, however because of the small size of MPs and the
limitations of current flow cytometry instrumentation in measuring
biological particles in these size ranges accurate measurements are
hampered by this methodology. We investigated whether the use
of flow cytometry combined with imaging, such as is possible with
the ImagestreamX imaging flow cytometer, would be a more robust
approach to characterizing circulating MPs. A comparison between
the two platforms was conducted utilizing latex nanoparticles (0.221.3u), liposomes (0.2u), and microparticle preparations stained with
Annexin V, CD41, CD45, and CD235a.
Methods: A human colon cancer cell line was modified to express
high TF density (>200,000 TF/cell) and generate tumor-MP with
extremely high TF-dependent procoagulant activity, as assessed
with a Factor Xa generation assay applied on MP purified by highspeed centrifugation steps (24,000g – 1h). After detachment these
CY-1 cultured tumor cells were spiked into citrate anti-coagulated
blood (transfusion bags from blood bank) at different levels (10 to
1,000 cells/µL) and incubated with gentle agitation for 3h at 37°C.
PFP were prepared by centrifugation (2500g 15mn twice), aliquoted
and frozen at -80°C for further analysis in either FCM or purified
MP-associated FXa generation assay. Standardized FCM analysis
was operated on a 3-laser Beckman-Coulter Gallios instrument
using a cut-off set at 0.3 µm-eq. with the help of Megamix-Plus FSC
beads (BioCytex). A 5-color IF protocol using minimal fluorescence
compensation was set-up to delineate MP subsets using AnnexinVFITC (for PS+ MP), CD41- PC7 (for PMP), CD235a-APC-A750
(with red laser excitation for Ery-MP), C15-Pacific Blue (with
violet laser excitation for myeloid Leu-MP) and the PE channel for
all PE-conjugated MAbs tested in parallel with the above-cited,
subset-specific, cocktails. Tested MAbs included HLA-DR, CD9,
11b, 24, 61 (GPIIIa), 66abe (NCA, CEA), 87 (uPAr), 142 (TF), 324
(E-cadherin), Epcam (CD326) as an epithelial origin marker and
EGFr as a tumor-associated marker.
Saturday,
18 May
Background: Microparticles (MPs) are submicron vesicles released
from cell membranes in response to activation, cell injury or
apoptosis. Circulating MPs in blood are believed to express proteins
and phospholipids associated with their parent cells of origin and
have become important biomarkers in many diseases. As such,
not only has the quantification of these MPs become of interest,
but also the characterization of their size and source has become
equally important. The clinical importance of evaluating MPs has
become increasingly recognized, although no standardized method
exists for their measurement and/or characterization.
oncogenic, angiogenic, procoagulant and/or matrix degradation
potential as well as cell-borne biomarkers for otherwise hidden
tumors. Since tumor-MP may harbour some but not all of the cellsurface antigens expressed by the tumor cells of origin, prior in
vitro studies using cancer cell lines may help optimizing detection
strategies before going to real blood/plasma samples from cancer
patients. We wanted to define whether tumor-derived MP appear as
an individualized MP subset or if they may mix-up with either PMP,
Ery-MP or Leu-MP.
Special
Lectures
Joanne Lannigan1, Christine Rudy2, Uta Erdbrugger2
Microbiology/Immunology/Cancer Biology, University of
Virginia, Charlottesville, VA, United States, 2Nephrology,
University of Virginia, Charlottesville, VA, United States
1
Congress
Overview
86
121
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
Our overall aim is to improve our basic understanding on EVs
by providing catalogs describing EVs’ size, morphology and
phenotypes in physiological and pathological situations, and to
develop reliable methods of EV detection and quantification.
In this initial study, we focused on EVs from platelet-free plasma
(PFP) samples of healthy donors and on EVs from activated platelets.
Cryo-Electron Microscopy (EM) was used to reveal the morphology
and size of the various types of particulate material present in
pure plasma. Gold nanoparticles functionalized with various types
of ligands were developed and used for labeling selected subpopulations of EVs, principally EVs exposing phosphatidylserine
(PS), platelet-derived EVs and red blood cell-derived EVs, using
Annexin-5 (Anx5), anti-CD41 and anti-glycophorin (Abs),
respectively. In parallel, flow cytometry analysis of PFP EVs was
performed, focusing on Anx5-binding EVs.
PFP samples contain three main morphological types of EVs,
consisting of spherical EVs, of tubular-shaped EVs and of large cell
fragments. Spherical EVs constitute about 75% of all EVs present
in PFP, most of them ranging in diameter from 50 nm to 500 nm.
Tubular EVs form about 15% of all EVs, with a lengthextending over
5 µm, while large cell fragments extend up to 10 µm. The majority
of EVs are smaller than 500 nm, while 10% to 20% EVs are larger
than 1 µm. Strikingly, we found that, in PFP, the sub-population
of EVs that expose PS and bind Anx5 comprises only 25% of all
EVs. We found also that about 20% of PFP EVs expose CD41,
while most of the large cell fragments derive from red blood cells.
Size histograms of the whole EV population, as well as of the subpopulations of PS-positive, CD41-positive and glycophorin-positive
EVs were determined, for the first time. In addition, the influence of
aging, centrifugation and freeze-thawing on EVs structure and PSexposure was investigated, showing a direct influence of some of
these parameters on PS-exposure.
This study provides novel basic knowledge on plasmatic EVs
and will serve as a reference for characterizing EVs in various
pathological situations.
89
High Throughput Drug Screening
J. Paul Robinson1, Valery Patsekin2, Padma Kumar Narayanan,
Nianyu Li3
1
BMS/BME, Purdue University, West Lafayette, IN, United
States, 2Purdue University, West Lafayette, IN, United States,
3
Amgen, Seattle, WA, United States
The core philosophy of high content screening (HCS) is the ability
to rapidly transform large quantities of raw data sets into highly
reduced representations of the original biological problem. Many
cell based screening assays currently involve automated image
based systems that use image analysis techniques to reduce
complex data into meaningful phenotypic representations of the
biological problem being screened. This is a complex processes
that uses best-fit equations or processes that are not excessively
computationally time consuming. The result is a large number of
derived parameters from which decisions are made.
Multiparameter drug screens of cells can be also be achieved using
high speed flow cytometry. It is in fact, highly competitive from a
time perspective – less than 10 minutes per 384 well plate with
data reduction taking a few minutes at most. Our laboratory has
developed a set of tools that are an essential paradigm shift for flow
cytometry analysis. The process is entirely graphical, interactive,
rapid and reductive producing very specific results. For example, if
the normal pipeline for a screen typically concludes with generating
an easy-to-interpret metric such as an IC50, this must be capable
directly from flow cytometry listmode dataset of any size (e.g. 96
well plate, 384 well plate, or 1536 well plate) in real time.
Using familiar concepts of automated data processing, high
throughput flow cytometry provides an opportunity to supplant
some HT imaging approaches by offering very high but different
122
content at the same or faster speed. When we add the automated
data analysis algorithms to multifactorial screen design, our
approach delivers a dramatic increase in the throughput and
information content. There are also new opportunities to explore
automated classification of subpopulations, and use of advanced
statistical machine learning that operate well within the new
paradigm. The result is rapid identification of phenotypic changes
in multiple cellular subsets producing traditional or new measures
of differentiation within a drug screening network.
90
"Deep Blue" Lasers for Detecting Low-Level Cyan
Fluorescent Protein Expression: An Example of
Optimizing Excitation Conditions for Maximum
Probe Sensitivity
William Telford1, Jane Hu-Li2, Veena Kapoor1, Nga Hawk1,
Brigitta Mester3, Alfonso Schmidt3, Ryan Kyle3, Kylie Price3,
Graham Le Gros3
1
National Cancer Institute, Bethesda, MD, United States,
2
National Institute of Allergy and Infectious Diseases, bethesda,
MD, United States, 3Malaghan Institute of Medical Research,
Wellington, New Zealand
Modern flow cytometers can be equipped with single wavelength
lasers than span the entire visible spectrum, theoretically allowing
the excitation of almost any fluorescent probe. Despite this
flexibility, most benchtop cytometers remain limited to four or
fewer excitation wavelengths, requiring some important fluorescent
probes to be excited under suboptimal conditions. A common
example of this problem are the cyan fluorescent proteins, which
are commonly used in combination with Green Fluorescent Protein
(GFP) and the red fluorescent proteins to measure multiple gene
expression events by flow cytometry simultaneously. Enhanced
Cyan Fluorescent Protein (eCFP), the Cerulean series and AmCyan
are several common examples. These proteins are traditionally
excited using a violet laser diode, emitting at approximately 405
nm. However, their excitation maxima lies in the 430 to 440 nm
range, suggesting that violet diodes actually make suboptimal
excitation sources for these proteins. Blue laser diodes, which
emit in the 440 to 450 nm range, have recently become available
as excitation sources on some high-end flow cytometers; however,
they remain uncommon despite their relatively low cost. Cyan
fluorescent proteins in fact have relatively low quantum efficiencies
and are not very “bright” relative to other fluorescent proteins;
using a spectrally optimal laser source should be crucial for good
detection, especially in systems with low expression levels.
Several previous studies have shown that blue laser diodes can
excite cyan fluorescent proteins at substantially higher levels than
violet diodes. In this study, we confirm that blue laser diodes excite
eCFP and Cerulean at levels 5- to 10-fold higher that power matched
violet laser diodes. DPSS deep blue lasers with a similar but slightly
longer emission in the 457 to 460 nm range were also evaluated
and also provide substantially better excitation than a violet source.
In two transgenic mouse systems with inducible expression of
very low-level Cerulean and AmCyan expression, the fluorescent
proteins were in fact not detectable at all using violet lasers, but
could be clearly distinguished using blue diode and DPSS sources.
Excitation and detection were therefore not merely improved, but
made detection of the cyan fluorescent protein possible.
While critical for success, an optimal excitation source must be
coordinated with the appropriate detection optics, and must take
into account the other elements of the flow cytometer. Detection
of cyan fluorescent proteins is complicated by a fluorescence
emission range (480-510 nm) that is close to the 488 nm laser
line, with elevated background noise in a region of the spectrum
that already possesses strong cellular autofluorescence. It is also
necessary to exclude the blue laser light from the detection optics,
and to minimize spectral overlap with GFP, which is also somewhat
ISAC 2013 Program and Abstracts
Results: For purposes of testing and comparison, we obtained
a series of anti-human-CD8 antibodies conjugated to 24 dyes
commonly used for immunofluorescence (26 reagents, 2 clones).
Using a set of reagents that label the same cell marker allows us to
compare saturation staining levels of different anti-CD8 conjugates
on cells with the signal tradeoff evaluations on the beads. We can,
for example, see if the density of binding sites on the beads is high
enough to lower the binding potential for antibodies conjugated
with bulky dyes like PE.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
How the reagent evaluations function in CytoGenie AutoColor will
be covered in a presentation by Wayne Moore.
Conclusions: Pending further evaluation of the variability of
the method, complementary binding provides a well-defined,
reasonably accurate and reasonably general way to assign
quantitative scales to measurements of fluorescent antibodies and to
use reagents with different dyes to evaluate and compare antibody
binding levels. This procedure should allow ordinary laboratories
using commercial reagents to make quantitative evaluations of any
antibodies bound by capture microspheres.
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Estimates of the typical brightness for reagents coupled to different
dyes (as photoelectron signal per antibody molecule) are needed
in the CytoGenie AutoColor system for predicting how well
various reagent-dye combinations will work in multi-color staining
experiments. Our evaluations are being run on an instrument with
known photoelectron scales on all fluorescence channels making
it possible to compare the effective brightness of similar amounts
of different reagents in terms of the numbers of photoelectrons
generated.
Poster
Session
Conclusion: Others have investigated expert systems approaches
to panel design and semi-quantitative methods for particular
instruments, but neither of these has resulted in a robust general
solution. Our method is completely general and draws on a
combination of specific data on instruments and dyes, user-supplied
information on cell populations of interest and user-supplied
estimates of marker levels in these populations to rank possible
reagent-dye panels for the specified application.
Wednesday,
22 May
Results: For each panel to be evaluated, we use the assembled
information to compute a predicted staining index for each
measurement on each cell population of interest and combine these
into a composite quantitative figure of merit for each proposed
panel. We have developed efficient computational methods of
scoring such proposed panels to extract the most promising and
rank them for investigators. It turns out that this ranking does not
depend on the absolute photoelectron and brightness scales and
thus it should be robust in the face of incomplete information if
reasonable relative estimates are available.
Methods: We have developed a procedure for quantitative
comparison of signals from different dye-conjugated antibodies
using complementary binding of test and reference reagents by antimouse Ig, kappa microspheres. By partially loading microspheres
with graded amounts of a test reagent and subsequently filling
the microspheres with a common, near-saturation dose of a
reference reagent labeled with a distinctly different dye, we obtain
a composite sample with bead populations representing different
tradeoffs of bead loading with the two reagents. Quantitative values
for the reference reagents (currently anti-CD8-FITC and anti-CD8APC), are anchored to the well-specified QuantiBRITE system by
running QuantiBRITE PE beads to calibrate the PE channel scale
and BD 1:1 anti-CD20-PE as a test reagent in complementation
bead stains with each of the reference reagents.
Tuesday,
21 May
Methods: We have developed a mathematical model of signal
detection in flow cytometers that predicts, for each channel in a
multi-color system, the separation between signal-of-interest and
overall background, including spectral overlap effects of other
dyes. This requires information on the instrument configuration,
photoelectron scales of the measurement channels, available
reagents, dye characteristics, cell populations of interest and
marker levels for these cell populations. Some of this information
is quantitatively specifiable, and some of it, particularly marker
levels and catalog reagent characteristics, will usually have to
be estimated. The CytoGenie AutoColor design tool has been
extended to assemble the necessary information. It allows users
to define a set of cell populations, the markers of interest and also
the expected importance and expression level of each marker.
CytoGenie includes an extensive catalog of commercially available
reagents and allows local stock reagents to be considered. It also
maintains information about dye brightness and spectra and specific
instrument configurations (laser wavelengths and power and optical
filter layouts that can be exported by some instrument software).
Background: Knowing how much of a dye-labeled antibody is
needed to produce a particular signal is important for quantitative
cytometry, but evaluating this for normal reagents has been
elusive. Comparisons of stain index values for different antibodydye conjugates have been published, but these are signal vs.
background evaluations that do not compare the actual signal
levels produced by equivalent amounts of different antibody-dye
conjugates.
Monday,
20 May
Background: With the proliferation of multi-laser cytometers and
new fluorochromes, reagent selection for high dimensional flow
cytometry staining panels has become a laborious and hit-or-miss
process requiring considerable expertise and often multiple trial
runs. Depending on the intrinsic brightness of the fluorochromes,
spectral compensation and on the cell populations and markers
involved, some reagent combinations are likely to work well while
others will fail. Furthermore, with the large number of available
reagents, the number of feasible combinations to analyze a given
set of makers has become much too large for direct human analysis.
David Parks1, Aaron B. Kantor2, Wayne A. Moore2, Stephen
W. Meehan3, Leonore A. Herzenberg2
1
Stanford Univ, Stanford, CA, United States, 2Genetics, Stanford
University, Stanford, CA, United States, 3Genetics, Stanford
University, Burnaby, BC, Canada
Sunday,
19 May
Wayne A. Moore1, Stephen W. Meehan2, Aaron B. Kantor1,
David R. Parks3, Leonore A. Herzenberg1
1
Genetics, Stanford University, Stanford, CA, United States,
2
Genetics, Stanford University, Burnaby, BC, Canada, 3Stanford
Univ, Stanford, CA, United States
Quantifying the Relationship between Antibody
Bound and Signal Observed for a Large Panel of
Dye-Conjugated Antibodies Using Complementary
Binding by Antibody Capture Beads and Use of
This Information in Cytogenie Autocolor to Predict
Optimal Multi-Color Stain Sets
Saturday,
18 May
A New Computational Method for Predicting
Optimal Reagent-Dye Combinations in Staining Panel
Design for High-Dimensional Flow Cytometry
92
Special
Lectures
91
Congress
Overview
excited in the blue range. Use of a 500/24 nm bandpass filter
with a high-specification 488 nm notch filter was found to be
optimal for cyan fluorescent protein detection, as was minimize
the intervening filters and dichroics in the emission signal path
to maximize sensitivity; detection at the “back end” of PMT
octagon or trigon cluster compromised sensitivity, while detection
at the “front end” with few or no non-essential optical elements
maximized it. Collectively, the optimal laser source and optical
configuration made a normally undetectable fluorescent reporter
system detectable.
123
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
93
95
DNA Sequencing Detects Residual Leukemia
Tissue Factor Bearing Microparticles Measured
by Impedance-Based Flow Cytometry Predict
Thrombosis in Cancer Patients
Brent Wood
Laboratory Medicine and Pathology, University of Washington,
Seattle, WA, United States
In acute lymphoblastic leukemia, the detection of residual disease
following initial chemotherapy has been identified as one of the
most important predictors of clinical outcome. The two principal
techniques currently used for monitoring residual leukemia are
flow cytometry and molecular genetic methods based on PCR
amplification of polymorphic loci such as the immunoglobulin
receptor and T cell receptor gene families. The former method
has the advantage of speed and low cost but is somewhat less
sensitive and requires subjective interpretation, while the latter
is more sensitive but significantly more expensive, laborious
and difficult to implement at early time points after therapy. In
particular, standard PCR methods require the identification of
patient specific DNA sequences and the generation and validation
of patient specific primers prior to evaluation of post-therapy
samples. Next-generation sequencing offers the promise of highsensitivity, objectivity, and rapid turnaround time at reduced cost
through simultaneous multiplexed sequencing of all VDJ family
members. This talk will discuss issues relating to the detection and
quantitation of residual disease in acute lymphoblastic leukemia
and present data demonstrating the application of next generation
sequencing to acute lymphoblastic leukemia of both B and T cell
lineages.
Tuesday,
21 May
Veronique Neumeister1, Kurt Schalper1, Allison England1,
Elizabeth Zarella1, Fabio Parisi2, Yuval Kluger2, David Hicks3,
David Rimm1
1
Pathology, Yale University, School of Medicine, New Haven,
CT, United States, 2Yale University, School of Medicine, New
Haven, CT, United States, 3Pathology, University of Rochester,
School of Medicine, Rochester, NY, United States
With increasing demands for personalized medicine and tailoring
therapy according to biomarker expression as well as their possible
changes in response to neo-adjuvant therapy, accuracy, objectivity,
reproducibility and dynamic range are important considerations
in assay selection. Also, it has been shown that tissue quality can
play a critical role in these assessments. Pre-analytical variables can
alter tissue quality such that results are inconclusive or inaccurate
and do not represent thein vivo status of the patient’s tumor. This
presentation will discuss the performance of quantitative IF (QIF) in
comparison to quantification of DAB based immunohistochemistry
(IHC). Then new methods will be presented that allow in situ
measurement of mRNA and microRNAs, which in combination
with protein assessment facilitate the construction of multi
parameter models in order to predict outcome or response to
therapy in cancer. Finally, measurement of several proteins and the
construction of an internally calibrated tissue quality index (TQI) as
a way to monitor tissue quality for immunological assessments will
be illustrated.
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Quantitative IF – A Molecular Tool for Assay
Development and Tissue Quality Assessment in
Cancer
Wednesday,
22 May
94
Jeffrey Zwicker
Beth Israel Deaconess/Harvard Medical School, Boston, MA,
United States
Background: Pathologic changes in microparticle populations
contribute to the hypercoagulability associated with various disease
states, including malignancy. Tissue factor bearing microparticles
promote thrombus propagation and fibrin deposition in vivo by
laser-induced vascular injury models and increased levels of
circulating tissue factor bearing microparticles have been measured
in cancer patients.
Methods: Acknowledging the limitations of light scatter flow
cytometry in the characterization of microparticle populations, we
utilized an impedance-based flow cytometer that was specifically
modified for microparticle measurement.
Results: We demonstrated in a case-control study that individuals
with cancer and acute venous thromboembolic events (VTE) were
4-times as likely to have elevated populations of tissue factor
bearing microparticles relative to matched cancer controls. The
median number of tissue factor-bearing microparticles in the cancer
VTE cohort (7.1x104 microparticles/uL) which was significantly
greater than both the idiopathic VTE and cancer-no VTE groups (P
=0.002 and P = 0.03, respectively). In order to evaluate the benefit
of primary thromboprophylaxis in cancer patients with high levels
of tissue factor bearing microparticles we enrolled 70 patients
cancer patients into a randomized, controlled-phase II study.
Individuals with high levels of tissue factor bearing microparticles
were randomized to low molecular weight heparin (enoxaparin
40mg once daily) or observation. Those patients with low levels
of tissue factor bearing microparticles were observed and not
randomized to low molecular weight heparin. For those enrolled
into the observation-only arms both patients and clinicians were
blinded to microparticle status. The cumulative incidence of VTE
at 2-months in the high tissue factor bearing microparticle group
randomized to enoxaparin (N=23) was 5.6% while the higher tissue
factor bearing microparticle group-observation (N=11) arm was
27.3% (Gray test P=0.06). The cumulative incidence of VTE in the
low tissue factor bearing microparticle was 7.2% (N=32). No major
hemorrhages were observed in the enoxaparin arm.
Conclusions: We conclude that increased numbers of circulating
tissue factor bearing microparticles detected by impedance flow
cytometry identify cancer patients with a high incidence of VTE.
Targeted thromboprophylaxis for high risk cancer patients appears
effective. We recently initiated a phase III clinical trial to evaluate
the efficacy of a novel oral anticoagulant to prevent VTE in high risk
cancer patients.
96
Design and Application of Receptor Occupancy
Assays Used to Measure Pharmacodynamic Response
to Treatment with Biologic Therapies
Virginia Litwin1, Cherie Green2, Marna Williams3, David
Wunderlich4, Meina Liang5, John Ferbas2
1
Covance, Inc., Chantilly, VA, United States, 2Amgen, Inc.,
Washington, DC, United States, 3Genentech, South San
Francisco, CA, United States, 4Pfizer Inc., Washington, DC,
United States, 5MedImmune, Gaithersburg, MD, United States
Speaker/Author
Index
Flow Cytometry is an increasingly valuable platform for assessing
pharmacodynamic (PD) measurements during discovery and
development of biological therapeutics. Examples of PD assays
include receptor occupancy (RO) assays for biologics targeting
cellular antigens as well as cellular depletion/repletion following
124
ISAC 2013 Program and Abstracts
Microvesicle Analysis
Silas Leavesley1, James Mansfield2
University of South Alabama, Mobile, AL, United States,
2
PerkinElmer, Waltham, MA, United States
1
101
In Pursuit of Immune Tolerance
Gerald Nepom
Immune Tolerance Network, Seattle, WA, United States
Background: The Immune Tolerance Network (ITN) is a NIAIDsponsored clinical trial organization that designs, conducts,
and analyzes trials using innovative immune interventions in
transplantation, allergy, and autoimmune disease. A primary
objective is to establish immune tolerance, defined as a durable
clinical remission with reprogramming or deletion of deleterious
immune responses. Investigation of immunological mechanisms to
inform clinical outcome, therapeutic effect, and individual response
profiles are paramount in the design of each trial.
Methods: Multiparameter and multidisciplinary analysis tools
are the foundation of our approach, supporting a philosophy
of broad interrogation of the immune response. This creates
challenges for data management and data analysis across multiple
platforms, but enables pooled analyses across multiple trials and
multiple diseases. The ITN developed an open-source web portal,
ITNTrialShare.org, designed to facilitate access and analysis to these
complex datasets for all interested investigators.
Conclusions: The ITN is committed to engagement and
collaboration with the cytometry community, and provides a
platform of clinical opportunity to explore innovative cytometry
applications and analysis.
ISAC 2013 Program and Abstracts
Speaker/Author
Index
Following on from the very successful careers workshop at CYTO
2012, this workshop will be a forum for cytometrists to talk about
career development and issues that we encounter trying to reach
our career goals.
Poster Session
Abstracts
Are you new to cytometry? Been doing cytometry for a few years?
Want a change in career? Want some help working in cytometry?
Results: Examples from ITN clinical trials will be presented
that illustrate how biomarker discovery and mechanistic insight
informed by cytometry analysis has been used by the ITN to make
clinical trial decisions, and to help interpret clinical outcomes.
Opportunities for investigators to work with the ITN on tool
development, as well as opportunities to utilize ITN data from
previous trials, will be highlighted.
Rachael Walker1, Andrew Filby2
1
Babraham Institute, Cambridge, United Kingdom, 2Cancer
Research UK, London, United Kingdom
Oral Session
Abstracts
Career Development: Cytometry STILL Needs You,
but do You Need Cytometry?
This workshop will present the latest technology advancements in
Cytometry Instrumentation. We will have specific focus areas on
the challenges and opportunities for chip-based cytometry, new
detection modalities, and implementation of force based particle
micromanipulation approaches in flow cytometry. New work in
these areas will be presented in short oral presentations. We will
also discuss future trends and challenges in these areas.
Commercial
Tutorials &
Exhibits
99
Steven Graves1, Giacomo Vacca2
1
Center for Biomedical Engineering, University of New Mexico,
Albuquerque, NM, United States, 2Kinetic River Corporation,
San Jose, CA, United States
Poster
Session
Spectral imaging is a set of techniques that offer significant
capabilities for image cytometry, especially for multiparameter
and multiplexed analysis. Tissue cytometry represents a subset of
image cytometry that has significant implications for diagnostic
pathology and personalized medicine. There are numerous
potential advantages for applying spectral imaging approaches for
tissue cytometry, including addressing autofluorescence and other
background issues as well as enabling higher levels of multiplexing.
In this workshop we will review the available and emerging
capabilities for spectral imaging, the state of the art in tissue
cytometry, present novel applications such as the enumeration and
phenotyping of subsets of immune cells and discuss the challenges
and opportunities in these areas.
Trends in Cytometry Instrumentation
Wednesday,
22 May
Spectral Imaging and Tissue Cytometry
100
Tuesday,
21 May
98
*= young denotes time as cytometrist not your age
Monday,
20 May
Small membrane vesicles released from cells (aka microvesicles,
exosomes, ectosomes, microparticles) are attracting increased
interest as diagnostic and therapeutic targets. Flow cytometry is an
attractive method for the analysis and separation of these biological
particles, but their small size and dim signals challenge the
sensitivity of commercial instruments. A consequence of this is that
many aspects of instrument set up, calibration, and experimental
design that are trivial for the measurement of large cells become
critically important for accurate microvesicle analysis. This also
helps define targets for improvements in flow cytometry and other
measurement technologies that are emerging as complementary
techniques for classification and characterization of microvesicles.
This workshop will address some of these issues through short
presentations and discussion.
This workshop is aimed for young cytometrists* and is being
organised and run by young cytometrists. It is also aimed at
technical staff, those new to cytometry and people interested in a
career change.
Sunday,
19 May
Nancy Fisher1, Joanne Lannigan2
1
University of North Carolina, Chapel Hill, NC, United States,
2
University of Virginia, Charlottesville, VA, United States
Saturday,
18 May
97
We aim in this workshop to discuss how to develop your career
in the various roles that use Cytometry, whether it be working in
a core lab, Pharma, being a student using Cytometry or working
for a commercial company. There will be brief talks on the various
cytometry-related career choices, which we hope will turn into a
lively debate on the demands of each career choice and how to
progress in each field. We will share experiences of working in
industry, core-facilities and jobs associated with cytometry. The
workshop aims to be interactive and we will be open to discuss any
issue that arise such as how to set up a lab from scratch, running a
core as a one (wo)man show, what you need to know to work in a
core facility, what should be put on your CV to get you the dream
job and how to develop your role.
Special
Lectures
This session will focus on RO assays including 1) overview of what
a flow cytometric RO assay is and the information that can and
cannot be obtained with this type of assay, 2) case studies of assay
development and validation, including technical challenges, 3)
modeling and correlation with PK and difficulties encountered for
data interpretation. Unique aspects of RO assays will be discussed
(eg, selection of competitive and non-competitive antibodies,
impact of increased soluble receptor, receptor internalization, and
receptor mediated clearance).
Congress
Overview
treatment with therapeutic compounds. Assessment of RO is
useful to confirm physical target coverage for biologics targeting
cellular antigens and support dose selection when combined with
pharmacokinetic (PK) modeling.
125
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
102
Integrating Flow Cytometry and Transcriptomics
Mario Roederer
ImmunoTechnology Section, Vaccine Research Center, NIH,
Bethesdsa, MD, United States
Background: The immune system is comprised of incredibly
diverse sets of cells, each programmed to carry out overlapping
sets of effector functions. Quantifying any one function provides
an incomplete view of the immune response, as information about
what other responses are generated is absent. Quantifying multiple
responses is far superior, but when carried out on a bulk level,
loses information about cellular heterogeneity, gene programs,
and a myriad of interactions that may occur at the single-cell level.
Since individual cells are the atomic unit of immune function, the
maximum information content is achievable only by measuring
these functions independently and simultaneously on a cell-by-cell
basis. For this reason, flow cytometry is a powerful technology
to assess immune function in settings like vaccination and
pathogenesis. Nonetheless, current flow cytometry technology is
limited to measuring the expression of ~16 proteins per cell.
Methods: To extend the multiplexing of gene expression
measurements, we combine single cell sorting (based on cell
surface phenotype) with highly-multiplexed qPCR for 96 or more
genes. We choose to quantify lymphocyte-centric genes, including
those encoding transcription factors, signaling molecules, effector
molecules, and regulatory molecules.
Results: On a single-cell basis, we can correlate protein expression
with gene expression. Discordant results for the same gene reveal
post-transcriptional regulatory mechanisms. We have identified
gene signatures associated with vaccine-elicited T cells as well as
with productively SIV-infected cells in vivo.
Conclusion: The combined technology gives us an unprecedented
view into the complexity and range of immunological functions
expressed by vaccine or virus-specific immune cells. Using
this approach, we can search for correlates of clinical outcome
based on either: quantitative gene expression; and/or cell
subset representation, enumerated by groups of cells sharing
gene expression profiles. These analyses give us new insights
into functional immune states in pathogenesis, treatment, and
vaccination.
103
An UV-C LED Sheath Fluid Desinfection Module for
Flow Cytometric Cell Sorting
Toralf Kaiser1, Jenny Kirsch1, Johannes Glaab2, Tim Kolbe3,
Michael Kneissl3,4, Hyun-Dong Chang1
1
DRFZ, Berlin, Germany, 2Ferdinand-Braun-Institut, LeibnizInstitut für Höchstfrequenztechnik im Forschungsverbund
Berlin e.V, Belrin, Germany, 3Institut of Solid State Physics,
Technische Universität Berlin, Berlin, Germany, 4FerdinandBraun-Institut, Leibniz-Institut für Höchstfrequenztechnik im
Forschungsverbund Berlin e.V, Berlin, Germany
Standard protocols for performing sterile sorts are based on
extensive washing procedures using ethanol or other sterilizing
reagents and have to be performed in a daily time-consuming
routine. However, sterilizing reagents are expensive and, if not
complete removed, can exert toxicity on subsequently sorted cells.
In addition, cell sorters are usually not in a sterile environment and,
therefore, the risk of recontamination is high. Thus, establishment
and maintenance of aseptic conditions for routine sorting remain
one of the biggest challenges in flow cytometry.
We have developed a flow through reactor which sterilizes
the sheath fluid at the point of use. The flow through reactor
is comprised of a 35 LED array positioned on top of three
concentrix channels through which the sheath fluid passes. We use
126
Galliumnitrid (GaN) based UV-C (220-290 nm) LEDs, which have
attracted increasing interest as novel UV-C radiation sources for
applications in water purification and desinfection. Compared to
conventional gas-discharge lamps, UV-C LEDs have a compact form
factor, operate at very low DC voltages, exhibit fast on/off switching
capabilities, and their emission wavelength can be tailored to the
optimum wavelength for germicidal effectiveness. The flow through
reactor is made of UV-reflecting polytetrafluorethylene to efficiently
distribute the UV optical power. A synthetic fused-silica window
sandwiched between the UV-C LEDs and the flow-through reactor
protects the LEDs from fluid contact and provides transmittance
of >90% in the UV-C spectral region, allowing the almost
interference-free irradiation of the sheath fluid as it flows through
the reactor. We have installed the module in a FACS Diva cell sorter
and have connected it to the sheath fluid tubing close to the nozzle.
At a sheath pressure of 30 psi, we were able to reduce the number
of microorganisms up to 1000-fold. The cell sorter remained free of
microorganisms for at least 7 days after the sterilization with bleach
was performed.
Thus, the module allows for the easy and continuous maintenance
of sterility in cell sorting. Due to the compactness and flexibility
of the UV-C LED module, it can easily be customized and
implemented to other existing and newly developed cell sorters.
104
Hollow Core Photonic Crystal Fiber (HC-PCF) Laser
Sources: Closing in on True Tunable Laser Sources for
Flow Cytometry
William Telford1, Veena Kapoor1, Nga Hawk1, Yingying Wang2,
Frederic Gerome2, Fetah Benabid2
1
National Cancer Institute, Bethesda, MD, United States, 2XLIM
Research Institute, Limoges, France
Modern high-end flow cytometers can be equipped with many
lasers of varying wavelengths (nine or more in some cases),
allowing excitation of a wide variety of fluorescent probes.
However, integrating many individual lasers into a single
cytometer is a brute-force approach to the problem of exciting
many fluorochromes, being both inefficient and costly. The
“ideal” laser for flow cytometry would produce multiple, discrete
tunable lines from a single laser source, allowing any wavelength
and combination of wavelength desired. Supercontinuum white
light lasers emit over most of the visible spectrum and have
been previously used in flow cytometry as tunable sources.
However, their continuous emission and relatively low power per
nanometer require optical filters with wide bandwidths to isolate
the wavelengths of interest with sufficient power to provide good
excitation. True tunable fiber lasers with discrete wavelength
emission (~1 nm) have also been demonstrated for cytometry but
only cover narrow regions of the visible spectrum.
Hollow core photonic crystal fibers (HC-PCFs) are specialized
fiber optics with a hollow structured core. These fibers can be
sealed and pressurized with various Raman gas mixtures at high
internal pressures. When optically pumped with a powerful
visible laser source, HC-PCFs emit a series of Raman laser lines
with Stokes shifts both higher and lower than the pump laser
wavelength. For example, a 532 nm pump laser coupled to a
HC-PCF pressurized with hydrogen gas produce discrete laser
lines at approximately 487, 502, 515, 550, 563, 587, 610 and
622 nm. This technology is related to the supercontinuum
phenomenon; unlike supercontinuum sources, however, HC-PCF
bands are discrete with bandwidths comparable to traditional single
wavelength lasers (~ 1 nm), and are powerful enough for use in
flow cytometry. The laser line arrays produced are quite relevant to
the excitation spectra of many traditional fluorochromes. Changing
pump laser wavelength and the Raman gas in the fiber produces
other arrays with varying wavelengths. For example, a 514 nm
pump laser exciting a hydrogen-pressurized fiber produces discrete
laser lines at 530, 546 and 564 nm. In this study, several HC-PCF
ISAC 2013 Program and Abstracts
106
Determining Intracellular Protein Localization with
Fluorescence Lifetime-Based Flow Cytometry
2
2
2
Methods: We used the scanning flow cytometry, which is based
on the measurement of angle-resolved light-scattering patterns
(LSPs) of individual cells and on the solution of the inverse lightscattering (ILS) problem. We measured LSPs with the Scanning Flow
Cytometer fabricated by CytoNova Ltd. (Novosibirsk, Russia, http://
cyto.kinetics.nsc.ru/).The solution ofILS problem is based on fitting
an experimental LSP with theoretical ones, modelingplatelets as
oblate spheroids.Thus volume and shape of individual plateletsin a
sample are determined.
Poster
Session
Commercial
Tutorials &
Exhibits
In addition to measuring platelet LSPs, we also performed antibody
(anti-P-selectin) labeling to correlate shape and surface antigen
expression of platelets. We used several agonists (ADP, collagen,
and thrombin) to induce platelet activation. Flow-cytometric
measurements were accompanied by microscopic observations and
Coulter analysesas independent controls.
Oral Session
Abstracts
Results: The study of platelets with the scanning flow cytometry
wasperformed for several donors. The platelets volume and
shape determined by the solution of the ILS problem were in
an agreement with the corresponding data from microscopic
observation and Coulter analyses. The determined platelet
shape showed a correlation with the P-selectin expression after
stimulation by several agonists.
Poster Session
Abstracts
Conclusions: The shape-based approach to detect activated platelets
is presented. It does not require labeling step, which facilitates rapid
tests in clinical setting. High precision in measurement of platelet
axis ratio and volume opens a way to study the kinetic details of
platelet activation. The results of the comparison with existing
methods are presented.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Wednesday,
22 May
Results: The fluorescence lifetime value of EGFP was changed when
localized into different subcellular regions of the MCF-7 cells. The
fluorescence lifetimes changed to a larger extentthan the average
fluorescence intensity because the average concentration of the EGFP
remained the same despite its location within the cell.When the
EGFP-LC3 protein localizedinto autophagosomesand formed punctate
regions during amino-acid starvation, the average fluorescence
lifetime became shorter compared to the protein in a diffuse
Background: Blood platelets are involved in many diseases,
including myocardial infarction, stroke, peripheral vascular
disease, cancer, and many infections. Their hyperfunction,
especially inappropriate platelet activation, plays a prime role
in the increasing heart-disease burden of society. It is, therefore,
important to assess platelet activation. There are several approaches
to detect activated platelets measuring the expression of surface
receptors with the flow cytometry. We propose an alternative
approach based on the measurement of platelet shape, also being a
marker of platelet activation. This measurement is possible with the
scanning flow cytometry and does not require labeling. Hence, it is
promising for clinical analyses due to performing the test instantly
after the venipuncture.
Tuesday,
21 May
Methods: Enhanced green fluorescent protein (EGFP) was chosen
for all protein localization studies. EGFP was expressed as a tagged
partner to different proteins using a breast cancer cell line(MCF-7).
The tagged constructs includedEGFP-LC3, EGFP-mLC3 (G120A,
a mutant protein that is unable to localize at autophagosome)
and EGFP-p27.To induce subcellular localization of the LC3 and
p27 proteins, amino-acid and serum deprivation was applied.
Also, nucleo-cytoplasmic, or otherdistribution of the LC3 and p27
proteins was confirmed through confocal microscopy and cellular
fractionation. The average fluorescence intensity and fluorescence
lifetime were measured with an Accuri flow cytometer as well as a
home-built fluorescence-lifetime based flow cytometer, respectively.
Finally, protein levels were measured by western blotting.
Alexander Moskalensky 1,2, Maxim Yurkin 1,2, Anastasiya
Konokhova1,2, Dmitry Strokotov1,2, Vyacheslav Nekrasov1,2,
Andrey Chernyshev1,2, Valeri Maltsev1,2
1
Laboratory of Cytometry and Biokinetics, Institute of
Chemical Kinetics and Combustion SB RAS, Novosibirsk,
Russia, 2Department of Physics, Novosibirsk State University,
Novosibirsk, Russia
Monday,
20 May
Background: An important role in the normal biological activity
of proteins is theirlocalizationto different regions inside the cell.
Protein localization can be readily measured with microscopy
using standard fluorescence or immunofluorescence-based
techniques. Flow cytometry can also detect protein presence,
howeversubcellular changes in protein location are difficult to
measurewithoutimage-based approaches. In this contribution,
we introduce fluorescence lifetime-based cytometry as a way of
indicating protein movement inside the cell at a high-throughput
level. That is, changes in the fluorescence lifetime of fluorescent
proteins are measured when bound to other proteins of interest,
whose subcellular locationsare altered. By detecting the
fluorescence lifetime changes, we are able to indirectly measure
two key protein localization and mislocalization events: (1) the
nuclear to cytosoliclocalization of the p27 cell cycle regulator,
an event which is correlated with cell cycle progression and
also, carcinogenesis; and (2) the localization of the LC3 protein
to particular regions of the cytosol to form autophagosomes, a
subcellular characteristic of autophagy.
A Label-Free Shape-Based Detection of Activated
Platelets with Scanning Flow Cytometry
Sunday,
19 May
Ali Vaziri Gohar , Ruofan Cao , Wenyan Li , Patrick Jenkins ,
Jessica P. Houston3, Kevin D. Houston4
1
Molecular Biology Program, New Mexico State University,
Las Cruces, NM, United States, 2Department of Chemical
Engineering, New Mexico State University, Las Cruces, NM,
United States, 3Molecular Biology Program and Department
of Chemical Engineering, New Mexico State University, Las
Cruces, NM, United States, 4Molecular Biology Program and
Department of Chemistry and Biochemistry, New Mexico State
University, Las Cruces, NM, United States
1
Conclusion: The fluorescence lifetime is a quite powerful
photophysical trait that indicates differen tchemical and
biochemical environmentsof fluorophores. In the context of
subcellular protein localization, we envision a powerful highthroughput technique for drug and target screening. Future
work will be to evaluate the lifetime sensitivity and resolution
and introduce other protein localization events. This study was
supported by a New Mexico State University Interdisciplinary
Research Grant by the Office of the Vice President for Research.
Saturday,
18 May
105
cytoplasmic state. We observed many fluorescence lifetime changes
(3-ns to 10-ns) for several different controls ranging from mutant
LC3, non-starved cells, cells with only EGFP, cells with no EGFP,
etc. Additionally, when the p27 protein localization was studied a
range of 3 to 4 ns fluorescence lifetime changes were observed.
Special
Lectures
While still an early technology, HC-PCFs have the potential to
produce many useful, discrete laser lines from a single laser source,
making them another step in the “any excitation wavelength, any
emission wavelength, any probe” paradigm of flow cytometer
design.
Congress
Overview
pump laser and gas configurations were assembled as excitation
sources on a BD LSR II and modified FACSort at both the NIH in the
USA and the XLIM Centre in France, and used to excite a variety of
fluorescent probes, including PE and APC on MESF microspheres,
and cell lines expression several red fluorescent proteins including
DsRed and tdTomato. In all cases, the laser lines produced from
HC-PCF sources gave equivalent excitation to conventional single
wavelength lasers.
127
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
108
Kinetics of Annexin A5 Interaction With Model
Membranes, Determined by Flow Cytometry
Novel Flow Cytometry Assay for Real-Time Detection
of Molecular Extension of Lymphocyte FunctionAssociated Antigen-1
Nicolas Arraud, Céline Gounou, Alain Brisson
CBMN, University of Bordeaux, Bordeaux, France
Annexin A5 (AnxA5) is a soluble protein that binds calcium and
negatively charged lipids, principally phosphatidylserine (PS)
with a sub-nanomolar dissociation constant (Kd)(Tait, Gibson,
and Fujikawa 1989). AnxA5 is a popular marker of apoptosis and
platelet activation, processes characterized by the exposure of
PS lipids on the outer leaflet of the plasma membrane. Despite
extensive studies, important questions concerning the elementary
interaction between AnxA5, calcium and PS are still unresolved,
principally the kinetic parameters of the interaction or the very
nature of the AnxA5 binding site.These questions deserve to be
answered in the context of the biotechnological or biomedical
applications of AnxA5.
As evidenced by the pioneer work of Sklar and Nolan, flow
cytometry is a unique method for investigating the thermodynamic
and the kinetics of molecular interactions, provided one of the
partner is fluorescently labeled and one of the partner is large
enough, or fluorescent enough, to be detected.We applied
to AnxA5 an approach previously developed by Gilbert and
coworkers for studying the binding of coagulation factors to model
membranes deposited on silica microspheres, called lipospheres
(Gilbert et al. JBC, 267, 15861-8, 1992). Factor VIII functions in
an enzyme complex upon the activated platelet membrane where
phosphatidylserine exposure correlates with expression of receptors
for factor VIII. To evaluate the specificity of phosphatidylserinecontaining membrane binding sites for factor VIII, we have
developed a novel membrane model in which phospholipid bilayers
are supported by glass microspheres (lipospheres. We produced a
mutant of AnxA5 that contains a single cystein located oppositely to
the membrane binding face, allowing the straightforward synthesis
of AnxA5 labeled with a single fluorophore, using maleimide
chemistry. The interaction between fluorescently labeled AnxA5 and
20% PS-containing lipospheres (1.5 µm diameter) was performed
by flow cytometry using a conventional Cytomics FC 500 (Beckman
Coulter).
Equilibrium experiments performed at various lipospheres and
AnxA5 concentrations show the very high sensitivity of this method,
as binding was detected down to tens of femtomolars of protein,
with a calculated Kd in the picomolar range. At low protein
concentration, binding took enough time to reach equilibrium so
thatbinding kinetics could be recorded, over time periods ranging
from few minutes to one hour. Kinetics data were extracted
using CXP software Automator. The initial binding rate was
linearly dependent on both AnxA5 and liposphere concentration,
indicating a bi-molecular reaction model. The dissociation process
was extremely slow, almost non detectable at constant calcium
concentration. The binding kinetic constants were determined.
Full kineticdata were fitted to calculate kinetic constants by an
independent method. This is to our knowledge the first analysis to
the kinetics of AnxA5 interaction with supported lipid bilayers using
flow cytometry.
The information provided by this study allows to describe
extensively and quantitatively AnxA5 binding to model membrane
lipospheres. It is thus possible to use these parameters to assay
picomolar concentrations of AnxA5 within minutes. We are
currently developing applications based on this fast and sensitive
AnxA5 assay, and we are extending our work for providing a
comprehensive model of AnxA5, calcium and lipid interactions.
Alexandre Chigaev1, Yelena Smagley2, Shilei Zhang3, Mark
Haynes2, Wei Wang3, Larry Sklar2
1
Pathology, Cancer Center, University of New Mexico,
Albuquerque, NM, United States, 2Pathology, Center for
Molecular Discovery, University of New Mexico, Albuquerque,
NM, United States, 3Chemistry and Chemical Biology,
University of New Mexico, Albuquerque, NM, United States
Background: Integrins are a large family of cell adhesion receptors
widely expressed on mammalian cells. Integrin-dependent cell
adhesion can be rapidly and reversibly modulated in response
to cell signaling without a change in receptor expression.In the
case of integrins cell adhesion is modulated through a series of
conformational changes within the molecule that include changes
in the affinity of the ligand binding pocket, molecular extension,
and clustering, as well as byother mechanisms.The ability to
detect these changes in real-time is critical for the understanding
of integrin physiology. Until recently, using flow cytometry for
real-time detection of integrin conformational and affinity changes
was limited to the alpha4beta1-integrin (VLA-4), the adhesion
molecule that is expressed on leukocytes, hematopoietic stem cells,
dendritic cells, and other cell types. This was achieved using a
unique set of assays, where a ligand-mimicking fluorescent probe
served as a donor in a fluorescence resonance energy transfer
(FRET)-based extension assay, and the fluorescent probe binding
kinetics was used to determine ligand binding affinity. Here we
present a set of data, where we used two novel probes specific
for another leukocyte integrin, namely Lymphocyte FunctionAssociated Antigen-1 (alphaLbeta2-integrin, LFA-1), to detect
real-time conformational extension on live cells at natural receptor
abundance.
Methods: One of the distinctive features of LFA-1 is the inability to
bind LFA-1 specific ligand in the absence of “inside-out” activation.
Therefore, it was impossible to use a ligand-mimicking probe
asa FRET donor as was previously done for VLA-4 integrin. We
took advantage of the two published structures of LFA-1-specific
allosteric antagonists that can bind to LFA-1 without cellular
activation (BIRT0377 and XVA-143). Fluorescent derivatives of these
molecules served as FRET donors and the red fluorescent lipid-like
molecule (PKH 26) served as the FRET acceptor.
Results: Because the distance between the two ligand-binding sites
for the two probes iscomparable to the Forster distance for the two
fluorophores, we detected a significant difference between the
quenching kinetics for BIRT0377- and XVA-143- based probes.
Very rapid signal quenching detected for the XVA-based probe
suggests thatat rest theXVA-143-ligand binding site is located in
close proximity to the membrane. Triggering the signaling pathway
frequently used for the T-cellactivation induced rapid unquenching
of the FRET signal consistent with previously reported rapid
extension of the LFA-1 molecule. Surprisingly, the change in the
signal was significantly larger than previously reported for VLA-4
integrin.
Conclusions: This novel assay for detecting of LFA-1 extension
can be used for real-time studies of the integrin physiology,
since molecular extension is believed to be a part of the normal
mechanism of integrin activation, which regulates cell recruitment
and mobilization.
Speaker/Author
Index
Poster Session
Abstracts
107
128
ISAC 2013 Program and Abstracts
Discovery of Regulators of Receptor Internalization
by High Throughput Flow Cytometry
Results: SERS-stained capture beads were measured using either
635 nm excitation in a conventional flow cytometer or 660 nm
excitation in a custom spectral flow cytometer. All SERS tags
showed similar binding behavior to the calibrated bead set: At
low capture densities, SERS intensity increased linearly, giving a
measure of the brightness per tag. At higher capture densities, SERS
intensity plateaued as steric hindrance prevented the binding of
additional tags. Using the per tag brightness estimates obtained
from low density beads, the total brightness at saturation, and
the surface area of the capture beads, we calculated the effective
binding foot print of the different SERS tags. We found that SERS
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Methods: We prepared beads (3 um diameter) with different
amounts of capture molecules (neutravidin or anti-IgG) and
used fluorescent ligands (biotin-PE or IgG-PE) and calibrated
intensity microspheres (QuantaBrite PE) to determine their binding
capacity. We prepared several different formulations of SERS
tags from nanorods or nanoshells of Au and/or Ag, all with a red
resonance (excitation maximum), and functionalized them with
biotin or IgG. These SERS tags were characterized by TEM and
nanoparticle tracking analysis (NTA), as well as Raman and UV/vis
spectroscopy. We than labeled the calibrated capture beads with
the functionalized SERS tags, and measured their SERS intensities
using conventional and spectral flow cytometry.
Oral Session
Abstracts
Interaction between antigen-specific T cells and antigen
presenting cells (APC) cognate ligand involve reorganization of the
cytoskeleton and recruitment of adhesive and signaling molecules
to the site of intercellular contact. Sustained adhesion of T cells
to APCs and formation of the immunological synapse after T cell
receptor stimulation are required for the antigen-specific response.
Introduction: Nanoparticle tags exhibiting surface enhanced Raman
scattering (SERS) offer an important complement to fluorescence
labels for analytical and cytometry applications. SERS tags can be
tuned to excite at specific wavelengths, encoded with distinctive
spectral signatures, and functionalized with recognition molecules.
Moreover, SERS tag photostability make them useful for imaging,
and their tunability in the near-IR make them attractive for in vivo
use. As for fluorophores, brighter SERS tags are nearly always better,
and designing and producing SERS tags with increased brightness
is an area of active research. A limitation in the field is a lack of
standardized and transportable methods for characterizing the
performance of SERS tags. Adapting an approach from fluorescence
flow cytometry, we prepared capture beads with known binding
capacity and used these to estimate the brightness and binding
footprint (size) of several different SERS tag varieties.
Commercial
Tutorials &
Exhibits
Haley Pugsley, Sherree Friend, Raymond Kong, Brian Hall,
Shobana Vaidyanathan, David Basiji
Amnis part of EMD Millipore, Seattle, WA, United States
John Nolan1, Erika Duggan2
La Jolla Bioengineering Institute, La Jolla, CA, United States
12
Poster
Session
Studies of Immunological Synapse Formation and
Downstream Signaling Events Using the FlowSight
and ImageStream Imaging Flow Cytometers
Development of Brighter Surface Enhanced Raman
Scattering Tags for Multiplexed Cytometry
Wednesday,
22 May
110
111
Tuesday,
21 May
Conclusions: These findings suggest that the platform is suitable to
search for receptor ligands with diverse and previously unknown
efficacies. This approach has also been advanced to simultaneously
monitor the trafficking of two receptors in a single cell, which
provides a unique toolset to study the behavior of receptor/coreceptor pair due to drug synergy effects.
Monday,
20 May
Results: Primary screening yielded several new compounds as
hits in addition to all known drugs for the β2AR in the library.A
series of biochemical and biological assays including the FAP
based approach was carried out to discriminate canonical and
non-canonical ligands and to investigate the signaling pathways
associated with these new hit compounds. Along with several
weak orthosteric agonists, a compound that regulated surface β2AR
expression via an unknown mechanism was identified. These
results are reported in a recent publication (Wu et al, 2012. Mol
Pharm, 82, 645-657).
Sunday,
19 May
Methods: We present a high-throughput flow cytometry
compatibleapproach to discover new ligands that regulate receptor
trafficking, and to identify their specific signalling pathways. The β2adrenergic receptor (β2AR) was chosen to be our model system.The
human β2AR with a fluorogen activating protein (FAP) tag at the
extracellular N-terminus was stably expressed in human monocyte
U937 cells that are accessible to the fluorogen Thiazole Organge
(TO1-2p). A high-throughput flow cytometry screen against the
Prestwick Chemical Library (PCL) containing ~ 1200 off patent
drugs was conducted
Saturday,
18 May
Background: Cell surface receptors such as G-protein coupled
receptors (GPCRs), receptors for tyrosine kinases (RTKs), and ion
channels are major drug targets because they play an important
role in both intercellular target activation and intracellular
communication. Ligand induced trafficking of these receptors can
serve as a useful therapeutic model. However, direct measurement
of plasma membrane protein trafficking by flow cytometry has been
challenging.
One way to measure an immunological synapse is by fluorescently
labeling the molecules that have been recruited to the synapse and
imaging by fluorescence microscopy. However, immunological
synapses are rare and therefore difficult to analyze objectively and
statistically by traditional microscopy methods. To overcome these
problems, we employed the Amnis imaging flow cytometers to
objectively collect imagery of large numbers of cells. We report
the percentage of T cells involved in an organized immunological
synapse, the recruitment of adhesion molecule LFA-1 and signaling
molecule Lck to the synaptic complex and subsequent translocation
of NFkB from the cytoplasm to the nucleus in the T cell. In this
study, Raji B cells loaded with Staphylococcal enterotoxin B (SEB)
were incubated with human T cells to create T cell-APC conjugates.
Cells were stained in various combinations for CD3, CD19,
Actin, LFA-1, Lck and NFkB. Results from the FlowSight and the
ImageStream imaging flow cytometers are compared. Using the
FlowSight imaging flow cytometer we demonstrate image-based
parameters that were used to assess the frequency of conjugates
with an organized immunological synapse in an objective and
statistically significant manner. Employing the ImageStream
imaging flow cytometer we further evaluate the specific location
of the adhesion and signaling molecules LFA-1 and Lck within the
immunological synapse complex in T cells and measure the nuclear
localization of NFkB in the T cell.
Special
Lectures
Yang Wu1,2, Phillip Tapia3, Gregory Fisher4, Alan Waggoner5,
Jonathan Jarvik5, Larry Sklar1,6
1
Pathology, University of New Mexico, Albuquerque, NM,
United States, 2Center for Molecular Discovery, University
of New Mexico, Albuquerque, NM, United States, 3UNM
Center for Molecular Discovery, University of New Mexico,
Albuquerque, NM, United States, 4Technology Center
of Netwarks and Pathways, Carnegie Mellon University,
Pittsburgh, PA, United States, 5Department of Biological
Science and Technology Center of Netwarks and Pathways,
Carnegie Mellon University, Pittsburgh, PA, United States,
6
Center for Molecular Discovery, University of New Mexico,
Albuqerque, NM, United States
Congress
Overview
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129
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
tags prepared from slightly aggregated nanoparticles or from
bimetallic nanoparticles were slightly larger on average (<2x)
and had a modestly increased binding footprint (~50% larger),
but were 5-10 fold brighter on a per tag basis than tags based on
monodisperse or single metal nanoparticles, and resulted in brighter
overall staining of cells.
allows us to precisely produce mono-disperse nanocrystals with
tunable sizes of 12 nm, 20 nm and 30 nm, shown in Fig 1A. Owing
to the exceptional brightness and the background-free conditions,
we have achieved the capacity to distinguish a single SUPER Dot
by wide-field microscopy imaging and within a fibre-dip sensor,
heralding a new era for optical sensing.
Conclusions: Using an approach adapted from fluorescence flow
cytometry, we used calibrated reagent capture beads to characterize
the performance of several different SERS tag preparations. We
were able to estimate the average brightness per tag, the average
binding footprint per tag, and the effective brightness (brightness/
footprint) of the different tag preps, giving a predictive indicator of
performance in actual flow or image cytometry. While we used a
custom spectral flow cytometer for these measurements, we also
show that similar results can be obtained with a conventional
benchtop flow cytometer, making this approach straightforward to
apply in most any modern research environment. Supported by NIH
EB003824.
SUPER Dots are upconversion nanocrystals, able to step-wise
absorb two or more near-infrared photons, and display intense
emission in the visible spectrum (Fig 1). In contrast to traditional
two-photon microscopy methods that require expensive femtosecond
lasers, this sequential photon conversion process can be readily
achieved by a compact near-infrared laser diode with low power.
Doped with various lanthanide ions (Tm3+, Er3+, or Ho3+), these
materials are photo-stable without photobleaching or blinking
problems, and are biocompatible, showing no cytotoxicity against
human osteosarcoma cells, or in-vivo in C.elegans and mice.
112
SUPER Dots: The Next-Generation Bio-Labels
Background: A fundamental problem in the detection of lowabundance molecules and rare-eventcells is the weak signal
strength of the commercially available probes as well as the natural
autofluorescence of cells such that it is difficult to discriminate the
labeled target cells from the background.
Methods: We employed the solvothermal method to synthesize
monodisperse NaYF4 nanocrystals in hexagonal phase with Tm3+
concentrations in the range 0.2~8 mol% codoped with 20 mol%
Yb3+ (Fig 1. A), attempting to enrich the emitter concentration by
high power irradiance.
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
Figure 1. SUPER Dots’ emission spectra excited at 980nm. A). TEM
images show size-tunable nanocrystals; B). Single SUPER Dots are
visible to naked eyes through a wide-field microscope
Results and Discussions: We have developed new nanocrystals,
which we have named SUPER Dots (Strong UP-conversion Encoded
nano-Radiators), that have a detection limit over three orders of
magnitude more sensitive than the commercial nanocrystals, such
as quantum dots. Our SUPER Dots are high-brightness fluorescent
nanocrystals that have broken down the fundamental scientific
barrier of so-called “concentration quenching” in lanthanide
nanomaterials. This allows us to enrich the emitters from a few
hundred to tens of thousands densely packed into a single dot.
Having so many emitters in each SUPER Dot significantly enhances
the upconversion efficiency and signal brightness of the nanocrystal
by a factor of over 70, shown in Fig 1. Our experience in synthesis
130
113
RNA Flow Cytometry for Multiplex Gene Expression
Analysis for Specific Intracellular mRNAs in
Individual Cells
Emily Park1, Woodraw Lomas1, Mary E. Hanley1, Dev Mittar1,
Nan Su2, Yuling Luo2, Vernon Maino1
1
BD Biosciences, San Jose, CA, United States, 2Advanced Cell
Diagnostics, Inc., Hayward, CA, United States
Recent advancements in molecular technologies for gene expression
analysis have facilitated our understanding of the intricate biology
of cells and tissues. However, most gene expression data are
derived from studies based on bulk measurements of heterogeneous
cell populations, obscuring information derived from rare or
specific cell types. The understanding transcription profiles at
the level of single cells is becoming an area of intense interest.
To date, the techniques for RNA detection from single cells have
been limited primarily due to the technical challenges of detecting
specific RNA species at the required sensitivity, since the majority
of functionally important mRNAs are present at low copies in
individual cells (<50 copies per cell).
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Jiangbo Zhao, Dayong Jin
Macquarie University, Sydney, Australia
Moreover, SUPER Dots emit bright light for an extended period,
in the order of tens of microseconds, far longer than conventional
fluorophores that emit for nanoseconds. Using this feature
we can reduce the background interference that is caused by
autofluorescence by time delayed gating, giving sufficient sensitivity
for single-nanoparticle imaging.
Flow cytometry is a powerful single cell analysis tool that allows
the analysis of multiple biomarkers simultaneously in individual
cells. To develop a method to detect intracellular RNAs in the
flow cytometry platform (RNAFlow), the RNAscope® technology
has been adapted applying its novel target-specific probe design
method. The developed RNAFlow has sufficient sensitivity to
distinguish cells containing a low abundance RNA transcript from
the negative cell population. Furthermore, multiple distinct RNA
targets were simultaneously detected with a high specificity in
individual cells. This method can quantify the frequency of cells
expressing specific RNA as well as the number of RNA copies in
each expression-positive cell.
We will discuss detailed technical data for the RNAFlow method
as demonstrated in a few specific applications, including HIV,
immune activation, and circulating tumor cell analysis. Examples
of combined RNAFlow with conventional flow cytometry such
as protein targets and cell cycle analysis will be discussed. The
RNAFlow assay, independently or as a combined assay with coprotein detection, represents a valuable research tool for the specific
and sensitive detection of multiple RNA transcripts in individual
cells in heterogeneous biological specimens. Overall, the developed
method will be useful for the analysis of functionally important RNA
species from single cells, even at very low copy numbers.
ISAC 2013 Program and Abstracts
Cytometry of Low-Cell-Count Samples: Chipcytometry
for Deep Immunophenotyping of Cerebrospinal Fluid
and Bronchoalveolar Lavage Cells
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Background: EuroFlow consortium has recently developed a
standardized approach to immunophenotyping of hematological
malignancies (Kalina et al., 2012; van Dongen et al., 2012). The
standardization is performed on both levels, uniform antibody
panels and uniform instrument settings, so that a computational
meta-analysis of the data is possible. However, due to availability of
the instruments at the project’s beginning in 2006, we have proven
the approach only on 8-color BD Biosciences digital instruments.
Lately, 8-color flow cytometry has become available on several flow
cytometry platforms of different makers. Thus, we pilot tested the
feasibility of standardized acquisition and merged analysis of data
across the platforms.
Poster Session
Abstracts
Background: Cytogenetic abnormalities are important factors in the
classification and prognosis in hematologic malignancies including
acute myeloid leukemia (AML). Detection of these abnormalities is
commonly performed by fluorescence in situ hybridization (FISH),
a slide-based molecular cytogenetic technique designed to detect
Tomas Kalina1, Michaela Nováková1, Marcela Vlková2, Daniel
Thürner1, Ester Mejstrikova1, Quentin Lecrevisse3, Ondrej
Hrusak1
1
Pediatric Hematology and Oncology, Charles University
Prague, 2nd Medical Faculty, Praha 5, Czech Republic,
2
Department of Clinical Immunology and Allergology, St.
Anne’s University Hospital and Faculty of Medicine, Masaryk
University, Brno, Czech Republic, 3Cancer Research Center
(IBMCC-CSIC), Department of Medicine and Cytometry
Service, University of Salamanca, Salamanca, Spain
Oral Session
Abstracts
Orla Maguire1, Kristen Humphrey1, Eunice Wang2, AnneMarie
Block2, Sheila Sait2, Paul Wallace1, Hans Minderman1
1
Flow and Image Cytometry, Roswell Park Cancer Institute,
Buffalo, NY, United States, 2Pathology and Laboratory
Medicine, Roswell Park Cancer Institute, Buffalo, NY, United
States
Pilot Investigation of Euroflow Standardized 8-Color
Panel on Different Flow Cytometry Platforms
Commercial
Tutorials &
Exhibits
Image Cytometry-Based Detection of Aneuploidy by
FISH-IS
116
Poster
Session
115
Conclusions: The present study demonstrates the applicability of
FISH-IS for detecting numerical chromosome aberrations detectable
by quantitative changes in hybridization signal (gains or losses),
establishes accuracy and sensitivity of detection compared to
conventional FISH, and also demonstrates the feasibility to study
procured clinical samples. The current FISH-IS protocol can be
modified for use in many other FISH applications.
Wednesday,
22 May
We invite the scientific community to join our consortium and
explore cellular diversity in other low cell count samples types.
Tuesday,
21 May
First datasets generated by measuring a standardized inital
markerset (Human Immunophenotyping Panel [http://bit.ly/ZclYkV],
22 markers per cell, seperating 48 celltypes and functional states)
already revealed very interesting and unexpected cytometric
phenotypes and new biomarker candidates being invisible for
conventional cytometry techniques.
Results: A combined spot count and fluorescence intensity
algorithm was necessary to account for overlapping spots and
accurately determine ploidy. In models of aneuploidy, the sensitivity
of detection of monosomies and trisomies among 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 from
10 AML patients with trisomy 8 (range 3-97% +8 with a mean of
62%), and a high reproducibility was also found (standard deviation
between 3 experiments ranging from 0.3-1.9%) in 5 AML patients
(range 3-94% +8 with a mean of 79%). Additionally, FISH-IS
analysis of samples procured at clinical remission demonstrated the
presence of residual trisomy 8 cells indicating that this approach
may be used to detect cytogenetic abnormalities associated with
minimal residual disease.
Monday,
20 May
The German Competence Network Multiple Sclerosis, the
German Center for Lung Research and Team Chipcytometry
recently have established a consortium providing sample logistics,
validated marker sets and a data analysis pipeline to perform deep
immunophenotyping of immune cells from CSF and BAL from
patients with neurological and lung diseases in multicenter studies.
As an additional feature, cell samples can be stored in novel
microfluidic chips using cell-attracting surfaces enabling for the first
time a true cytometric clinical biorepository. Currently, sample and
biomarker stability has been validated for 16 month.
Sunday,
19 May
Chipcytometry is an emerging, image-cytometry based technology
enabling analysis of a virtually unlimited, flexible set of biomarkers
per cell (PMID 19006067). Due to its cell-saving, sample preserving
nature, Chipcytometry is especially suitable for the analysis of rare
patient samples with very low cell numbers.
Methods: The slide-based FISH protocol was amended and
optimized for FISH-IS and hybridized cells in suspension were
analyzed by ImageStream cytometry. Male and female healthy
donor peripheral blood mononuclear cells hybridized with
combinations of enumeration probes for chromosomes 8, X and
Y served as cell models with different ploidy (disomy, monosomy,
and trisomy) to determine sensitivity and specificity of the assay. The
FISH-IS protocol was then compared with conventional FISH for 10
AML patients with known trisomy 8.
Saturday,
18 May
Localized cellular immune responses play an important role in
many disease areas like oncology, autoimmune diseases and
infectious diseases. It has been shown that analysis of the local
immune response provides much more accurate data about
pathological processes when compared to measuring biomarkers
in the peripheral blood. It is expected that this will lead to the
discovery of new, highly sensitive and specific biomarkers.
However, due to very low cell numbers in body fluids like
cerebrospinal fluid (CSF - typically 1000-5000 cells/sample),
bronchoalveolar lavage (BAL) or joint fluid, cellular immune
responses have not been accessible for in-depth analysis using
standard cytometry techniques.
and locate specific nucleic acid sequences. Sensitivity of this
microscopy-based method is limited by the abundance of abnormal
cells among the 200-1,000 cells analyzed and it is therefore not
applicable during minimal residual disease (MRD) stages. 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. Due to the high throughput nature of this approach, it
has the potential to enable molecular cytogenetic analysis of MRD.
Special
Lectures
Christian Hennig1, Anja Mirenska1, Martin Stangel2,3, Gesine
Hansen4,5
1
Team Chipcytometry, Hannover Medical School, Hannover,
Germany, 2KKNMS (German Competence Network Multiple
Sclerosis), Hannover Medical School, Hannover, Germany,
3
Department of Neurology, Hannover Medical School,
Hannover, Germany, 4DZL (German Center for Lung Research),
Hannover Medical School, Hannover, Germany, 5Department
of Pediatric Pneumology, Allergology, and Neonatology,
Hannover Medical School, Hannover, Germany
Congress
Overview
114
131
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
Methods: We have set the PMT voltage using hard-dyed 8-peak
Rainbow beads to reach common target channel values. Where
needed, target values were re-scaled to 18-bit common scale. We
have stained peripheral blood of three healthy donors with modified
EuroFlow Lymphocytosis Screening Tube to obtain discrete positive
lymphocyte subset in all 8 channels. After staining we split the
samples and acquired on BD FACS Canto II, BC Navios, BC Cyan
ADP and Miltenyi MACSQuant Analyzer. Next, we re-scaled to 18bit, merged and analyzed in Infinicyt software.
Results: Data obtained from the same sample on different cytometry
platforms were very similar. After gating lymphocytes on FSC and
SSC (scatter parameters were not standardized), we could apply
the same gating strategy (gate position) on all samples. When we
analyzed MFI of gated positive subsets, the values were distributed
with average CV of 18,8% (range 7% - 32%), which presents
variability that is lower than both the inter-individual and interlaboratory variability based on the previous quality control rounds
on BD instruments only. Background staining levels (negative
lymphocytes) were also comparable. However, we did see
systematic platform related differences, which can be attributed to
different filter sets used in collection optics.
(P≤0.001) correlation between the percentage of sickled cells and
the percentage of SCA blood spiked into the normal control at 3%
oxygen.The percentage of fetal hemoglobin in the samples, known
to reduce sickling, correlated significantly to the percentage of
normal red blood cells after deoxygenation (R=.609; P=0.027).
Ex vivo treatment with the candidate anti-sickling agent Aes103 showed a clear dose response relationship between the
concentration of Aes-103 and the number of sickled cells (R
= -0.766; P<0.0001) and normal red blood cells (R=0.658;
P<0.0001).
Conclusions: We have utilized imaging flow cytometry as a rapid
and automated assay to classify and quantitate sickled red blood
cells in deoxygenated blood from sickle cell anemia patients and
to follow the dose response to an anti-sickling agent.This method
provides a highly efficient quantitation with high specificity and
favorable statistical power.
Conclusions: We show that polychromatic flow cytometry protocols
can be standardized even across different flow cytometry platforms
developed by different makers. This opens an opportunity to data
exchange, inter-laboratory collaborations and computational
data analysis of multi centric studies regardless of flow cytometry
equipment used. Variability of the data introduced by instrument is
lower than sample preparation variability. Further improvement may
be reached by using fluorochrome matching reference standard.
Supported by UNCE 204012, P/302/12/G101, NT/12425-4, T.K. is
supported as ISAC Scholar
117
Use of Imaging Flow Cytometry as an Assay for
Sickling Capacity in Patients with Sickle Cell Anemia
Leigh Samsel, Eduard van Beers, Laurel Mendelsohn, Rehan
Saiyed, Philip McCoy, Gregory Kato
NHLBI, NIH, Bethesda, MD, United States
Background: Sickle Cell Anemia (SCA) is a monogenetic
disorder causing red blood cells to obtain a “sickled” shape after
deoxygenation. Preclinical and early phase trials in SCA use the
percentage of sickled cells as an outcome variable. Limitations
to current assays include operator bias, low sensitivity, and
high variability.Imaging Flow Cytometry (IFC)provides rapid
morphological characterization of large numbers of cells. We have
employed IFC as an assay to distinguish sickled red blood cells from
normal red blood cells, and to follow the dose response relationship
between the number of sickled cells relative to treatment with an
anti-sickling agent.
Methods: Peripheral blood obtained from sickle cell patients
wasincubated for one hour with the candidate anti-sickling agent
Aes-103 (5-hydroxymethyl-2-furfural or 5-HMF)at 37°, diluted
1:100 with HemOx buffer (TSC Scientific Corp.), aliquoted into a
96 well plate, and either kept at normal oxygen levels or placed in
a glovebox with 2% oxygen for 2 hours. After incubation, samples
were fixed with glutaraldehyde, washed, and kept on ice until
acquisition on an ImageStreamx imaging flow cytometer (Amnis
Corporation) at 60X magnification. Using Amnis’ IDEAS 5.0 analysis
software, various masks and features were tested on brightfield
imagery to best distinguish normal from sickled red blood cells.
Results: The shape ratio feature (minimum thickness ofinput
imagery divided by its length) calculated on customized userdefined masksprovided a distinction between normal and sickled
red blood cells (Figures A and B, respectively), and a continuum
between the two morphological extremes. Cut off values were
selected to enable classification and quantification. Spiking
experiments demonstrated a strong (R >0.925) and significant
132
Figure A.Brightfield imagery of normal red blood cells (high shape
ratios).
Figure B. Brightfield imagery of sickled red blood cells (low shape
ratios).
118
Kinetic Study of Morphological Changes in Human
Lymphocytes during Early Stages of Apoptosis Using
Scanning Flow Cytometry
Irina Polshchitcina1, Dmitry Strokotov1,2, Valery Maltsev1,2
Institute of Chemical Kinetics and Combustion, SB RAS,
Novosibirsk, Russia, 2Novosibirsk State University, Novosibirsk,
Russia
1
Background: Apoptosis is the process of programmed cell death
that is morphologically characterized by a decrease in the cell size,
chromatin condensation and chromosomal DNA fragmentation. At
the end of this process the cell is fragmented into separate apoptotic
bodies that are phagocytized by macrophages. Apoptosis in the
human body is involved in inherited and acquired humoral and
cellular immune system response.The purpose of this research is to
study the kinetics of morphological changes in human lymphocytes
during early stages of apoptosis.
Usually the early stages of apoptosis are indentified by biochemical
or immunological methods. However such methods may have
an undesirable effect on the object studied. On the other hand
apoptosis is accompanied by significant morphological changes of
cellular nucleus which can be measured with non-invasive optical
methods. Therefore this work is devoted to development of a new
fluorescence-free technique for the kinetic study of the early stages
of apoptosis by means of measuring morphological changes of
mononuclear cells population with a scanning flow cytometer.
Methods: Experimental basis of this work is scanning flow
cytometry technique that allows one to study the light scattering of
individual cells. Solving the inverse light scattering problem onecan
obtain some characteristics of cell. In our work in flow cytometric
ISAC 2013 Program and Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Cancer is often viewed as a caricature of normal development,
but the extent to which its cellular heterogeneity recapitulates
and depends on the normal multilineage differentiation processes
remains unknown. The classical adult differentiation scheme,
as exemplified by 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. In the liver, pancreas and the lungs, mature functional
cells appear to conditionally differentiate to a transit-amplifying
progenitor state under conditions that summon tissue repair.
Further, experiments from nuclear-somatic cell transfer as well
as the creation of induced pluripotent stem cells emphasize
the concept that stemness is inducible and may be viewed as a
cell state rather than 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 re-expressed when differentiation signaling
Poster Session
Abstracts
These results suggest that oRG cells are probably present in all
mammals and are not a specialization of a larger brain with
Vera Donnenberg, James B. Hicks, Albert D. Donnenberg
Univ of Pittsburgh, Hillman Cancer Center, Pittsburgh, PA,
United States
Oral Session
Abstracts
We have recently found that cells resembling oRG cells are
present in mouse embryonic neocortex, and arise from asymmetric
divisions of ventricular radial glia. Time-lapse imaging reveals that
the cells undergo self-renewing asymmetric divisions to generate
neurons directly.This contrasts with human oRG cells that produce
neurons indirectly through production of transit amplifying cells.
Genomic and Phenotypic Pedigree of Breast Cancer
Cell Subsets
Commercial
Tutorials &
Exhibits
We have begun to characterize the types and locations of
progenitor cells responsible for human cortical development. We
found that large numbers of radial glia-like cells and intermediate
progenitor cells populate the human OSVZ. The OSVZ radial
glia-like cells, termed oRG cells, have a long basal process but,
surprisingly, do not have basolateral polarity and lack contact
with the ventricular surface. Using real-time imaging and clonal
analysis, we demonstrate that the oRG cells undergo self-renewing
asymmetric divisions to generate daughter neuronal progenitor cells
that can further proliferate. The daughter cells undergo multiple
rounds of symmetric division before generating neurons, suggesting
that they are a transit amplifying cell population. The oRG cells are
also gliogenic, supporting their classification as a form of neural
stem cell.
121
Poster
Session
Recent insights gained from studies of the developing cerebral
cortex are illuminating potential evolutionary steps that contributed
to structural and functional features of the human brain. Radial
glial cells (RG) undergo self-renewing, asymmetric divisions to
generate neuronal precursors that can further proliferate in the
subventricular zone (SVZ) to increase neuronal number. Unlike the
developing rodent cortex, the developing human cortex contains a
massively expanded SVZ (OSVZ) that is thought to account for the
bulk of cortical neurogenesis.
AML is a heterogeneous disease where different genetic and
epigenetic alterations can contribute to therapeutic outcome.
AML therapy has remained the same for over three decades, most
patients die despite achieving initial complete remission. Even
under very aggressive multi-agent chemotherapy regimens and
myeloablative allogeneic stem cell transplantation, relapse rates
are high. Increasing evidence suggests that AML is originated and
maintained by a subpopulation of leukemic stem cells (LSCs),
which are resistant to standard chemotherapy and thereby provide
a reservoir of cells that drive disease relapse. Importantly, high LSC
fractions correlate with poor disease outcomes. Flow cytometric
methods to identify LSCs from their normal counterparts have made
possible to identify targets to ablate LSCs while sparing normal
hematopoietic stem cells (HSCs). Cell surface markers currently
used to isolate and detect LSCs will be discussed as well as novel
approaches used to target LSCs with minimal toxicity to HSCs.
Furthermore, flow cytometry is being utilized for the development
of novel approaches for predicting a patient’s response to antileukemia stem cell therapies that may allow clinicians to properly
stratify patients into groups.
Wednesday,
22 May
Neural stem and progenitor cells in human cortical development
and evolution
Monica Guzman
Medicine, Weill Cornell Medical College, New York, NY,
United States
Tuesday,
21 May
Arnold Kriegstein1, Jan Lui2, David Hansen3
Neurology, University of California, San Francisco, San
Francisco, CA, United States, 2Neurology, UCSF, San Francisco,
CA, United States, 3UCSF, San Francisco, CA, United States
1
Identification and Targeting of Leukemia Stem Cells
Monday,
20 May
Neural Stem and Progenitor Cells in Human Cortical
Development and Evolution
120
Sunday,
19 May
119
The diversity of neural stem and progenitor cells observed during
human cortical development, consisting of ventricular RG, oRG
cells, intermediate progenitors of the inner SVZ, and transit
amplifying cells of the OSVZ raise the question of whether a
similar diversity of stem and progenitor cells are present during the
differentiation of human stem cells toward forebrain neurons. The
answer may be important for modeling human neurodevelopmental
diseases that affect the cortex ranging from cortical malformations
such as microcephaly and lissencephaly to more subtle disorders
such as autism and schizophrenia.
Saturday,
18 May
Conclusions: This new fluorescence-free method allowsone to
determine such lymphocytes characteristics as the size of cells and
cell nuclei before and after the initiation of apoptosis, the refractive
index of the cytoplasm, the refractive index of the nucleus, the
characteristic time of the apoptosis lag phaseand the fraction of
apoptotic cells, that are important for medical diagnostics. Also,
this information may be useful in the assessment of individual
sensitivity of tumors to chemotherapy drugs. The advantage of this
fluorescence-free technique is a high speed of cell analysis which
provides high statistical accuracy.
increased cortical area. Instead, an evolutionary increase in the
number of oRG cells and their transit amplifying daughter cells
likely amplified neuronal production and contributed to increased
cortical size and complexity in the human brain.
Special
Lectures
Results: The flow cytometric experimental data demonstrated the
s-shaped dependence of the nuclear cells volume on time during
early stages of apoptosis. Applying the function proposed by us for
the processing of the experimental kinetic data we calculated such
parameters of apoptosis as the characteristic time of the apoptosis
lag phase, the fraction of apoptotic cells and the nucleus size.
Congress
Overview
study we used lymphocyte samples obtained with a densitygradient separation procedure from the whole human blood. We
applied abilayer sphere as an optical model of a lymphocyte. This
model has four parameters: cell diameter, ratio between nucleus
and cell diameter, the refractive indices of the nucleus and the
cytoplasm. We measured the distribution of lymphocytes in these
parameters from light scattering profiles of the cells. Lymphocytes
were analyzed before induction of apoptosis and during 3 hours
after induction.
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Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
The goal of this presentation is to demonstrate how to combine
multiparameter flow cytometry and sorting with immuno-histology,
in vivo xenografting and molecular techniques to determine the
phenotype and the genomic pedigree of breast cancer subsets.
342
Poster Locator
The first set of numbers listed references the
program number. The second set of numbers
listed following the letter "B" signifies the
poster board number/location in Hall GH.
For example, B122/B1:
122= Program number
B1 = Poster board location in Hall GH
Cell Based Assays in Drug Discovery: Changing the
Hts Paradigm
Hakim Djaballah
HTS Core Facility, Memorial Sloan-Kettering Cancer Center,
New York, NY, United States
High content assays (HCA) have successfully evolved over the
course of nearly two decades now, enabling us to perform highly
complex cellular based screens, sometimes involving primary
and/or human embryonic stem cells. With this development and
acceptance as a screening platform, came data explosion requiring
special logistics typically not associated with screening operations.
I will present examples of how these cell based assays have allowed
us to screen for modulators of different biologies and therapeutic
areas notably not achievable through the classical target based
approaches. I will also discuss the symbiotic relationship between
flow cytometry and automated microscopy based assays across the
drug discovery paradigm.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
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.
134
ISAC 2013 Program and Abstracts
Congress
Overview
Multimedia Poster Abstracts
122/B1
Population-Based Study of Automated Dna Image
Cytometry as a Screening Method for Cervical
Cancer in Rural Areas of China
97.5%
Histological
LBC
Automated DNA
ICM
# of positive
cases detected
by automated
DNA ICM
%
Negative
345 4
1
3
76
261
23
322
9,536
5.4%
CIN1
206 0
2
19
50
135
30
176
CIN2
42
6
6
5
15
10
23
19
CIN3
38
14
7
2
4
11
28
10
Inv cancer
4
2
1
0
0
1
4
0
26
17
29
145
418
108
527
1.7%
862
28.5%
0.06%
94
83.9%
LSIL
1,134
0.6%
930
82.0%
HSIL
312
0.2%
308
98.7%
Total
181,455
100%
11,730
6.5%
The correlation between histological findings and LBC and
automated DNA ICM results is being analyzed.
Total
HSIL ASC-H LSIL ASCUS
NILM Positive Negative
Speaker/Author
Index
3,025
112
Dx
Poster Session
Abstracts
ASC-US
ASC-H
ISAC 2013 Program and Abstracts
Table 1. 635 women with Histological follow-up results associated
with LBC and DNA ICM
Oral Session
Abstracts
176,872
%
Commercial
Tutorials &
Exhibits
Negative
# of cases
Results: A total of 9,261 women from rural areas in Wuhan were
screened using both LBC and automated DNA ICM. 399 (4.3%)
ASC-US, 34 (0.37%) ASC-H, 124 (1.3%) LSIL, 47 (0.5%) HSIL, and
8677 (93.5%) negative cases were determined. Positive results from
automated DNA ICM werefound in 38.5% of ASC-US cases, 85%
of ASC-H cases, 87% of LSIL cases,100% of HSIL cases, and 2.5%
of cases with negative Pap cytology. Histological follow-up results
were obtained for 635 women including 84 cases of CIN2 or higher
grade lesions (Table 1). If LBC were to be used to refer all cases of
HSIL, ASC-H, and LSIL to colposcopies and biopsies, 43 lesions that
required removal would have been discovered (17 CIN2, 23CIN3,
and 3 cancers), for a sensitivity of 51% and a specificity of 94%. If
automated DNA-ICM were to be used instead, and all positive cases
were to be referred to colposcopy and biopsy, then 55 lesions that
required removal would have been discovered (23 CIN2, 28 CIN3,
and 4 cancers), for a sensitivity of 68% and a specificity of 90%.
Poster
Session
Pap test results
Materials and Methods: Cervical samples from the women were
collected by a brush and fixed. Two slides were prepared from
each sample using a cytospin. The Papanicolaou method was
used to stain one slide for manual cytology examination based on
TBS criteria, while the other slide was stained with Feulgen for
automated DNA ICM analysis. Cervical histological biopsies were
performed on women whose Pap tests showed LSILs and above and/
or when automated DNA ICM analyses reported at least 3 cells with
abnormal aneuploidy (≥5C). These histological follow-up findings
were compared and analyzed.
Wednesday,
22 May
Table 1. Comparison of automated DNA ICM and LBC methods.
Introduction: Results fromautomated DNA image cytometry (DNA
ICM) analyses were compared with liquid-based cytology (LBC)
results for women who had cervical histological biopsies. The
purpose of the study was to determine whether automated DNA
ICM can serve as a primary or contesting method for cervical
cancer screening in China.
Tuesday,
21 May
Results: A total of 181,455 women from rural areas in Wuhan were
screened using both LBC and automated DNA ICM. The mean age
of the women was39 years, and the age rangewas 35 to 45years.
We compared the results of automated DNA ICM to those of LBC.
The rate of positive detection by automated DNA ICM was 5.4%
in women with negative Pap tests and 98.7% in women with HSIL
Pap tests (Table 1). 1,498 women had cervical histological followups. Of these women, CIN1+ lesions were diagnosed in 525 cases
(35.1%), including 7 cases of invasive cervical cancer (0.47%).
Xiaorong Sun1, Hau Li2, Min Zhang3
Wuhan Landing Medical High-Tech Co., Ltd., Wuhan, China,
2
Hubei Zhong Shan Hospital, 3Department of Cytology, Hubei
Zhong Shan Hospital, Wuhan, China
1
Monday,
20 May
Materials and Methods: Our population-based screening program
was made possible by the Wuhan government. 15 rural areas in
Wuhan were selected for the study. Cervical samples from the
women were collected by a brush and placed into a cytofixative
solution. Two slides were prepared from each sample using a
cytospin. The Papanicolaou method was used to stain one slide for
manual cytology examination based on TBS criteria, while the other
slide was stained with Feulgen for automated DNA ICM analysis.
Cervical histological biopsies were performed on women whose
Pap tests showed LSILs and above and/or when automated DNA
ICM analyses reported at least 3 cells with abnormal aneuploidy
(≥5C).
Study of Sensitivity and Specificity of Dna Image
Cytometry in Cervical Squamous Lesions
Sunday,
19 May
Introduction: Worldwide, cervical cancer is the third most common
cancer among women and over 85% of cervical cancer occurs in
developing countries. It is estimated that China accounts for 14%
of the world’s annual incidence of cervical cancer and 12% of the
world’s annual mortalities related to cervical cancer. There is no
national screening program for cervical cancer in China.Screening
remains opportunistic and is centered in large cities. 60% of the
Chinese population resides in rural areas, where 90% of incidents
of cervical cancer cases might occur. These areas lack sufficient
cytopathologists and cytotechnicians to interpretate Pap cytology
specimens. The purpose of this study is to compare automated DNA
Image cytometry (DNA ICM) and liquid-based cytology (LBC) as
primary screening methods for cervical cancer and precancerous
lesions.
123/B2
Saturday,
18 May
Xiaorong Sun1, Hau Li2, Min Zhang2
1
Wuhan Landing Medical High-Tech Co., Ltd., Wuhan, China,
2
Department of Cytology, Hubei Zhong Shan Hospital, Wuhan,
China
Conclusion: The preliminary results suggest that automated DNA
ICM might be used in countries where it would be difficult to
introduce population based cervical cancer screening due to the
lack of cytopathologists and cytotechnologists.
Special
Lectures
Automated Microscopy (B1 – B3)
Automated DNA ICM positive: ≥3 aneuploid cells (>5C)
135
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
Conclusion: BothLBC and automated DNA-ICM have low sensitivity
and high specify for detectingcervical squamous lesions.Combining
the two may increase the sensitivity in detecting cervical lesions.
This approach may be appropriate in current China. Further
detailed study is needed.
124/B3
Simultaneous Recording of Action Potentials
and Calcium Transients from Stem-Cell Derived
Cardiomyocytes: Applications for Cardiotoxicity
Testing
Ross Whittaker1, Raquel Vega1, Randy Ingermanson1, Fabio
Cerignoli2, Rob Towart3, David Gallacher3, Mark Mercola4,
Jeffrey H. Price1
1
Vala Sciences Inc., San Diego, CA, United States, 2Vala
Sciences, Inc., San Diego, CA, United States, 3Center of
Excellence for Cardiovascular Safety Research, Beerse,
Belgium, 4Bioengineering, Sanford-Burnham Med Res Inst. and
Univ. Calif., San Diego, La Jolla, CA, United States
Current methods for preclinical cardiotoxicity testing generally
examine the effects of candidate compounds on the activity of
single ion channels using manual or planar patch clamp methods.
Limitations in these assays require that the tests be performed with
cell lines which stably express the ion channel of interest. This
reductionist approach grossly underestimates the complexity of
cardiomyocyte excitability and physiology. We have developed a
new automated image cytometer and associated software which
facilitates a more physiologically relevant test of compound effects
on cardiomyocyte excitation-contraction coupling. This new
approach utilizes a dual channel automated Image cytometer that
allows for simultaneous measurement of the cardiomyocyte action
potential and calcium transient using voltage and calcium sensitive
dyes. By using these advanced imaging techniques this system
frees the need for the assay apparatus to interact physically with
the cells, allowing the use of a wide array of cell types including
more clinically relevant models such as cardiomyocytes derived
from human induced pluripotent stem cells (hIPSC). Here we
demonstrate the application of this system to a small scale screen of
known cardioactive compounds in hIPSC derived cardiomyocytes.
Our results suggest that the ability to identify perturbations in
the cardiomyocyte action potential and/or calcium transient due
to exposure to cardioactive compounds is on par with existing
technologies. However, this system demonstrates a much higher
throughput than existing systems and provides a more complete
analysis of compound effects on exicitation-contraction coupling in
the cardiomyocyte.
Cell Proliferation and Death (B4)
125/B4
High Resolution Cell Cycle and Apoptosis Analysis in
a Two Color Fluorescence Plot
Olivier Herault1,2, Christine Vignon2
Department of Biological Hematology, University Hospital of
Tours, Tours, France, 2LNOx team, CNRS UMR 7292 GICC,
Tours, France
1
Background: An optimal technology for cell cycle analysis would
allow measuring concomitantly apoptosis, G0, G1, S, G2 and M
phases in combination with cell surface phenotyping. We propose
an easy method in flow cytometry allowing this discrimination in
an only two color fluorescent plot. It is based on the concomitant
use of 7-aminoactinomycin D (7AAD) and antibodies anti-Ki67 and
antiphospho(Ser10)-histone H3 conjugated to Alexa Fluor® 488 to
discriminate G0 et M phases, respectively. The proposed method
is particularly valuable in a clinical setting as verified by analyzing
136
human leukemic cells from marrow samples or exposed to cell
cycle modifiers.
Methods: The method was established using the human KG1a
leukemic cell line and was applied to characterize the cell cycle
and apoptosis of fresh human marrow leukemic cells. The cells
were permeabilized with 1 mL of ice cold ethanol (1h, 4°C).
Following two washes with PBS, 1% FBS and 0.25% Triton X100
(PFT), the cells were stained in 200 μL of PFT for 30 min at
room temperature in the dark with 1 μg 7AAD (Sigma-Aldrich),
5 μL Alexa Fluor® 488-conjugated antihuman Ki67 mAb (B56,
Becton-Dickinson) and 3 μL Alexa Fluor® 488-conjugated
antiphospho(ser10)-histone H3 polyclonal antibody (Cell Signaling
Technology). A control tube was prepared with 1 μg 7AAD and 5 μL
of mouse IgG1 Alexa Fluor® 488 (BectonDickinson). After 2 washes
with PFT, the cells were stained with 10 μL of APC-Cy7conjugated
anti-CD45 (A20, Becton-Dickinson) followed by incubation for 20
min at 4°C. Cells were then washed twice with PBS, centrifuged for
5 min at 500 g and resuspended in 300 μL of PBS.
Results: This flow cytometric method allows for a precise analysis
of the impact on the cell cycle of various functional modulators.
It was applied to analyze the proquiescent effects of contact with
human bone marrow mesenchymal stem cells (MSCs) as well as
the apoptosis induction and mitosis inhibition of the human KG1a
cell line by camptothecin. The cell cycle characteristics of untreated
KG1a cells were clearly quantified as follows: 0.4% subG1, 0.8%
G0, 67.9% G1, 14.9% S, 14.2% G2 and 1.8% M phase. The
contact with marrow MSCs during 72h induced an increase in G0
phase and a decrease in M phase (5.3% and 0.4%, respectively).
We verified the antiproliferative and proapoptotic effects of 24h
exposure to camptothecin, which induced a decrease in S, G2 and
M phases (6.1%, 6.2% and 0.4%, respectively) and an increase in
sub-G1 phase (1.7%). Moreover, it is interesting to note that the
staining protocol preserved the integrity of the plasma membrane
and allowed for the analysis of heterogeneous cell populations,
as verified by analyzing leukemic blast cells (CD45int SSClow) in
bone marrow samples.
Conclusion: We document here the successful utilization of this
method to discriminate concomitantly apoptosis and all the cell
cycle phases in a model of leukemic cells exposed to inducers of
cell cycle perturbations and in human leukemic marrow samples.
Cell Sorting and Selection (B5)
126/B5
Optimization of Flow Cytometric Detection and
Sorting of Transgenic Plasmodium Parasites by
Selection of Optical Filters
Ivan Vorobjev 1,2, Kathrin Buchholz 3, Prashant Prabhat 4,
Natasha Barteneva5
1
Biological faculty, Moscow State University, Moscow, Russia,
2
A.N. Belozersky Institute, Moscow State University, Moscow,
Russia, 3Harvard School of Public Health, Boston, MA, United
States, 4Semrock, Inc., Rochester, NY, United States, 5Boston
Childrens Hospital, PCIMM, Harvard Medical School, Boston,
MA, United States
Background: Malaria remains a major cause of morbidity and
mortality worldwide. Flow cytometry-based assays that take
advantage of fluorescent protein (FP)-expressing malaria parasites
have proven to be valuable tools for quantification and sorting
of specific subpopulations of parasite-infected red blood cells.
Identification of subpopulations of parasites expressing green
fluorescent protein (GFP) is complicated by autofluorescence (AF)
of red blood cells and low signal from transgenic parasites. It has
been suggested that cell sorting yield could be improved by using
filters that selectively match the emission spectrum of GFP.
ISAC 2013 Program and Abstracts
Mass cytometry (CyTOFTM) is a novel cell analysis platform that
allows precise and simultaneous quantification of up to 100
antigenic parameters on individual cells using panels of antibodies
conjugated to rare earth metal isotopes. By comprehensive
single-cell proteomic profiling, this innovative technology has
wide-ranging applications in various areas, including diagnostic
pathology, tumor immunology, and biomarker discovery. Cells
in a single tube are simultaneously labeled with lineage-specific
antibodies that identify cell types of interest in heterogeneous
mixtures (such as blood, bone marrow, and body fluids), as well
as antibodies towards key regulatory proteins within activated
signal transduction pathways. Signaling pathway deregulation
due to underlying molecular defects can be dissected to generate
activation profiles of theoretically all cell types within the biologic
ecosystem. Further, in vitro perturbations with growth factors can
unravel abnormalities such as cell-cycle progression independent of
external stimuli in oncogenic states.
High-dimensional cell type-specific data generated from such
experimentation can pose analytical challenges. We propose an
algorithmic approach to data analysis as follows. First, cells are
grouped by strategic gating based on phenotypic similarities. Cells
with abnormally high or low activity levels are identified and
characterized by their surface marker expression profile. Then,
levels of various effector proteins are quantified in the individual
groups of cells for the various conditions tested. Next, cell-specific
effects of test compounds on growth factor-induced changes
in activity of key signaling proteins are measured to generate
biomarker response profiles that predict therapeutic efficacy and
off-target liabilities. Data sets across different patient samples are
compared to generate phosphoproteomic signatures associated with
various clinical scenarios such as complete response, suboptimal
response, loss of response, non-adherence, and disease progression.
Ultimately, real-time monitoring of diseased cells with abnormal
biologic activity can guide optimal therapeutic approach. The
cell-specific signaling profiles are seamlessly integrated with other
pathologic data to predict clinical outcomes.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Jitakshi De
Deepath Medical, Palo Alto, CA, United States
Poster
Session
Background: Flow cytometry data are most commonly managed
simply by a file system and naming conventions for the raw FCS
files. When a relational database is used, it is typically for managing
secondary data, such as the results of manual gating or the output
of a clustering algorithm. Numerous clustering programs have
been developed for automated flow cytometry analysis in the
last half-decade. Many of these have appeared in the FlowCAP
competition, where they have been evaluated against manual
gating and clinical diagnostic criteria. However, it has been
appreciated in the computer science community that no generalpurpose, perfect clustering algorithm exists. Furthermore, it is also
important to remember that the true quantity of interest – biological
populations – are actually latent variables (unobservable), and that
the observed clusters are surrogates for the underlying biological
populations. Observed clusters are the result of the underlying
biology, experimental manipulations, data transformations, choice
of clustering algorithm, and choice of algorithm’s input parameters
(possibly including implicit parameters such as a random number
seed). To overcome the bias introduced by a particular clustering
algorithm or by its input parameters, it is necessary to use more
than one algorithm and parameter set. The goal of overcoming
algorithm bias will be greatly facilitated by using a relational
database with data extending all the way down to individual events.
Multidimensional Data Visualization Tools for Highly
Multiparametric Analysis of Signaling Pathways by
Mass Cytometry
Wednesday,
22 May
James Cavenaugh1, Andy Straw2, Ted Pawlicki3, Jonathan
Rebhahn1, Tim Mosmann1
1
Center for Vaccine Biology & Immunology, University of
Rochester, Rochester, NY, United States, 2Dept. of Biostatistics
and Computational Biology, University of Rochester, Rochester,
NY, United States, 3Dept. of Computer Science, University of
Rochester, Rochester, NY, United States
128/B7
Tuesday,
21 May
An Event-Level Relational Database for Flow
Cytometry Data
Diagnostics (B7)
Monday,
20 May
127/B6
Conclusions: The ELRDB described herein has significant
improvements over a file system based data management insofar as
expediting comparisons based on different data transformations and
clustering approaches. This ELRDB is particularly well suited for the
results of both hard and soft clustering algorithms.
Sunday,
19 May
Computation and Informatics (B6)
Results: The use of both horizontal and vertical tables balances
storage efficiency, flexibility, and performance. Queries identifying
events in overlapping clusters with fractional memberships allow
consensus clusters using different approaches to be quickly
identified. We have also developed several convenience features for
data exchange.
Saturday,
18 May
Discussion: Filter optimization is particularly important for
applications where the fluorescent signal and percentage of
positive events are relatively low, such as analysis of parasiteinfected samples within the intention of gene-expression profiling
and analysis. The approach outlined here results in substantially
improved yield of GFP-expressing parasites, and requires decreased
sorting time in comparison to standard methods. It is anticipated
that this protocol will be useful for a wide range of applications
involving rare events.
Methods: A schema for an event-level database (ELRDB) was
developed using MS Access and later was ported to PostgreSQL.
This schema uses tables for the experiment level data (e.g., panel
design and instrument conditions), FCS TEXT-level metadata,
raw data, transformations, clustering algorithms metadata, and
clustering results data. A reader for FCS files was developed using
Java and was used to populate the ELRDB. Suitable queries to
facilitate multi-model inference are being developed.
Special
Lectures
Results: A new approach to evaluate filter performance in flow
cytometry using two-dimensional dot blot was developed. Filter
selection was performed in two steps. First, dichroic filter that
transmits whole spectrum of GFP emission was selected (502LP
and 466LP) and replaced standard dichroic from the FL1 channel.
Second, by selecting optical filters with narrow bandpass (BP)
having GFP emission peak close to the center of the filter
transmission band in the FL1 channel (510/20, 512/20 and 517/20),
AF was decreased and signal to background ratio improved. Sorting
of GFP-expressing parasite populations from infected red blood
cells at 90 or 95% purity with these filters resulted in 50-150%
increased yield when compared to the standard filter set-up. The
purity of the sorted population was confirmed using imaging
cytometry and microscopy of cytospin preparations of sorted red
blood cells infected with transgenic malaria parasites.
Congress
Overview
Methods: Detection of transgenic Plasmodium falciparum parasites
expressing either tdTomato or GFP was performed using FACS Aria
II cell sorter with interchangeable optical filters. Parasitaemia was
evaluated using different optical filter sets and, after optimization of
optics, the GFP-expressing parasites were sorted and analysed by
microscopy after cytospin preparation and by imaging cytometry.
To tackle large datasets generated from high-throughput
cytometry experiments, data visualization tools were developed
137
Congress
Overview
Special
Lectures
which allowed representation of parameters of interest in
certain cell-types for the various test conditions and samples
tested, enabling comparison of activated signaling pathways in
heterogeneous pathologic samples. Data visualization tools to
support multidimensional cell-specific proteomic profiling will be
presented.
Facility Management (B8)
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Alan Graham Pockley1,2
1
John van Geest Cancer Research Centre, Nottingham Trent
University, Nottiingham, United Kingdom, 2Chromocyte
Limited, Sheffield, United Kingdom
Background: The increasing complexity of flow cytometry
instrumentation and the move towards customizable and ‘nonstandard’ configurations is making it progressively more timeconsuming and difficult for users to identify combinations of
fluorochromes that are appropriate for their specific instrument
and identify the suppliers of these. Furthermore, many Principal
Investigators with no previous experience in multi-parameter
flow cytometry are now realising the benefits and strengths of the
technique. These issues haveprompted the need for resources that
allowflow cytometrists to search for antibodies that are suitable
for their own particular instrument, identify the suppliers of these
and provide a comprehensive listing of flow cytometry-related
material and resources. Chromocyte (www.chromocyte.com) has
been developed as such a resource and its aim is to improve flow
cytometry knowledge and practice globally.
Chromocyte is a free online global resource which comprises
four principal environments: CALCULATE, LOCATE, EDUCATE
and COMMUNICATE. Using a series of dropdown menus in
CALCULATE, users can select and configure their instrument, and
registered users can save these configurations.A new development
allows instrument configurations that have been provided and
saved by Core Directors to be made available to other Users via
Chromocyte’s ‘Facility Management’ interface. Once configured,
the specificity of antibodies required can be defined using an
interface which returns the maximum number of matches and the
antibody-fluorochrome conjugates that are consistent with the
configured instrument are listed. Antibodies can be selected from
this panel and the suppliers of these are provided. Results can be
downloaded as a spreadsheet.
The intention of Chromocyte is not to replace the need for
understanding flow cytometry. Indeed, its guiding principle is to
enhance the practice of flow cytometry and facilitate its adoption
into laboratories that have no, or limited, prior experience of the
technique.
configure instrument(s)
design antibody panels
find suppliers
educate
•
•
•
•
training resources
cell‐based assays
latest news and activities
Product Focus pages
search for antibodies and isotype controls
find products, services and resources
communicate
•
•
•
•
Introduction: Acoustic focusing in cylindrical capillaries has been
shown to be effective for flow cytometry. However, these capillaries
necessitate precise coupling between a cylindrical capillary and
a rectangular flow cell for sensitive optical analysis. Additionally,
cylindrical systems preclude the use of most microfab approaches.
It is highly desirable to develop acoustic focusing approaches for
flow cytometry that work in square or rectangular channels. Such
systems must focus particles in two dimensions (height and width)
to mimic the axial focusing found in hydrodynamic and cylindrical
capillary acoustic flow cytometers.
Methods: Here we have constructed an acoustic focusing flow cell
constructed from a ~500 µm thick silicon wafer with a ~500 µm
wide channel etched entirely through it to create a square channel
that is ~500 µm on a side. The top and bottom of the channel are
created byanodically bonding the waferbetween two optically clear
glass slides. By etching a channel completely through the wafer
the channel depth is known, uniform, and completely transparent.
We have attached a piezoelectric drive (PZT) to the silicon wafer
and drivenit at a frequency equal to a ½ harmonic for the channel,
which is predicted to create matching standing waves both across
the width and height of the channel. We have added multiple
inlets and outlets to these channels to enable their use in both
conventional flow cytometry and applications that require particles
to be moved between different fluid streams.
Results: Video analysis demonstrated that the acoustically driven
square channel provides for central positioning across the height
and between the side-walls, as the acoustic wave is matched in
both horizontal and vertical dimensions. Inconsistencies between
the channel height and the etched channel width can cause
focusing issues. Theseissues can be corrected for by quickly
sweeping between two close frequencies, less than 100 KHz, to
better focus particles in two dimensions. Using this technique
of matched channel dimensions of width and height and swept
acoustic standing waves for particle focusing, we have constructed
a flow cytometer that is capable of making measurements of
conventional flow cytometry particles and on particles thatare too
large for conventional flow cytometers.We have also demonstrated
the ability of our microfabricated multiple inlet systems to perform
rapid particle switching necessary for many applications.
Conclusion: The fabrication approaches shown here enable simple
integration of these acoustic focusing planar devices into custom
microscope based flow cytometers. Additionally, these systems
are easily microfabricated for more complex sample handling
applications. Finally, the ability of these flow cells to reliably
place cell sized or large particles in both horizontal and vertical
dimensions of a wide channel will make these approaches very
valuable to future flow cytometry analysis.
locate
•
•
Microfabricated Square Channels for Two
Dimensional Acoustically Focused Flow Cytometry
exchange information
‘top tips’
view and post protocols
‘Panel of Experts’
Acknowledgements: Chromocyte is free to use due to the
invaluable financial support of our Corporate Sponsors.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Chromocyte: An Online Resource for the Flow
Cytometry Community
•
•
•
130/B9
Travis Woods1, Steven W. Graves2
1
Center for BioMedical Engineering, University of New Mexico,
Albuquerque, NM, United States, 2University of New Mexico
129/B8
calculate
Flow Cytometry Instrumentation
(B9 – B12)
138
ISAC 2013 Program and Abstracts
Using the Flexibility of the Bd Influx™ Platform
for the Development of a Stream Monitoring and
Correction Solution
Results: The LMM-DS is capable of combining two fluids and
imaging objects suspended in them in a hollow slide at 4X, 10X
and 40X magnification in bright-field, phase contrast, dark-field and
fluorescence illumination using its own novel illuminator-condenser
with no moving parts. Experimenter operations may be programmed
or controlled from the ground. Images are preserved via the
LMM computer. The MFC consists of two major compoenents, a
novel single-use multichannel microfluidic cartridge that includes
volumetric mixing of sample and reagent solutions and a novel
miniature electronic reader (with maximum dimension <20 cm)
that facilitates magnetic cell selection for analysis and achieves
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Methods: Three novel generic systems have been developed to
address this problem: a dynamic microscopy stage (“LMM-DS”)
with a stopped-flow cell and built-in illuminator-condenser, a
microfluidic flow cytometer (“MFC”) with semiconductor electrooptics and single-use channels, and a space-qualified fluid
analytic contained transfer tool (“ACT2”) for safely obtaining and
introducing liquid specimens into analytical devices on orbit.
Oral Session
Abstracts
In recent years flow cytometric instruments have become more
automated, and at the same time increasingly used in routine
environments. Larger numbers of samples are being run, and with
these increasingly large multiparameter datasets, the result is that
post-run data analysis is becoming a bottleneck in the workflow.
Background: Serious sample-transfer issues impede the practice
of standard microscopy (and therefore cytometry) on crewed
space vehicles. Currently the International Space Station (ISS) has
the equivalent of two dissecting microscopes inside gloveboxes
and one highly automated Leica RXA microscope known as
the Light Microscopy Module (LMM). The latter is installed
on the U. S. module within an equipment rack known as the
Fluids Integrated Rack (FIR) and has been dedicated to the study
of colloid phenomena such as self-assembly. The lack of bioanalytical capabilities on ISS has stimulated the development of
instrumentation for quantitative microscopy of cellular systems and
model organisms.
Commercial
Tutorials &
Exhibits
Katherine Gillis, Eric Santarelli, Arni Barican, Patrick de
Borja, David Luong, Asima Khan, Julie Clor, Rick Pittaro,
Ray Lefebvre
EMD Millipore, Hayward, CA, United States
Paul Todd1, Michael Kurk1, N. Samuel Logan2, Scott Moyers2,
John Vellinger1, Teimour Maleki Jafarabadi3, James F. Leary4
1
Techshot, Inc., Greenville, IN, United States, 2Techshot, Inc.,
Greenvile, IN, United States, 3Birck Nanotechnology Center,
Purdue University, W. Lafayette, IN, United States, 4Purdue
University
Poster
Session
Simplifying Multiparametric Analysis Further on
Microcapillary Flow
Cytometry and Microscopy aboard the International
Space Station
Wednesday,
22 May
132/B11
133/B12
Tuesday,
21 May
Monday,
20 May
Results & Conclusions: The stream monitoring software
implemented can reliably acquire the stream video feed, analyse
the stream image and update the cytometer’s amplitude to minimise
variation in the gap between the stream and first detached droplet.
The stream monitoring software has maintained breakoff stability in
a number of sort attempts both in testing and during user sorts (6+
hours) with different nozzles and with different pressures. The BD
Influx™ architecture provides for a platform where ideas be rapidly
implemented via software and/or hardware modification.
Sunday,
19 May
Methods: A 10 laser BD Influx™ (355nm, 405nm, 445nm, 488nm,
514nm, 532nm, 561nm, 594nm, 635nm, 786nm) with a 6 way
sort module installed (5kV deflection plates, increased vertical
displacement collection chamber), small particle option and
polarization sensitive detectors located at the Advanced Cytometry
Facility at the Centenary Institute, Sydney, Australia was used. A
generic BNC video capture card was installed into the computer
and the stream monitoring, pinhole and waste bucket video feeds
were directed into the card. Monitoring software was compiled in
Matlab for x64bit operating systems. This software runs alongside
BD FACS™ Sortware and the Influx architecture allows both
applications to concurrently interface with the cytometer controller.
Parameters were coded to allow visualisation and user control of
stream breakoff location, droplet gap, Δamplitude changes, initial/
current amplitude and video feed settings.
Saturday,
18 May
Background: The BD Influx™ is part of BD’s newer generation
fluorescence activated cell sorters utilising a flexible and modular
architecture. This platform allows user customisation to perform
non-standard functions opening up this cytometer to innovative
modifications. We describe the utilisation of this architecture in the
design, test and implementation of software to visualise, track and
correct the stream on the BD Influx™.
In addition, experiment set-up is often cumbersome and timeconsuming, requiring multiple steps for instrument configuration
and specialized data analysis for each specific assay. From its
inception, the guava easyCyte platform has always been focused
on ease-of-use, and the novel software strategies described here
aim to further simplify the process of data acquisition and analysis.
New application-specific software templates paired with optimized
FlowCellect reagents and protocols allows greater ease of use for
the customer and increases the utility and value of flow cytometric
analysis. These assay-specific templates and in addition to new
features such as, gain independent compensation (GIC), updated
gating strategies, custom statistics expressions, contour plots,
histogram smoothing, 21 CFR 11 compliance enabling features, and
user interface refinements greatly amplify the utility of the platform.
The data presented here will highlight how the design of InCyte™
software allows the user to seamlessly move from acquisition to
analysis. Combined with existing features such as six-parameter
heat-mapping, integrated IC50/EC50 curve generation, and dragand-drop gating, InCyte offers sophisticated data analysis in just
a few steps, with “instant updates” in real time and easy export of
data in a variety of formats. With these new software strategies,
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 InCyte™ software simplify the analysis
of multi-parametric cellular data providing faster and more accurate
results.
Special
Lectures
Suat Dervish, Steven Allen, Frank Kao, Adrian Smith
Advanced Cytometry Facility, Centenary Institute, Sydney,
Australia
Congress
Overview
131/B10
139
Congress
Overview
Special
Lectures
Saturday,
18 May
4-color fluorescence counting using superluminescent LED’s and
avalanche photodiodes. The ACT2 accommodates 3 sizes of
off-the-shelf plastic syringes within a fully contained cylindrical
holder and transfers suspended cells and other solutions between
any contained-sample vessel (such as a culture chamber) and any
storage, fixation or analysis system including the LMM-DS. All of
these products are designed to function in weightlessness.
Conclusions: The LMM-DS is a self-contained cytometry platform
due to arrive at ISS in 2014. The MFC is a hand-held cytometer
applicable to the counting of subsets of blood cells. The ACT2
transfers contained fluids safely from sample vessel to analytical
tools and will be added to ISS toolkits in 2014. Acknowledgments.
Research was supported by NASA contracts NXX12CE76P,
NNX11CF84P, NNX11CF85P.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Immunology (B13)
134/B13
Apoptosis as Modulating Factor of CD8+ T
Lymphocytes in Human Cutaneous Leishmaniasis
Results: Ex vivo experiments showed that the PDT have a higher
percentage of effector CD8+ T lymphocytes compared to HS
and to PAT, but a higher percentage of these cells undergoing
apoptosis. Moreover, after in vitro stimulation, it was observed an
increase in frequency of effector CD8+ T cells in both groups of
patients as well as apoptosis of these cells, thus demonstrating an
antigenic specificity. Our results showed that the occurrence of
apoptosis in effector CD8+ T cells (CD45RA+CD27-) may reflect a
downregulation in their functional activity and may contribute to
the persistence of disease, even if the treatment has already started.
The elevated apoptosis-rate observed in effector CD8+ T cells of
PDT, when stimulated by LbAg, suggests that the specific antigenic
stimulus could be associated to the process of activation-induced
cell death (AICD).Thus, despite a lower percentage of effector CD8+
T lymphocytes in AT patients, the highest percentage of viable and
functional cells may be associated with the control of infection and
healing of the lesions in these individuals.
Conclusion: Our data suggest that the immune change in the host
response to parasite, promoted by antimonial therapy between
tenth (PDT) and eighty days (PAT) of starting treatment, include the
modulation of CD8+ cells by apoptosis and characterize immune
and clinical patterns associated to the process of healing. Using
a multiparametric flow cytometric, we showed, for the first time,
the immunological patterns of patients during the treatment, which
could be a new approach to improve the knowledge of immune
response involved in progression and cure of leishmaniasis.
Raquel Nogueira1,2, Clarissa Cunha1, Adriano Gomes-Silva3,
Alda Maria Da-Cruz3, Armando Schubach4, Maria Inês
Pimentel4, Sérgio Mendonça5, Marcelo Lyra6, Álvaro Luiz
Bertho5,7
1
Immunoparasitology Lab, Oswaldo Cruz Institute - Fiocruz,
Rio de janeiro, Brazil, 2Flow Cytometry Core Facility, Oswaldo
Cruz Institute - Fiocruz, Rio de janeiro, Brazil, 3Interdisciplinary
Laboratory for Medical Research, Oswaldo Cruz Institute Fiocruz, Rio de janeiro, Brazil, 4Reference Center for
Leishmaniasis, Evandro Chagas Clinical Research Institute, Rio
de janeiro, Brazil, 5Immunoparasitology Lab, Oswaldo Cruz
Institute - Fiocruz, Rio de Janeiro, Brazil, 6Reference Center
for Leishmaniasis, Evandro Chagas Clinical Research Institute,
Rio de Janeiro, Brazil, 7Flow Cytometry Core Facility, Oswaldo
Cruz Institute - Fiocruz, Rio de Janeiro, Brazil
Microelectro-Mechanical Systems
(MEMS) and Microfluidics
(B14 – B15)
Background: American tegumentary leishmaniasis was registered
in all states of Brazil and is endemic in Rio de Janeiro, where it is
caused mainly by Leishmania braziliensis. The immune response
is mainly mediated by T lymphocytes and the evaluation of the
immunological characteristics has been based on the determination
of the frequency of CD4+ and CD8+ T lymphocytes and cytokine
production. Previous data from our laboratory indicate that the
active disease and the spontaneous cure of human cutaneous
leishmaniasis (CL) have been associated with higher or lower
levels of apoptotic CD8+ T cells, respectively, at least within the
lesions environment. Some authors have shown that apoptosis
in cells of immune response may be directly related to the
immunopathogenesis of some disorders such as Dengue, Chagas
disease and AIDS. Despite these studies, the role of apoptosis in
immune response of CL remains uncertain. CD8+ T lymphocytes
have heterogeneous functional profiles and the involvement of
effector, naïve and memory CD8+ T-cell and an association between
apoptosis and functionally-defined circulating CD8+ T-lymphocyte
subsets in CL patients still remains undefined.
Background: Liposomes are used for targeted drug delivery. When
made using simple methods such as sonication, they exhibit wide
variations in size (10’s of nm to µm). Liposome size determines
the volume of drug carried, the residence time in the circulatory
system, and the kinetics of drug delivery. Monodisperse liposomes
are made using microfluidic or extrusion approaches. However,
these remain time consuming and could be improved with regards
to throughput and precision. Here, we explore separation of
liposomes into monodisperse populations using acoustophoresis.
As acoustic waves position particles depending on their density and
compressibility compared to their surrounding medium, liposomes
represent a challenging target. However, we have previously
developed high frequency acoustic devices in microchannels
for parallel flow cytometry. As acoustic energy increases with
frequency, these devices have shown promise in manipulation of
liposomes.
Methods: We used a multiparametric flow cytometry protocol
which included 7-AAD and anti-CD3, anti-CD8, anti-CD45RA,
anti-CD27 monoclonal antibody. This protocol allowed us
to identify subsets of CD8+ T lymphocytes such as effector
(CD45RA+CD27-) cells. Peripheral blood mononuclear cells were
obtained from CL patients with active disease during the treatment
(PDT); CL patients cured after the treatment (PAT); and healthy
subjects (HS). The stained samples were analyzed ex vivo and after
in vitro L.braziliensis-antigen (LbAg) stimulation using Kaluza 1.2
software.
140
135/B14
Acoustic Manipulation of Liposomes
Pearlson P. Austin Suthanthiraraj, Steven W. Graves
Center for Biomedical Engineering, Department of Chemical
and Nuclear Engineering, University of New Mexico,
Albuquerque, NM, United States
Methods: To demonstrate acoustophoresis of lipid solutions
that include polydisperse liposomes, we centrifuged lipids from
2% milk, labeled them using Nile Red and focused them in our
13-channel acoustic flow cell that operates at 4.5 MHz. We have
also prepared liposomes using conventional sonication procedures
on purified phospholipids, suspended them in Tris buffer, labeled
them using Nile Red, and focused them in the same acoustic flow
cell. The images of the focused streams were recorded using an
emCCD camera integrated into an epifluorescence microscope.
We will further demonstrate separation of the focused streams of
both lipids as well as size-selected liposomes in acoustic flow cells
incorporating separation channels to collect the focused streams.
ISAC 2013 Program and Abstracts
Poster
Session
Commercial
Tutorials &
Exhibits
138/B17
Background: Flow cytometry has been considered a quantitative
platform that usually required long acquisition time where it was
challenging to discriminate surface expressed target protein from
randomly expressed proteins. Ligand induced trafficking of cell
surface receptors such as G-protein coupled receptors (GPCRs),
Speaker/Author
Index
Conclusion: This uFIC platform, by providing a more efficient
and objective way to evaluate the cellular responses, should have
significant implications as a future high throughput and high
content screening (HT-HCS) platform for cytotoxicity assays and
drug screening.
Poster Session
Abstracts
Yang Wu1, Phillip Tapia2, Gregory Fisher3, J. Jacob Strouse4,
Peter Simons4, Alan Waggoner3, Jonathan Jarvik3, Larry Sklar2
1
Pathology, University of New Mexico, Albuquerque, NM,
United States, 2University of New Mexico, 3Carnegie Mellon
University, 4UNM Center for Molecular Discovery, University
of New Mexico, Albuquerque, NM, United States
Oral Session
Abstracts
Discovering New Ligands with Diverse Signaling
Pathways for Old Receptors
Results: Throughout these studies, we have shown that the uFIC
was able to provide comparable experimental data with those of
conventional in vitro assays using plate reader and flow cytometry.
Moreover, the uFIC platform can also provide further advantages
over conventional instruments, such as higher throughput, lower
assay cost, less generation of toxic waste, and etc., which should
have significant implications in pharmaceutical and biological
applications as a future high throughput cell cycle analysis platform.
ISAC 2013 Program and Abstracts
Wednesday,
22 May
Methods: Microfluidic devices with concentrantion gradient
generator and cell culture chambers/channels were designed,
fabricated, and applied for the assessment of various cellular
responses. In this uFIC platform, all the treatment, measurement,
and analysis of adherent cells were performed in a precisely
controlled microenvironment with minimum disturbances.
Absorbance or fluorescence images were acquired using an
inverted type fluorescence microscope equipped with a cooled
CCD camera. The acquired images were further analyzed to
determine the number of cells as well as individual cellular
absorbance or fluorescence information using Java-based image
processing and analysis software. Then, combinatorial analysis
of cellular responses, such as changes in cellular morphology,
mitochondrial enzyme activity, and cellular DNA contents were
performed.
Bispecific antibodies that engage CD3epsilon on T cells and a
cell surface antigen on tumor cells have been shown to result in
redirected lysis of the tumor cells. These molecules have been
used in the oncology field to cause directed killing of CD19+ cells
in B cell non Hodgkin’s lymphoma (NHL) and B-precursor acute
lymphocytic leukemia (ALL) (Nagorsen and Baeuerle, 2011. Exp.
Cell Res.). Key attributes of these bispecific antibodies include:
target cell-dependent activation of T cells through engagement
of the T cell receptor component, CD3epsilon, highly potent
redirected lysis of a target cell, serial lysis by activated T cells and
amplification of the response through activation of the T cells.
Here we describe a method to visualize the cytolytic synapse
that occurs between the T cell and the tumor cell. The synapse
is composed of a collection of signaling molecules as well as
cytoskeletal molecules that result in a synaptic complex between
2-5 micron in size. Once a synapse forms between the T cell and
the target cell, the T cells release cytolytic granules resulting in
tumor cell cytotoxicity. The necessity to retain a synapse in situ
makes confocal microscopy an ideal technology to visualize these
very small structures. Confocal microscopy works by collecting
a single plane of focus within a sample while eliminating the
out of focus fluorescence in the sample resulting in very clear
images of the synapse in high resolution. Furthermore, using
confocal microscopy allows for potentially multiplexing many
cytolytic synapse markers with different fluorescent tags. Here
we show that the localization of PKCtheta (a ubiquitous marker
of the immunological synapse) and CD45 (to identify the T cells)
work well to identify the synapse using high resolution confocal
microscopy. In these studies, an anti-CD3epsilon/anti-tumor
protein bispecific antibody was used to engage the anti-tumor target
expressing adenocarcinoma cell line with freshly isolated human
T cells. The visualization of the cytolytic synapse as well as the
proteins present in the synapse can help characterize the stability
of the synapse and the resulting potency of the killing effect. In the
future, this assay may also be used to stain tissues from preclinical
or clinical models treated with bispecific molecules serving as
biomarkers for activity in vivo.
Tuesday,
21 May
Background: The miniaturization, integration, and automation of
cell-based assays are one of the most important tasks for enhancing
efficiency and reducing costs of many in vitro cytotoxicity assays.
Among the numerous efforts, the microfluidic image cytometry
(uFIC) platform, which is a novel approach for in situ cytotoxicity
assessment of the cells cultured and treated within microfluidic
channels under a precisely controlled chemical environment, is
considered as one of the most promising approach. Here, following
our recent works on morphology- (Lab Chip 2010;10:415-417),
MTT- (Cytometry Part A.;2012;81A:691-697), and DNA contents(Cytometry Part A. 2013; in press) based uFIC, we present our
recent efforts to develop a uFIC platform based on microfluidic
technology and image cytometry.
Susan Ludmann1, Margaret Weidner2, Nianyu Li3, Cindy
Afshari4, Padma Narayanan3, Kathleen Keegan2
1
Discovery Toxicology, Amgen, Inc., Seattle, WA, United States,
2
Therapeutic Innovation Unit, Amgen, Seattle, WA, United
States, 3Discovery Toxicology, Amgen, Seattle, WA, United
States, 4Amgen
Monday,
20 May
Tae Hyun Yoon, Jonghoon Park
Hanyang University, Seoul, Korea, Republic of (South)
Development of a Confocal Imaging Based
Immunological Synapse Formation Assay to Visualize
Bispecific Antibody-Mediated Tumor Cell Killing
Sunday,
19 May
Microfluidic Image Cytometry (uFIC): Modernizing in
Vitro Cell-Based Assays with Microfluidic Technology
and Image Cytometry
137/B16
Saturday,
18 May
136/B15
Multidimensional Image Cytometry
(B16 – B19)
Special
Lectures
Conclusion: The aim of this work is to demonstrate that acoustic
focusing technique can manipulate liposomes that range widely
in size and offer an approach for rapid separation of liposomes
of a specific size. Preliminary work suggests that highly sizepolydisperse liposomes can be successfully separated using
acoustophoresis. As acoustophoresis can process high volumes of
liquid sample, this approach may offer a rapid method for selection
of precisely sized liposomes for drug delivery in the treatment of
many diseases.
Congress
Overview
Results: We successfully focused lipids and liposomes from milk
into focused streams in our acoustic flow cell. Due to relative lower
density of the lipids in comparison to distilled water, the lipids
focused into the antinodal positions. In our trials with liposomes
from purified lipids, we also observed focusing to antinodal
streams. We assume that these streams consist of the free lipids in
solution and the liposomes. However, a comprehensive analysis of
the focused streams will help to verify such assumptions. Hence,
our future experiments will focus on collecting liposomes from
these focused streams in a size dependent manner.
141
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
receptors for tyrosine kinases (RTKs), and ion channels represent
important therapeutic targets. Quantitative and high-throughput
assays have the potential to expedite the search for new ligands
associated with receptor trafficking. We therefore explored new
approaches to high throughput flow cytometry, andhave adapted a
newly available reporter protein tag for the direct measurement of
protein trafficking in real time.
Methods: We present a highthroughput flow cytometry
compatibleapproach to discover new ligands that regulate receptor
trafficking, and to identify their specific signaling pathways. The
β2-adrenergic receptor (β2AR) and C-C motif chemokine receptor
CCR5 were chosen for this study.The human β2AR with fluorogen
activating protein (FAP) tag AM2.2, andmurine CCR5 with FAP
tag MG13 fused at the extracellular N-terminus, were stably
expressed in human monocyte U937 cells either individually or
together in a single cell. The FAP tag AM2.2 is accessible to the
fluorogen Thiazole Organge (TO1-2p), and MG13 is accessible
to the fluorogen Melachite Green (MG-2p).A high-throughput
flow cytometry screen against the Prestwick Chemical Library
(PCL) was conducted. A set of follow-up experiments including
counter- screen, dose response screen, and secondary assays were
performed to study the mechanism of action of lead compounds.
Results: The hybrid platform combining highthroughput
flow cytometry and FAP technology was used to identify all
known ligands of human β2AR from PCL. Along with several
weak orthosteric agonists, a compound that regulated surface
β2AR expression via an unknown mechanism was identified
unexpectedly. In addition, the platform has been extended to
monitoring the trafficking of two receptors in the same cell. Both
receptors function similarly to their wild type counter parts, and
no cross-talk between the receptors was observed. These results
are reported in two recent publications (Wu et al, 2012. Mol
Pharm, 82, 645-657; Wu et al, Cytometry Part A, DOI: 10.1002/
cyto.a.22242).
Conclusions: The reported findings suggest that the platform is
suitable to search for receptor ligands with diverse and previously
unknown efficacies. The hybrid platform is robust, sensitive,
and easily adaptable to other cell surface receptors, and can
contribute to the de-orphanization of orphan GPCRs. The success
in simultaneously monitoring the trafficking of two receptors in
a single cell demonstrates the potential of this unique toolset in
studying the behavior of receptor/co-receptor pair due to synergistic
drug effects.
139/B18
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Quantification of Protein Aggregates with FlowSight
Imaging Cytometer
Christine Probst, Brian Hall, David Basiji
Amnis, EMD Millipore, Seattle, WA, United States
Aggregation may affect the stability and immunogenicity of proteinbased drugs. Thus, methods to quantify and characterize protein
aggregates are necessary for the optimization of pharmaceutical
formulations. Micro-flow imaging (MFI) is the current goldstandard for analysis of protein aggregates. MFI enables robust
statistical morphological analysis of protein aggregates as large
numbers of events can be imaged. However, as MFI is limited to
bright-field (BF) imaging, it is difficult to classify a given event as
protein, debris, or artifact. Consequently, MFI suffers from large
numbers of background counts, low signal-to-noise ratios, and high
measurement uncertainty.
The FlowSight offers a powerful alternative to MFI for protein
aggregate analysis. FlowSight simultaneously collects fluorescent
and BF images for each event, thus enabling the use of fluorescent
stains specific to proteins. In this experiment, we were able to
quantify the number of aggregated protein particles in various lots
of soy culture media using the FlowSight imaging cytometer at
20x magnification. Nile Red (NR), which binds the hydrophobic
142
regions of protein aggregates, was used as the protein-specific
fluorescent stain. Ultimately a combination of features derived
from BF and fluorescent images was used to classify each event as
protein or non-protein. Proteins were further characterized by size,
and concentration measurements were reported for each sample.
As we noticed high protein concentration measurements for media
prior to addition of soy particles, we prepared additional samples
using media filtered with a 0.2 um membrane. In contrast to BF
imagery alone, we found this method greatly reduced the number
of background counts to yield more reliable measurements.
140/B19
Optogenetic Coupling for Calcium Transient Analysis
and Cardiotoxicity Testing
Fabio Cerignoli 1,2, Saugata Ray 1, Patrick McDonough 2,
Mercola Mark1,3, Jeffrey H. Price1,2
1
Sanford-Burnham Medical Research Institute, La Jolla, CA,
United States, 2Vala Sciences Inc., San Diego, CA, United
States, 3Jacobs School of Engineering, University of California,
San Diego, La Jolla, CA, United States
Light-activated ion channel Channelrhodopsin-2 (ChR2) is a
new tool that allows control of excitable cells though light pulse
instead of electrical stimulation. However, due to its permeability
to cations that are relevant to the genesis of the membrane
action potential and to cardiac contraction (Ca2+, Na+ and K+),
direct expression of ChR2 in cardiomyocytes would impair the
physiological relevance of the whole approach. Here we present
the development of a co-culture system where the ChR2 gene is
expressed in an immortalized epithelial cell line (HeLa) that is
cultured in presence of Neonatal Rat Ventricular Myocytes (NRVMs)
or hiPSC-derived Cardiomyocytes. Light stimulation induces
membrane depolarization in ChR2 expressing cells and triggers
action potentials and calcium transients in the electrically coupled
surrounding cardiomyocytes. Calcium transients are recorded using
fluorescent calcium indicators and fluorescent microscopes. The
co-culture system allows simultaneous recording and analysis of
single-cell calcium transients from hundred of cardiomyocytes that
are stimulated with a single light pulse or paced to record multiple
transients. We have tested several ion blockers affecting calcium
handling (e.g. verapamil) or susceptible of causing arrhythmia (e.g.
sotalol) and compared the alteration of calcium transient dynamic
to electrical stimulation, measuring similar dose-response curves
in the two set ups. Furthermore, cells were still active after several
cycle of optical stimulation in opposition to cell death caused
by electrical field stimulation. The absence of electrodes also
simplifies the development of the platform for high throughput
setups. Overall, our data suggest that optogenetic coupling is a
valid alternative to electrical stimulation for drug screening in
cardiomyocytes, reducing toxic effects due to electrical stimulation
and simplifying the hardware configuration.
Other Biological Applications (B20)
141/B20
Automated Imaging of Cell Sorter Aerosol
Containment Test Samples: A Dual Bead Method
Kimberlyn J. Acklin, Veena Papanna, Kathryn E. Ruisaard,
Karen Ramirez, Karen Clise-Dwyer
South Campus Flow Cytometry & Cell Sorting Core Facility,
UT MD Anderson Cancer Center, Houston, TX, United States
Aerosols generated through cell sorting may contain infectious
organisms, which may pose a hazard to nearby personnel. To
ameliorate these risks, cell sorters are equipped with aerosol
containment systems that evacuate aerosols from the sort chamber.
ISAC recommendations for cell sorting biosafety were originally
published in 1997, and updated in 2007. Testing methodology has
ISAC 2013 Program and Abstracts
The use of three-dimensionalin vitro culture models during drug
discovery and preclinical development has become increasingly
useful in assessing potential tissue-specific on/off-target mechanism
of these agents. Three-dimensional pulmonary tissue models
obtained from human donors can thus be used to address questions
related to Chronic Obstructive Pulmonary Disease (COPD) in a
longitudinal manner. Individuals with COPD have increased mucus
production due to an increase in the number and/or activity of
goblet cells (the mucus producing glands in the airway epithelium).
Imaging lung tissue allows for observation of potential changes in
goblet cell numbers, mucus secretionin situ, and characterization
of ciliated epithelium on the apical surface. When there is an
overproduction of mucus, ciliary function is often compromised.
Here we describe a confocal microscopy study where we
performed a phenotypic evaluation of the 3D EpiAirwayTM Culture
Model (MatTek Corporation) using a number of tissue preparation
techniques and image acquisition methods to observe mucus and
cilia. EpiAirway tissue samples (from a normal donor or a donor
with COPD) were either untreated or were treated with IL-13for 7
days to stimulate mucus production. Samples were prepared using
a standard formalin fixed paraffin embedded method or fixed as a
whole mounted tissue (to be able to preserve the mucus production
on the apical surface of the tissue) and stained for mucin 5AC and
mucin 5B. Mucin distribution on the outside of the apical surface
as well as 15mm into the sample could be resolved well using
the whole mount procedure. Acetylated alpha tubulin was used
to observe cilia on fixed tissue, which not only allows for clear
distinction of the apical surface but is also an appropriate marker to
observe global changes in cilia number and/or length in this model.
Finally, a live cell imaging method was developed where images
were acquired every 36ms allowing for ciliary movement to be
observed. This procedure could potentially be advanced to study
variations in ciliary beating patterns/frequency in disease tissues or
in normal tissues post drug treatment.
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Traditional software user documentation generally takes the form
of a many-paged tutorial, which is daunting in its breadth of time
commitment. Video provides a means of interactive instruction
and is a very flexible medium. Having the ability to stop, start
and rewind is absolutely invaluable. It provides the option to
stop each video and challenge users to predict the outcome of a
demonstration, and elaborate on, or debate a point of reference.
You also have the option to rewind a section of the video to review
a segment to ensure that the user understands a key concept. We
sought to address user training, leveraging social media and its
inherently quantitative analytical environment. To that end, we
generated YouTube videos that teach the program interactively
and allow the user to follow step by step instructions relating to
sections in FlowJo that people are interested in. In addition, we
integrated our video organization and content using YouTube
analytics to get a record for our internal use. This allows us to use
FlowJo.com and YouTube to gather feedback on these videos and to
Market our material in our program as well as in YouTube search.
We present methods for teaching analysis processes and propose
recommendations for managing and developing video content to
support education in flow cytometry.
Susan Ludmann 1, Kimberly Jen 1, Anna Pirrone 1, Kathy
Rohrbach1, Nianyu Li1, Cindy Afshari2, Esther Trueblood1,
Padma Narayanan1
1
Amgen, Seattle, WA, United States, 2Amgen, Thousand Oaks,
CA, United States
Monday,
20 May
Matthew Aranda1, Emily Hodges1, Aaron Lewis2, Michael
Stadnisky3, Adam Treistar1
1
Engineering, Tree Star Inc., Ashland, OR, United States,
2
Video, Tree Star Inc., Ashland, OR, United States, 3Application
Scientist, Tree Star Inc., Ashland, OR, United States
Phenotypic Characterization of Polarized Epithelial
Cells in a 3D EpiAirway Culture Model Using
Confocal Microscopy
Sunday,
19 May
Using Videos to Teach Software
143/B22
Saturday,
18 May
142/B21
Tissue Cytometry/Morphometry
(B22)
Special
Lectures
Other Technology Advances (B21)
Congress
Overview
been improved over these years, moving from using bacteriophage
collected on bacterial lawns to 0.75 micron polystyrene YG beads
(PolySciences) collected via cyclex-d single stage impactors (EMS)
as indicators of aerosol escape. The present study presents the
results of series of refinements of the latter method, adapted to
our cell sorting facility. This Core facility includes a spinning disk
automated confocal system (BD Pathway 435) as the primary
fluorescence microscope used for bead counting.We have adopted
a method in which 0.75 micron YG beads are applied to all
slides and used for image-based autofocus, while the sort sample
consisted of 0.7 micron red fluorescent beads (Thermo Fisher).
Cyclex-dimpactors contain coverslips that allow air sampling of
bead-containing aerosolsto a single spot on the coverslip, which
can be imaged in its entirety in an 8x8 montage using a 4X
objective.Testing is applied to a wide variety of sort platforms in the
Core, including the Sony Biotechnology’s [Synergy] SY3200 andBD
FACS Jazz (beta edition) enclosed in Class II BSC in our BSL2 Cell
Sorting Suite, as well as the freestanding AMO-equipped BD Influx,
and 2 AMO-equipped BD Aria IIu’s used for BSL1 sort samples. All
sorters were tested in normal operation and in “failure mode” to
simulate a nozzle clog.
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
143
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Antigen-Specific Immune Responses
(B23 – B24)
144/B23
CD4+ T Cells are Source of Antigen Specific IFN-γ
Production in Whole Blood of Patients with Visceral
Leishmaniasis
Om Prakash Singh1, Rajiv Kumar1, Shalini Gautam1, Neetu
Singh1, Sussanne Nylen2, David Sacks3, Shyam Sundar1
1
Department of Medicine, Institute of Medical Sciences,
Banaras Hindu University, Varanasi, India, 2Tumor and Cell
Biology, Karolinska Institute, Stockholm, Sweden, 3Laboratory
of Parasitic Diseases, NIAID, National Institute of Health,
Bethesda, MD, United States
Introduction: Visceral Leishmaniasis (VL), also known as Kala-azar,
is a fatal chronic disease caused by protozoan parasite leishmania
and characterized by prolonged fever, spleno-hepatomegaly,
pancytopenia and leads to death if left untreated. One of the
key immunological features of VL patients is that their peripheral
blood mononuclear cells (PBMCs) do not proliferate and produce
interferon-gamma (IFNγ) and/or IL-10 in response to leishmanial
antigen as they have depressed cell mediated immunity. Employing
a whole blood assay (WBA), we have recently reported, the
surprising result that active VL patients secrete significant levels of
IFNγ in response to soluble Leishmania donovani antigen (SLA).
In the present study, we aimed to address the cellular source of
antigen driven IFNγ and the factors that drive this response in
stimulated whole blood.
Results: Depletion of CD4+ cells from whole blood reduced the
antigen driven IFNγ to the basal level. Depletion of CD8+ cells did
not show no any effect while depletion of CD15+ cells appeared
to reduce the IFN-γ to a lesser extent. By flow cytometric analysis,
CD4+ cells appeared to be main source of SLA induced IFNγ in
the WBA. Replacement of autologous plasma with HI-autologous
plasma and HI-FBS did not shown any effect on SLA induced IFN-γ.
Conclusion: We conclude that CD4+ cells are the main source of
antigen driven IFN-γ, though CD15+ (neutrophils) also contribute to
the gamma production in WBA. Importantly, soluble complements
factors, antileishmanial antibodies in whole blood do not play any
role in antigen specific cytokines production.
145/B24
Simultaneous Assessment of CMV Specificity and
Functional Response CD8+ T Cells from Bone Marrow
Transplant Recipients
Orla Maguire1, George Chen2, Kieran O'Loughlin1, Theresa
Hahn2, Philip McCarthy2, Paul Wallace1, Hans Minderman1
1
Flow and Image Cytometry, Roswell Park Cancer Institute,
Buffalo, NY, United States, 2Medicine, Roswell Park Cancer
Institute, Buffalo, NY, United States
Background: Human cytomegalovirus (CMV) infects 50 to 80%
of individuals worldwide. Infection in healthy individuals is
asymptomatic as a result of effective immune surveillance by
virus specific T cells but in immuno-compromised patients such
as hematopoietic cell transplant (HCT) recipients, life threatening
reactivation and clinical disease can occur despite therapy. Clinical
CMV reactivation presents with considerable heterogeneity in
conventional correlative parameters of CMV immunity such as the
number of CMV-specific T cells in the periphery or their functional
response with regards to cytokine production or proliferative
capacity. A source for the heterogeneous correlates may be that the
assessment of the CMV specific phenotype is performed separately
from any functional assessment.
Methods: In the present study, the potential of CMV-specific MHCmultimers to be used not only for detection and enumeration of
CMV-specific T cells but additionally to simultaneously serve as
a vehicle for antigen presentation to trigger a functional response
was assessed in peripheral blood samples from allogeneic HCT
recipients. The functional response in this case was determined by
time-kinetically quantifying the nuclear translocation of NFAT1 by
means of ImageStream cytometry.
Results: Our results demonstrate a strong correlation between the
enumeration of CMV specific T cells following CMV dextramer
binding assessed by conventional flow cytometry and ImageStream
cytometry. An increase in the nuclear localization of NFAT1 was
detectable in the CMV specific T cells following the dextramer
exposure and could be observed as early as 10 minutes following
exposure. No effect of the dextramer exposure was observed in
the dextramer negative cell populations. Both inter- and intrapatient heterogeneity was observed with regards to the number of
responding dextramer positive cells. In contrast, exposure to PMA/
ionomycin resulted in a homogeneous NFAT1 nuclear translocation
in both the dextramer-positive and –negative T cells.
Conclusions: These data thus suggest that a specific T cell response
could be generated in the CMV specific T cell population via
antigen presentation using CMV dextramers and that this approach
could enable the simultaneous assessment of antigen specificity
and a functional response. Having demonstrated the feasibility of
this approach we are currently investigating the effects of peptide
density on the MHC-dextramers with regards to the efficiency of
triggering the NFAT1 nuclear translocation.
This work was partially funded by NIH 4R33 CA12667 (H.M.)
Speaker/Author
Index
Poster Session
Abstracts
Commercial
Tutorials &
Exhibits
Methods: CD4+, CD8+ and CD 15 + cells were depleted from
whole blood of active VL patients using magnetic column and
beads. Whole blood and whole blood depleted of CD8+, CD4+and
CD15+ were cultured with soluble SLA for 24 hours. supernatant
was collected for the analysis of antigen specific IFN-γ by ELISA.
Whole blood intracelular staining was also perfomed to know the
cellular source of antigen driven IFNγ by flow cytometry. To know
the effect of antileishmanial antibody and complements, autologous
plasma were replaced with equal volume of Heat Inactivated (HI)
autologous plasma and HI-Fetal Bovine Serum (FBS) after washing
the whole blood cells with PBS and cultured with SLA for 24 hours.
Oral Session
Abstracts
Poster
Session
Poster Abstracts
144
ISAC 2013 Program and Abstracts
146/B25
Poster
Session
Here we report progress in the development of an epi-fluorescent,
continuous scanning HCS image-based cytometer that utilizes time
delay integration (TDI) imaging and reflective position focus control
in parallel, resulting in possible throughput rates of ~100,000 cells/s
(10X in cell monolayer slide format) with truly simultaneous three
color excitation and emission and 1.2 micron/pixel resolution.
This throughput and resolution compare favorably to flow and
laser scanning cytometers, respectively. Performing autofocus in
parallel with continuous motion imaging allows for a much larger
percentage of the overall scanning time to be devoted to light
collection, thereby removing many of the inefficiencies inherent in
currently available instruments and making image brightness and
signal-to-noise a function of light source and assay brightness.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Performing autofocus in parallel with continuous motion imaging
allows for a much larger percentage of the overall scanning time
to be devoted to image collection, thereby removing many of
the inefficiencies inherent in currently available instruments and
making image brightness and signal-to-noise a function of light
source and assay brightness.
Wednesday,
22 May
Here we report significant progress in three color, epi-fluorescent,
continuous scanning HCS , utilizing time-delay-integration (TDI)
imaging and reflective position focus control in parallel, making
screening rates of greater than 250,000 full wells/day (4.3min per
384- or 8.5min per 1536-well plate) a reality.
speed. Current imaging instruments typically scan at peak speeds
of up to 100,000 wells per day (corresponding to ~ 7400 cells/s at
10X in a 1536 well plate, assuming a cell monolayer), in contrast
with ìHTS plate readers capable of hundreds of thousands of wells
per day. This limitation is far more prohibitive in the screening of
assays where a small population of screened cells is expected to be
responsive. In these screens, a large number of images would need
to be collected for each condition in order to obtain statistically
relevant data. The time required to collect such large amounts
of data slows the screening down to impractical limits which
often disqualifies the assay from screening of large libraries of
compounds. The dependency of the screening speed on the amount
of data collected is largely due to the fact that the acquisition is
done field-by-field, where the automated microscope moves to an
area of the sample, stops, collects images then moves to the next
area and repeats. Here, we propose a continuous scanning imagingbased cytometer where images are acquired simultaneously with
the movement of the sample at a given speed. By eliminating the
need to stop and start movement, scan times significantly improve.
Furthermore, since images are collected at all times during the
movement, the overhead of screening large number of cells per
condition is minimized.
Tuesday,
21 May
The dependency of the screening speed on the amount of data
collected is largely due to the fact that the acquisition is done fieldby-field, where the automated microscope moves to an area of the
sample, stops, collects images then moves to the next area and
repeats. Previously we proposed a continuous scanning method
where images are acquired simultaneously with the movement of
the sample at a given speed. By eliminating the need to stop and
start movement, scan times significantly improve. Furthermore,
since images are collected at all times during the movement,
the overhead of screening large number of cells per condition is
minimized.
High content screening (HCS) is a powerful tool used
predominately in academic research for large-scale cellular
biology, for cDNA and RNAi screens, and in pharmaceutical
companies for early drug discovery using large compound libraries.
HCS can be described as a multi-tiered approach to acquire and
analyze large volumes of biologically relevant data captured from
automated microscopes in various imaging modes (e.g., confocal,
epifluorescent, reflective, and transmitted). HCS combines modern
cell biology, automated microscopy, and robotic handling to
produce spatially and temporally resolved datasets that contain
important phenotypic and morphological parameters in the cellular
systems under investigation. However, large-scale screening of
tens of thousands to millions of compounds and potential clinical
diagnostic applications (e.g., rare cancer cell detection in blood) is
largely limited by image acquisition
Monday,
20 May
However, the large-scale screening of tens-of-thousands to millions
of compounds and potential clinical diagnostic applications (e.g.,
rare cancer cell detection in blood) is largely limited by image
acquisition speed. Current HCS imaging instruments typically scan
at peak speeds of up to 100,000 wells per day, in contrast with
μHTS plate readers capable of hundreds of thousands of wells
per day. This limitation is far more prohibitive in the screening of
assays where a small population of screened cells is expected to be
responsive. In these screens, a large number of images need to be
collected for each condition in order to obtain statistically relevant
data. The time required to collect such large amounts of data slows
the screening down to impractical limits which often disqualifies
the assay from screening of large libraries of compounds.
Gregory Gemmen1, Behrad Azimi1, Ramses Agustin1, Mirco
Guigli2, Jeffrey Price1,2
1
Vala Sciences, Inc., San Diego, CA, United States, 2Sanford
Burnham Medical Research Institute, La Jolla, CA, United States
Sunday,
19 May
High content screening (HCS) is a powerful tool used
predominately in academic research for large-scale cellular biology
and cDNA and RNAi screens, and in pharmaceutical companies
for early drug discovery using large compound libraries. HCS a
multi-tiered approach that acquires and analyzes large volumes of
biologically relevant data captured from automated microscopes
in various imaging modes (e.g., confocal, epifluorescent, reflective,
and transmitted). HCS combines modern cell biology, automated
microscopy, and robotic handling to produce spatially and
temporally resolved datasets that contain important phenotypic
and morphological parameters in the cellular systems under
investigation.
Ultra High Throughput Image Cytometry via TDI
Scanning
Saturday,
18 May
Mirco Guigli 1, David Charlot 2, Behrad Azimi 3, Ramses
Agustin3, Gregory J. Gemmen3, Albert L. Kellner1, Jeffrey
Price1,3
1
Sanford Burnham Medical Research Institute, La Jolla, CA,
United States, 2Bioengineering, University of California San
Diego, La Jolla, CA, United States, 3Vala Sciences Inc, San
Diego, CA, United States
147/B26
Special
Lectures
Ultra High Throughput Image Cytometry Using Time
Delay and Integrate CCD Imaging and Reflective
Positioning Autofocus
Congress
Overview
Automated Microscopy (B25 – B26)
Speaker/Author
Index
ISAC 2013 Program and Abstracts
145
Congress
Overview
Special
Lectures
Biomarkers (B27 – B38)
148/B27
A Quantitative Flow Cytometry Assay Applied to
Receptor Occupancy Measurements Helps for
Preparation and Pharmacodynamic Monitoring of
Clinical Trials for Targeted Drugs
Philippe Poncelet1, Marie-Laure Ozoux2, Maxime Moulard1
R&T, BioCytex, Marseille, France, 2DSAR, sanofi, Vitry-surSeine, France
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
1
Background: Antibody-based drugs used for cancer treatment are
quite different from standard cytotoxic drugs: i) they are generally
less toxic and show a very wide safety margin, ii) treatment effects
may occur at doses much lower than the minimal tolerated dose
(MTD) iii) their antineoplastic effects may be cytostatic rather
than cytotoxic, iv) they may be combined with other therapies at
optimal doses. An early evaluation of the available target sites and
their occupancy prior to and following drug infusion may help
optimize targeted drug dosing, especially during early clinical
trials. Most often, target antigen/receptor expression is enhanced
on tumor cells but it may also be also present on normal tissues
and/or peripheral blood cells. Then, receptor quantitation and
occupancy on these easily available surrogate biomarkers may
bring invaluable information on the pharmacodynamics of the
candidate drug. Several candidate antibody-based drugs have
been monitored these last years using receptor occupancy assays
based on the use of two complementary reporter murine MAbs
and standardized quantitative flow cytometry (QFCM) protocols.
These antibody-based drugs initially included ReoPro® (Eli Lilly),
directed against platelets CD41 (GPIIb /IIIa) and later involved an
anti-CD33 immuno-conjugate for treatment of AML (AVE 9633),
an anti-CD221 (IGF1-R) naked antibody (AVE1644) and an antiCD38 naked antibody (SAR650984), all three from Sanofi-Aventis in
collaboration with Immunogen.
Methods: For each antigen/receptor system, 2 reporter murine
MAbs were searched to provide differential recognition of 2
different epitopes on the target antigen, one being drug-binding
dependent and the other remaining independent of bound drug. A
whole blood, no wash, multi-color, indirect immunofluorescence
(IF) QFCM assay (CellQuant Calibrator) was used to provide
absolute numbers of cell-bound MAb molecules per cell and thus
expression levels for each (targeted and non targeted) epitope.
Staining and fixing process have been validated which permitted
QFCM analysis in a core-lab up-to 3 weeks from the date of blood
sampling and treatment. In the case of the CD33 immuno-conjugate
AVE9633, using MAb against the cytotoxic moiety permitted
evaluation of the in vivo fate of the conjugate.
Results: Receptor occupancy could be monitored on surrogate
blood cells during monitoring of early Phase I clinical trials in
3 systems including CD41 (1), CD33 (2) and CD221/IGF1-R.
Hallmark of the IgF1-R model was the low albeit consistent
expression level on the normal blood cells (500-4,000 molecules/
cell on granulocytes and monocytes depending on individual
donor). This low level did not preclude PD monitoring which
showed IGF1-R down-modulation after drug infusion. As for CD33
(2), IGF1-R target epitope occupancy and down-regulation of total
receptor were measured in patients after drug infusion. In the CD38
system, only ex vivo pre-clinical experiments (Phase 0) will be
presented which aimed at i) checking assay robustness and possible
circadian rhythms in antigen expression and ii) at providing in vitro
data on human normal versus cancer cells to support the choice of
drug starting dose in future Phase I studies. Conclusion: Antigen &
receptor occupancy quantitation based on QFCM analysis can be
applied with sufficient reliability to be used for pharmacodynamic
monitoring in clinical studies of candidate targeted drugs. 1) Quinn
et al, Circulation 1999 2) Lapusan et al., Invest New Drugs 2012
146
149/B28
Withdrawn.
150/B29
Cell Type-Specific Analysis of Oncogenesis
David Galbraith1, Ning Weng2, Tom Doetschman2, Roger
Lasken3, Rashel Grindberg3
1
Bio5 Institute, and School of Plant Sciences, Univ Arizona,
Tucson, AZ, United States, 2Bio5 Institute, Univ Arizona,
Tucson, AZ, United States, 3J. Craig Venter Institute, San Diego,
CA, United States
Background: The earliest events in oncogenesis represent a valuable
source of potential cancer biomarkers that would be relevant both
in diagnosis and for therapy. We are establishing a GeneticallyEngineered Mouse Model (GEMM) to define such events in
pancreatic cancer, a particularly devastating and lethal disease.
Methods: The GEMM design involves four elements: (i) an open
reading frame encoding a chimeric GFP-histone fusion which,
when expressed, targets GFP to the nucleus, (ii) a floxed-stop
separating this ORF from a constitutive promoter, (iii) a mouse line
transgenically expressing the Cre-recombinase under the control of
the Pdx promoter, and (iv) a second mouse line expressing a floxstopped K-ras oncogene. The strategy is to produce a transgenic
mouse line containing elements (i) and (ii), and to combine this
genetically with the two further mouse lines, as well as with
other available cre-expressing lines for validation. Recombinasemediated expression of the GFP-histone fusion should result in
green-fluorescent nuclei, which then can be flow sorted from tissue
and organ homogenates, and profiled through RNA-seq of nuclear
polyA+ transcripts. Ultimately, identification of nuclear transcripts
co-expressed with the K-ras oncogene should define early cancer
biomarkers.
Results: The genetic elements described in (i) and (ii) have
been assembled and transgenic mouse lines produced using
established methods. Founders have been identified through
PCR-based analysis of genomic DNA extracted from tail-clips,
and characterization of changes in marker expression associated
with presence of active recombinase are underway. Methods
for characterization of gene expression from polyA+ transcripts
extracted from flow sorted, GFP-accumulating nuclei have been
defined and validated.
Conclusions: This strategy has the potential for broad applicability
in the study of mammalian development, under normal conditions,
in response to imposed perturbations, and in disease states. It
requires simply a mouse line expressing the cre-recombinase in
some specific developmental, cell type-specific, or disease context
of interest. This approach should therefore be of general interest to
the biomedical community.
151/B30
Analysis of Immunohistological Images of Stromal
Cell Populations in Ultrasound Guided Biopsies
of Synovium to Help Predict Patient Outcomes in
Rheumatoid Arthritis
Debbie Hardie, Jason Turner, Karim Raza, Christopher
Buckley, Andrew Filer
Immunity & Infection, University of Birmingham, Birmingham,
United Kingdom
Background: Early treatment in inflammatory arthritis has been
shown to be beneficial yet not all patients with early inflammatory
episodes go on to develop chronic rheumatoid arthritis (RA). Early
identification of patients who develop RA would provide a valuable
tool in deciding treatment and would greatly benefit patients.
We have previously identified a stromal cytokine profile in the
synovium of patients who subsequently developed RA and we have
ISAC 2013 Program and Abstracts
Conclusions: When using CD144 to identify EMPs, 99% of
observed particles were free of LMPs, GMPs, PMPs, and other
nucleated small particles (7AAD). This compares to a purity of only
60% when using a combination of CD31 and CD51. More research
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Results: Based on our replicated experiments, EMPs were best
identified by cell-surface CD144 expression relative to other
commonly used EMP markers (CD31 & CD51). When EMPs were
identified using CD31/CD51 there was contamination of the
EMP fraction with LMPs (CD45), GMPs (CD66b), and/or PMPs
(CD42b). This contamination was not observable on traditional flow
cytometry dot plots and could only be visualized in the FlowSight
image gallery.
Poster Session
Abstracts
All markers were stable at ambient temperature for 4 days in
peripheral blood collected in Cyto-Chex BCT tubes. The percentages
Oral Session
Abstracts
Our objective was to assess the stability of surface (CD3, CD4,
CD25, and CD127) and intracellular (Foxp3) Treg markers in cell
preservative, Cyto-Chex BCT. We have used this technology to
develop and validate an assay to enumerate human regulatory
T cells in whole blood and set the acceptability criteria for use
in clinical sample testing. The method presented here is a rapid
6-color flow cytometry method that enumerates Foxp3+ Tregs in
peripheral blood in an extended stability collection format (CytoChex BCT). The method was validated using blood from normal
subjects, Streck CDChex Normal controls, and Streck CDChex
Normal controls spiked with MT-2 cells.
Methods: The purpose of this study was to use imaging flow
cytometry to modify existing methods of identifying EMPs based
on cell-surface receptor expression and visual morphology. Venous
blood, treated with sodium citrate was collected from donors and
tested individually as well as pooled. Platelet poor plasma (PPP)
was isolated in a two-step serial centrifugation process (10-min at
400 x g; 30-min @ 10,000 x g). Prior to testing, all antibodies were
titered to determine the lowest quantity that yielded the brightest
staining. A diluted volume of the following antibodies was used to
label PPP (100 uL): CD31, CD42b, CD45, CD51, CD62E, CD66b,
CD105, CD144, and 7AAD. PPP fractions were labeled for 30-min
in dark on ice. A minimum of 20,000 EMP events were acquired
on each sample. The cell-surface markers used in this study
were selected based on those that are commonly reported in the
literature. After labeling, samples were acquired on a MilliporeAmnis FlowSight (60 mW 488 nm & 100 mW 642 nm lasers).
Analysis and compensation was completed post-acquisition using
Amnis IDEAS software (v.5.0.385).
Commercial
Tutorials &
Exhibits
A functionally committed Treg subset expressing the Foxp3 protein
comprises 1–10% of CD4+ T cells in peripheral blood. Almost all
current methods that enumerate Tregs require cryopreserved PBMC,
which modulate Treg markers or fresh specimens within 24-48
hours of collection. In most clinical studies, blood samples are
shipped to a centralized testing laboratory and the necessity to test
samples within a short time frame after blood collection has limited
the testing of regulatory T cells in whole blood by flow cytometry.
Background: Atherosclerosis is a chronic disease partially
characterized by the presence of lesions along the vascular
endothelial wall. Current clinical techniques lack the ability
to measure very early changes in endothelial cell health.
When endothelial cells are damaged, they release endothelial
microparticles (EMP) into circulation. Thus, blood EMP
concentration may represent a useful cardiovascular disease
biomarker. Despite the potential value of EMPs, current flow
cytometry techniques are unable to fully distinguish EMPs from
other small cell particles. Other small cell particles include
leukocyte (LMP), granulocyte (GMP), and platelet (PMP)
microparticles.
Poster
Session
Regulatory T cells (Tregs) determine the outcomes of protective
immunity to a spectrum of foreign antigens while maintaining
tolerance to self antigens and suppressing excessive inflammation
that can cause pathology. The presence of Tregs may help or
hinder immunotherapeutic approaches to treating cancer,
autoimmunity, and transplantation. Analysis of Tregs is becoming an
increasingly important consideration in the development of novel
immunotherapeutic strategies, particularly where the therapeutic
strategy is targeting Tregs.
Adam Venable, Randall Williams, Brian McFarlin
Applied Physiology Laboratory, University of North Texas,
Denton, TX, United States
Wednesday,
22 May
Ramesh Janani, Genet Fesseha, Terry Robins
Quest Diagnotics Lab, Valencia, CA, United States
Novel Method for the Identification of Endothelial
Microparticles Using Image-Based Flow Cytometry
and CD144
Tuesday,
21 May
A Flow Cytometry Based Method for Enumeration of
Foxp3+ Regulatory T Cells in Blood Samples Collected
and Stabilized in Cyto-Chex BCT Suitable for Use in
Central Laboratories
153/B32
Monday,
20 May
152/B31
The 6-color flow cytometry method for enumeration of Foxp3+
Tregs is an easy and sensitive method to detect regulatory T cells in
peripheral blood.
Sunday,
19 May
Conclusions: The presence of high levels of GP38 and FAP were
indicators of rheumatoid arthritis in very early disease. CD90 was
also associated with early disease.
The advantages of this assay over current methods include small
sample volume required for testing, longer sample stability, and
minimum sample manipulation before testing. This assay will
simplify clinical trial immune monitoring of regulatory T cells and
can provide crucial data on patient Treg numbers before, during,
and after therapeutic interventions.
Saturday,
18 May
Results: There was no difference between the fractional expression
of the sublining layer marker CD248, the generic stromal marker
PDI, lining layer markers CD55 and VCAM-1 and the endothelial
cell marker CD31 between very early outcome groups, normal
controls (knee pain) or longer duration RA. CD90 (sublining stromal
and endothelial cells) was expressed at higher levels in both ¡Ü3
month groups (P<0.05). GP38/podoplanin was expressed at higher
levels in RA of all durations (p<0.05) compared to normal, while
FAP was expressed at a higher levels in all inflamed groups (P<0.01)
but at highest levels during the first 3 months of RA (p=0.0007).
of CD3+CD4+Foxp3+CD25+CD127-/low Tregs were comparable
in whole blood collected in Cyto-Chex BCT, K2EDTA and densitygradient separated PBMC. The assay was robust and reliable for
enumeration of the low frequency Tregs, with CV's for inter-assay
precision of <25%. The CV's for total CD4+ T lymphocytes was
<5% for inter-assay precision, providing further evidence for
reproducibility. The lower limits of detection for these populations
were 200 events out of 50,000 CD3+ T cell events collected.
Special
Lectures
Methods: Ultrasound guided biopsies of synovial tissue samples
from patients with symptoms <3 months having a) early
inflammation that resolved; b) early inflammation that developed
RA; c) knee pain but no other symptoms and d) ACR/EULAR 2010
criteria for RA with symptoms >3 months, were compared. Stromal
markers CD248, fibroblast activation protein (FAP) and GP38/
podoplanin that are associated with inflammation were compared
with other stromal markers. The amount of fluorescence staining
in acetone fixed cryo-sections of these markers was quantified and
compared.
Congress
Overview
provided evidence for the importance of stromal cell populations in
inflammation. Non-haematopoietic stromal cells such as fibroblasts
are being investigated as new therapeutic targets. We therefore
investigated whether distinct stromal cell populations were present
in and predictive of patients who develop RA.
147
Congress
Overview
is needed to under how our modified EMP detection technique can
be used in experimental models of atherosclerosis or other forms of
chronic disease.
Global Implementation of Flow Cytometry
Instrument Standardization by Covance Central
Laboratory Services
Linsen Du1, Valerie Glutz2, Vincent Holl2, Virginia Litwin3,
Mark Edinger4, Jorg Hildmann4, Joan Batchelder4
1
Covance (Asia) Pte Ltd, Singapore, Singapore, 2Covance
(Geneva) Inc., Geneva, Switzerland, 3Covance (Indianapolis)
Inc., Indianapolis, IN, United States, 4BD Biosciences, San
Jose, CA, United States
Flow cytometry has become an increasingly important biomarker
assay platform for clinical evaluation of new therapeutic
compounds. In order to generate the high quality, longitudinal
biomarker data required to support global, multi-site clinical trials
all aspects of flow cytometry testing must be harmonized. The use
of common standard operating procedures (SOPs), centralized data
analysis, and instrument-to-instrument standardization represent
three ways to increase multi-site harmonization and provide
consistent longitudinal data.
In collaboration with Becton Dickinson, Covance Central
Laboratory Services (CCLS) has implemented an instrument-toinstrument standardization process globally. To date, instruments in
Geneva, Indianapolis, and Singapore have been standardized.
One of the initial steps of the process was to define a single
standard configuration across the instruments. CS&T baseline
reports from 10 FACSCanto™ II Flow Cytometers at different CCLS
locations were compiled to create a “virtual” flow cytometer.
Linearity and the Electronic Noise Robust SD (rSDEN) values from
each detector was compared in order to define the predicate values
for the “virtual instrument”. Then the target MFI value for the bright
CS&T bead was generated on a single instrument, with lyse/washed
whole blood samples. The same lot of CS&T beads were run on
each instrument in order to achieve the same target MFI value with
the bright beads; cytometer application settings were then saved.
Instrument performance monitoring and the challenge in
implementing instrument-to-instrument standardization across three
continents will be discussed in the poster.
155/B34
Sirosh Bokhari1, Wen-Rong Lie2, Ted Baginski1
1
Discovery & Development Solutions, EMD Millipore
Corporation/ Merck KGaA, St. Charles, MO, United States,
2
EMD Millipore Corporation/ Merck KGaA, St. Charles, MO,
United States
Background: Mutations in the EGFR (Epidermal Growth Factor
Receptor) gene that lead to over expression and activity of the
receptor have been associated with a number of cancers including
Non Small Cell Lung Carcinomas (NSCLC). EGFR receptor tyrosine
kinase regulates cell proliferation, survival, differentiation and
migration through downstream signaling of ERK 1/2, Akt and STATs
andmutations in the gene result in dysregulated function and
constitutive activation of the receptor and its downstream signaling
cascade promoting tumor development and proliferation. The
constitutive activation of EGFR has prompted the development of
EGFR tyrosine kinase inhibitors (TKIs) which can cause regression
in NSCLC cells. Targeted inhibition of activated protein kinases
using small molecule drugs or antibodies is an effective approach
to cancer therapy. Exon 19 deletions and L858R mutation constitute
the most common mutations associated with EGFR TKI sensitivity
Speaker/Author
Index
Poster Session
Abstracts
Commercial
Tutorials &
Exhibits
A Cross-Platform Validation Study of EGFR-Activating
Mutations in Non-Small Cell Lung Carcinoma Cells
Oral Session
Abstracts
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
Special
Lectures
154/B33
148
and are also associated with a greater than 70% response rates
in patients treated with the TKIs erlotinib or gefitinib. T790M
substitution and exon 20 insertions, on the other hand, are
responsible for acquired resistance to TKIs. The mechanisms behind
the role of EGFR mutations in NSCLC development, however, are
not fully understood.
Methods: NSCLC cell lines harboring various EGFR mutations
were used to determine EGFR expression and activation as
well as apoptotic gene expression following Gefitinibtreatment.
Moreover, a cross-platform validation was carried out to correlate
the results byflow cytometric analysis and MILLIPLEX® MAP using
the Luminex® technology. NSCLC cell lines either harbouringthe
L858R / T790MmutationsortheExon 19 deletionalong withan
NSCLC cellline wild-type for EGFR were used for the study.
Results: The L858R / T790Mcell line expressed higher levels of
EGFR mRNA andboth mutant cell lines demonstrated higher levels
of total and phosphorylated EGFR protein. EGFR-mutant cells
showed induction of several apoptotic genes following treatment
with Gefitinib. Moreover, these cellsshowinga higherconstitutive
expression of the prosurvival marker BCL2, also demonstrated a
significant decrease in pBAD, another prosurvival marker, after
Gefitinib treatment.
Conclusions: Cross platform analyses between qPCR, flow
cytometry and MILLIPLEX® MAP helped correlate gene regulation
at transcriptional and post-transcriptional levels. Such analyses will
be instrumental for understanding drug effects and devising specific
treatments and therapeutic targets.
156/B35
A Flow Cytometric Approach to Monitoring Cell
Health and Productivity of a Recombinant Human
Cell Line in Bio-pharmaceutical Development
Megan Gottlieb1, Jui Chang Chuang2, Ferenc Boldog2
Cell Culture Process Development, Shire HGT, Lexington,
MA, United States, 2Cell Culture Process Development, Shire,
HGT, Lexington, MA, United States
1
Purpose: Over the past several years, the use of flow cytometry in
cell line development in the biopharmaceutical arena has become
a well established technology platform. The advantages of using
this technique include an increase in cellular productivity, as
well as, a decrease in the time it takes to develop a stable, high
producing cell line. In addition to using flow cytometry in cell line
development, we describe here the utility of using a panel of flow
cytometry-based assays to monitor cell health during a DoE study.
Our primary goal in this work is to develop flow cytometry-based
assays to monitor the cell culture process and provide diagnostic
tools during process development.
Methods: A recombinanthumancell line was cultured under a
variety of process conditions in scale-down perfusion bioreactors.
Cell samples were obtained at several time points during the
production phase of cell culture. Cells were assessed by flow
cytometry for cell cycle, cellular reactive oxygen species (ROS), and
for different cell death modes (senescence, autophagy, apoptosis
and necrosis).
Results: The dataa) demonstrate differences in cell proliferation
among bioreactors, b) provide evidence that cellular ROS plays a
role in cell death, and c) eliminate apoptosis as a major factor in
cell viability decline.
Conclusions: We continue to expand and optimize this panel of
flow cytometric cell health assays as part of process development
for perfusion bioreactors.
ISAC 2013 Program and Abstracts
Implementation of a Flow Cytometric Eosinophil
CD11b Expression Assay for Clinical Application
Poster Session
Abstracts
Conclusion: We identified a new strategy for robust cellular-level
sensing of the free radicals with dramatically improved sensitivity,
stability, and specificity.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Results and Discussions: We found this core-shell architecture
forms an efficient donor-to-acceptor system via Luminescence
Resonance Energy Transfer (LRET) scheme, since the blue
upconverting emission band matches well with the optimum
absorption bands of the Ru(II) complexes, shown in Fig 1b. Both
the emission intensity and lifetime of UCNPs - Ru(II) complexpair
respond to the presence of various amount of specific ROS.
Moreover, owing to the upconversion sensitization, the new
biosensors have the advantages of beingnon-blinking, nonbleaching, and background-free. Furthermore, the near-infrared
(NIR) light excitation of the UCNPs endows the new responsive
biosensors deep penetration for luminescence imaging of ROS in
living systems. We also found this strategy is universal for designing
a series of molecularly-specific biosensors to probe biomedical
important molecules and ions, such as reactive oxygen/nitrogen
species, cations, and anions.
Oral Session
Abstracts
The development of bead-based multiplexed immunoassay
technologies has facilitated the rapid and thorough assessment
of clinical specimens for a large array of cytokines, chemokines,
growth factors, as well as many other relevant biomarkers
especially for specimens available in limited quantities. A major
Method: Here, we report a simple and universal strategy for
ROS detection by assembling the responsive Ru(II) complexes to
upconversion nanocrystals (UCNPs). The UCNPs are able to stepwise absorb two or more near-infrared photons at 980 nm, and emit
blue emissions at 478 nm. As shown in Fig 1a, the Ru(II) complex
can be encapsulated onto the UCNPs surface through the alphacyclodextrin assisted hydrophobic interaction.
Commercial
Tutorials &
Exhibits
Christina Baker, Shelley Secor-Socha, David Roumanes, Tim
Mosmann, Sally Quataert
University of Rochester, Rochester, NY, United States
Background: The Reactive oxygen species (ROS) have attracted
significantattentions in thebroad fields ofbiochemistry, cell biology
science, and medical research, since they are thought to be
associated with various pathological conditions and disorders.
Specific fluorescent biosensors have been demonstrated as powerful
means for detection of ROS due to their high sensitivity, rapid
response time, and high spatial and temporal resolutionswith the
aids of microscopic imaging techniques. Thus the field seeks robust
rational design to produce stable high-contrast responsive sensors to
each specific ROS molecule.
Poster
Session
A Micromethod for Bead-based Microarray
Immunoassays Enabling Clinical Translational
Research Studies
Jiangbo Zhao1, Run Zhang1, Lixin Zhang1, Yiqing Lu2, Ewa M.
Goldys2, Dayong Jin1
1
Macquarie University, Sydney, Australia, 2 Macquarie
University
Wednesday,
22 May
158/B37
Lightening up the Cellular-Level Reactive Oxygen
Species Using Upconversion Sensitized Ruthenium
Biosensors
Tuesday,
21 May
Conclusions: This work outlines the resources and infrastructure
required to implement flow cytometric cellular biomarker assays
in the clinical development process. Furthermore, we demonstrate
how allocation of resources in the field of sample handling and
standardization of data acquisition andanalysis minimizes assay
variability.
159/B38
Monday,
20 May
Results: The data from the stability assessment indicate <4 hour
post-collection stability, while the processed sample stability is
15 days when stored and shipped at 4ºC. Intra-subject variability
(biological variability) was determined to be < 20%CV. As such a
mitigation plan was developed to minimize the assay variability.
To overcome the limited stability of blood samples post-collection
(<4hrs), clinical specimens were collected and processed at the
clinical site. On-site training of clinical site analysts was absolutely
required in order to achieve a passing proficiency assessmentin a
pilot study measure of intra-assay (<20% CV), inter-assay (<30%
CV) and inter-analyst variability (<30% CV). To minimize effects of
variation during instrument set-up and data analysis, a validated
sample acquisition and gating strategy was employed.Upon sample
receipt and data analysis, intra-assay comparison between duplicate
samples (<20% CV) and inter-laboratory assessment (<30% CV) all
fell within the acceptable range.
Sunday,
19 May
Methods: A flow cytometric assay protocol was developed and
analytically validated in whole blood to measure CD11b antigen
expression on the cell surface of eosinophils. Briefly, blood from
healthy volunteers was collected into sodium citrate vacutainer
tubes and stimulated with DK-PGD2ex vivo. Clinical samples were
lysed, fixed and stained with an APC-conjugated CD11b antibody.
Eosinophil cell surface expression of CD11b was reported as mean
APC equivalents (MESF). As a biological control, PBS stimulated
blood was used for each clinical sample to determine basal CD11b
expression. For assay performance characterization and validation,
measurements of assay precision, sample stability as well as the
analysis of biological variability were evaluated. Pilot studies were
arranged to assess clinical site readiness in sample preparation
before initiating clinical studies.
Saturday,
18 May
Background: Flow cytometric monitoring of cellular immune
responses is becoming an increasingly important tool in the clinical
development process. Overcoming logistical challenges such as
limited sample stability and variation between labs and instruments
is essential however forthe implementation of sensitive, robust
and reproducible flow cytometric biomarker assays in clinical
applications. Herein we detail performance characteristics and
outline the validation and clinical implementation plan for an ex
vivopharmacodynamic assay that measures the inhibition of cell
surface CD11b expression on eosinophils in whole blood by flow
cytometry.
obstacle to the adoption of such technologies for use in clinical
translational studies within an academic setting is that the high cost
of the multiplexed bead array kits is prohibitive when processing
large numbers of samples. To encourage the use of this advanced
technology, a micromethod version of the xMAP™ (Luminex Corp.)
has been developed utilizing a fraction of the reagents proscribed
when performing this assay per manufacturer’s recommendations.
Additionally, the use of super-chemiblock was beneficial for serum
samples that contained high levels of non-specific antibodies that
directly bind the beads. The micromethod multiplex assay has been
optimized to maintain assay sensitivity, precision and accuracy,
and at the same time reduce non-specific background signals
while increasing the number of samples able to be processed using
a single kit by 4 fold. Importantly, sample sizes as little as 5 µL
of serum, plasma or tissue culture supernatant can be used. The
micromethod multiplex assay offers investigators an alternative
approach for assaying a multitude of analytes and provides
academic scientists effective ways to screen for biomarkers in
discovery research.
Special
Lectures
Richard Wnek , Michelle Tseng, Tajneen Natasha, Diana
Gallagher, Caroline Cilissen, Russell Weiner, Dianna Wu
Merck, Rahway, NJ, United States
Congress
Overview
157/B36
149
Congress
Overview
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
Special
Lectures
were re-stimulated ex vivowith KLH. Comparison of pre-test and
post-immunization sample resultsshowed KLH-specific increases
inthe expression of CD69, IFNγ, and TNFαin cynomolgus
CD4+CD8-T cells following antigen-specific stimulation. Moreover,
antigen-specific T-cell responses were greater in T cells from KLHimmunized monkeys treated with immuno-stimulatory agents,
compared to T-cells from KLH-immunized monkeys administered
vehicle control.These data demonstrate that this method is robust
for evaluating the effects of immuno-modulatory test articles on
ex vivo antigen-specific T-cell responses and can be used to assess
immunotoxicity and/or intended pharmacology in a non-human
primate model system.
Cell Proliferation and Death
(B40 – B49)
161/B40
Figure 1. The schematics of upconversion sensitized ruthenium
biosensors based on LRET scheme.
Biopharmaceutical Applications
(B39)
A High-Throughput Flow Cytometry-Based Method
for the Detection of Antigen-Specific T Lymphocyte
Activation in Non-Human Primates for Pre-Clinical
Drug Development Assessments
Matthew Bernard1, Gautham Rao1, John Loffredo2, Anish Suri2,
Helen Haggerty1, Wendy Freebern1
1
Immunotoxicology, Bristol-Myers Squibb, New Brunswick,
NJ, United States, 2Immunosciences Discovery Biology, BristolMyers Squibb, Princeton, NJ, United States
Adaptive immunity is acritical component of host defense,
protecting organisms against invading pathogens. T lymphocytes, in
particular, are central for potentiating adaptive immune responses.
Thus, understanding how their activity can be influenced by
pharmaceuticals is an important component of drug development.A
high-throughput, flow cytometric-basedactivation marker
characterization and intracellular cytokine staining (ICS) method
was developed to characterize ex vivoantigen-specific T-cell
activation in non-human primate peripheral blood mononuclear
cells (PBMCs).In brief, PBMCs isolated from peripheral blood
of monkeys immunized with keyhole limpet hemocyanin
(KLH), Engerix B (Hepatitis B vaccine), and tetanus toxoid (TT)
werestimulated ex vivowithin 4 hours of blood collection with the
protein antigensand anti-CD28/49d. After 4hours of stimulation,
Brefeldin A was added to block extracellular transport of cytokines
and PBMCsare incubated for an additional 16-20 hours. PBMCs
were then immuno-stained to assess surface expression of CD3,
CD4 and CD8, while Live/Dead Aquawas used to characterize
cell viability. Intracellular immuno-staining was subsequently
performed to assess cytokines, TNFα and IFNγ, and the activation
marker, CD69. Fixed immuno-stained samples were analyzed on
a BD FACSCanto II flow cytometer. This method was implemented
on atoxicity study in which cynomolgus monkeys treated with
vehicle control or proprietary immuno-modulators with potential
immuno-stimulatory activitywere immunized with KLH. PBMCs
isolated from monkey whole blood pre-test (prior to dosing and
immunization), or 22 and 57 days post primary KLH immunization
Oscar Onyema1, Ivan Bautmans1, Marc De Waele2, Joeri
Aerts3, Tony Mets1
1
Gerontology, Vrije Universiteit Brussel, Brussels, Belgium,
2
Hematology, Universitair Ziekenhuis Brussel, Brussels,
Belgium, 3Immunology and Physiology, Vrije Universiteit
Brussel, Brussels, Belgium
The expression of CD57 and the absence of CD28 on the surface of
circulating T-lymphocytes have been used as “senescence” markers.
The association of these surface markers with characteristics of
senescence that emerge during in vitro cellular aging is, however,
not clear. Using flow cytometry, polymerase chain reaction,
and proliferation assay, we have analyzed CD8+ T-cells in old
and young human subjects for their expression of CD57 and
CD28, and tested these subpopulations for proteins that play
different roles in cellular senescence and, T-cell homing and
differentiation. Significantly higher proportions of CD28+CD57+
and CD28-CD57+ cells were found in old subjects. Also these
two subpopulations from the elderly showed complete lack of
proliferation.Further characterization of the cells from the old
subjects showed that the expression of p21 and CD95 was highest
in CD28+CD57+ cells, as was the frequency of CD95, CD45RO
and CCR5 expressing cells, with most of CD28+CD57+ cells
expressing the surface markers. These three surface proteins are
known to be highly expressed by cell populations believed to be
dominated by senescent cells. The parallelism in the expression
of p21 and CD95 among the different subpopulations and
their highest level in CD28+CD57+ cells portends a possible
modulatory role of CD95 on p21 expression, independent of p53
that showed no difference in its level of expression among the
different subpopulations, and favours a likely role of these proteins
in promoting senescence in these cells. Notably, our observation
of a subpopulation of CXCR2+ cells among CD28-CD57+ cells
might emphasize the heterogeneity of CD28-CD57+ cells and the
possibility of a subpopulation of CD28-CD57+ cells that would
represent their senescent phenotype. We conclude that among
CD8+ T-lymphocytes, characteristics that correspond to a senescent
cell type are most prominent in CD28+CD57+ cells. These results
provide further evidence for the existence of senescent cells in vivo
and their possible importance in ageing.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
160/B39
Differential Homing and Senescene of CD8+
T-Lymphocytes from Elderly Humans
150
ISAC 2013 Program and Abstracts
Induced Germination of B. atrophaeus Spores by
Singlet Oxygen-Sensitizing Cationic Oligo-Phenylene
Ethynylenes (OPEs)
Background: In mixed 2D co-cultures, irradiated cells stimulate
proliferation of bystander cells via direct (physical) intercellular
contacts; however, neither gap junctional intercellular
communication nor long-range factors released into the medium
appear to be involved (Cytometry 2003; 56A: 71−80).The present
work is aimed to elucidate the effect of bystander cells onirradiated
cells, namely on their proliferation.
Methods: Confluent monolayers of rat liver epithelial cells (WBF344) were acutely exposed to137Cs γ-rays at dose of 5 Gy. To
test whether proliferation of irradiated cells is affected by the
number of adjacent bystander cells of the same type, irradiated
cells were plated together with unirradiated cells in different
proportions followed 24 h later by two-scheme FCM analysis of cell
proliferation proposed by Gerashchenko and Howell (Cytometry
2003; 54A: 1−7). By the time of analysis the vast majority of cocultured cells should physically contact each other. Also, the impact
of cell proximity on proliferation of irradiated cells was determined
by co-culturing different densities of irradiated and unirradiated
cells that were plated and mixed at ratio1:1. Thepopulations of
co-cultured cells were distinguished by using cell tracker dye
(carboxyfluorescein diacetate, succinimidyl ester).
Poster
Session
Commercial
Tutorials &
Exhibits
Results: Increasing the fraction of unirradiated bystander cells
relative to irradiated cells led to a decrease in proliferation of
irradiated cells. In co-cultures, in which 6% of irradiated cells were
initially mixed with 94% of unirradiated cells, proliferation ratio for
irradiated cells was 1.3-fold lower than that for irradiated cells in
co-cultures, in which 75% of irradiated cells were mixed with 25%
of unirradiated cells (P < 0.05). However, proliferation of irradiated
cells was not affected by cell density factor.
Oral Session
Abstracts
Conclusion: The fact that the number of bystander cells, but not
cell proximity, affects proliferation of irradiated cells prompted us to
assume that the long-range soluble factors are involved.
Poster Session
Abstracts
Methods: In this study, we developed a novel automated method
for the quantification of yeast budding percentages using
Cellometer image cytometry. The automated method utilizes a
dual-fluorescent nucleic acid dye (acridine orange and propidium
iodide) to specifically stain live cells for imaging analysis of unique
morphological characteristics of budding yeast. In addition, cell
cycle analysis using propidium iodide to measure DNA content
is performed as an alternative method for budding analysis. The
methods were compared directly to manual counting of yeasts to
identify budding populations.
Bogdan Gerashchenko1, Roger Howell2
R. E. Kavetsky Institute of Experimental Pathology, Oncology
and Radiobiology, Kyiv, Ukraine, Kyiv, Ukraine, 2New Jersey
Medical School Cancer Center, Newark, NJ, USA, Newark,
NJ, United States
1
Wednesday,
22 May
Background: The measurements of concentration, viability, and
budding percentages of Saccharomyces cerevisiae are performed on
a routine basis in the biofuel and brewing industries. Generation
of these parameters is of great importance in a manufacturing
setting, where they can aid in the estimation of product quality,
quantity, and fermentation time of the manufacturing process.
Specifically, budding percentages can be used to estimate the
reproduction rate of yeast populations, which directly correlates
with metabolism of polysaccharides and bioethanol production,
and can be monitored to maximize production of bioethanol during
fermentation. The traditional method involves manual counting
using a hemacytometer, but this is time-consuming and prone to
human error.
Probing the Impact of Bystander Cells on Irradiated
Cells in Mixed Co-cultures
Tuesday,
21 May
Leo Chan1, Daniel Laverty1, Alexandria Kury1, Dmitry Kuksin1,
Alnoor Pirani2, Kevin Flanagan3
1
Technology R&D, Nexcelom Bioscience LLC, Lawrence,
MA, United States, 2Applications, Nexcelom Bioscience
LLC, Lawrence, MA, United States, 3Software Development,
Nexcelom Bioscience LLC, Lawrence, MA, United States
164/B43
Monday,
20 May
Automated Quantification of Budding
Saccharomyces cerevisiae Using an Image Cytometry
Method
Sunday,
19 May
163/B42
Conclusions: Since concentration, viability, and budding
percentages can be obtained simultaneously using image cytometry,
the automated method can be integrated into the fermentation
quality assurance protocol, which can improve the quality and
efficiency of the bioethanol production process. The developed
method can be directly beneficial to both biofuel and brewing
fermentation process.
Saturday,
18 May
Cationic oligo-phenylene ethynylenes (OPEs) are quickly gaining
consideration as an alternative to traditional antibiotics, as they
have been shown to exhibit superb biocidal activity against
both Gram negative and Gram positive strains of bacteria. To
better understand the mechanisms by which a specific class of
oligomer—herby referred to as EO-OPE-Th-C2—interacts with
spore-forming Bacillus atrophaeus, flow cytometry is implemented.
Using SYTO 21 and Propidium iodide as live and dead stains,
respectively, patterns of fluorescence are evaluated to analyze
the capacity by which the EO-OPE-Th-C2 affects the viability of
B. atrophaeus, both as vegetative cells and spores. The bacteria
were exposed to varying concentrations of oligomer, both in the
presence and the absence of ultraviolet light. Results indicate that
appropriate concentrations of the oligomer facilitate germination
of B. atrophaeus spores under specific conditions. As this class
of oligomer has previously been found to act as a singlet-oxygen
sensitizer upon the exposure to UV light, it is hypothesized that the
resulting conditions encourage the germination of spores; however,
the resulting viability of the germinated spores remains to be
studied.
Results: We were able to show comparable yeast budding
percentages between manual and automated counting method, as
well as cell cycle analysis. Budding percentages were measured
from 0.5, 1, 2, 4, 6, 8, 10, 24, and 30 hours of standard liquid
culture of Saccharomyces cerevisiae. The results were comparable
at each time point. In addition, the automated image cytometry
method was used to analyze and characterize viability and budding
population of corn mash biofuel samples directly from fermenters
during standard fermentation at 1.5, 8, 23, 39, and 54 hours. The
results were directly correlated between viability and budding, as
expected.
Special
Lectures
Harry Pappas, David Whitten
Department of Biomedical Engineering, University of New
Mexico, Albuquerque, NM, United States
Congress
Overview
162/B41
Speaker/Author
Index
ISAC 2013 Program and Abstracts
151
Congress
Overview
Special
Lectures
Bradykinin Functions in Bone Marrow Metatastis
Amira Zarrouk1,2, Hayet Iddir3, Meriem Yousfi3, Thomas Nury3,
Catherine Gondcaille3, Mohamed Hammami1, Gérard Lizard4
1
Laboratoire de Biochimie - UR ‘Nutrition humaine et désordre
métabolique’, Université de Monastir, Faculté de Médecine,
Monastir, Tunisia, 2EA7270 (Biochimie du Peroxysome,
Inflammation et Métabolisme Lipidique), Université de
Bourgogne / INSERM / Faculté des Sciences Gabriel,
Dijon, France, 3EA7270 (Equipe Biochimie du Peroxysome,
Inflammation et Métabolisme Lipidique), Université de
Bourgogne / Faculté des Sciences Gabriel, Dijon, France,
4
EA7270 (Equipe Biochimie du Peroxysome, Inflammation et
Métabolisme Lipidique), Université de Bourgogne / INSERM
/ Faculté des Sciences Gabriel, Dijon, France
In Alzheimer’s disease, some lipid alterations point towards
peroxisomal dysfunctions (Lizard et al., J Alzheimers Dis. 2012;
29(2): 241-54). An accumulation of C22:0 and saturated very
long chain fatty acids (VLCFAs: C24:0 and C26:0), substrates for
peroxisomal β-oxidation, has been found in cortical lesions of AD
patients.
Human neuronal cells (SK-N-BE) were treated with C22:0, C24:0
and C26:0 (0.1 - 20 µM; 48 h). The impact of these fatty acids (FAs)
on cell death induction was evaluated by various flow cytometric
and microscopical methods. Flow cytometric analysis were
performed to quantify dead cells and the cells in SubG1 (apoptotic
cells) after staining with propidium iodide (PI). The impacts of FAs
at the mitochondrial and lysosomal levels were evaluated with
(DiOC6(3), JC1) and Acridine Orange, respectively. By conventional
fluorescence microscopy associated or not with confocal
microscopy, the effects of FAs on the cytoskeleton (neurofilaments,
tubulin, and actin), on peroxisomal components (ABCD1 and
ABCD3 transporters, catalase) were studied by using fluorescent
probes or specifically dedicated antibodies. The morphological
aspect of SK-N-BE cells was also determined after Hoechst staining
in order to distinguish between viable, apoptotic and necrotic cells.
Oxidative stress was estimated by flow cytometric analyses with
different fluorescent probes (H2DCFDA, DHE, DHR123, MitoSox,
and DAF), and by fluorescence microscopy in the presence of
monochlorobimane for GSH quantification. Lipid peroxidation was
quantified by flow cytometry with an antibody reacting with 4-HNE.
With C22:0, C24:0 and C26:0 (1 to 20 µM), an induction of cell
death was observed. It was characterized by higher percentages of
dead cells with depolarized mitochondria and altered lysosomes.
No sign of apoptosis was observed with Hoechst staining (no
increase of cells with condensed and/or fragmented nuclei) and
by analysis of SubG1 cells (no increase of the percentage of
cells in SubG1). An overproduction of reactive oxygen species,
including superoxide anions and H2O2, was also revealed, no or
slight overproduction of NO was found, and a decrease of GSH
associated with an increased lipid peroxidation was detected.
Thus, C22:0, C24:0, and C26:0 are able to induce neuronal
damages. Consequently, increased levels of these FAs in cortical
lesions of Alzheimer patients might favor the development of
Alzheimer’s disease.
Henning Ulrich1, Claudiana Lameu2, Mariusz Ratajczak3
Depto de Bioquímica, Instituto de Química, Univ of Sao
Paulo, Sao Paulo, Brazil, 2Departamento de Bioquímica,
Instituto de Química, Universidade de São Paulo, São Paulo,
Brazil, 3Stem Cell Institute at James Graham Brown Cancer
Center, University of Louisville, Louisville, KY, United States
1
Background: Many primary human tumors form metastasis in the
bone marrow. This type of metastasis is present in approximately
350,000 patients that die annually in the U.S. alone. It has been
shown that the stromal cell-derived factor-1 (SDF-1)-CXCR4
axis plays an important role in bone metastasis, as well as in
neuroblastoma (NB) and other tumors. This mechanism is similar
to the mechanism of "homing" of haematopoietic stem cells to
the bone marrow. Although many advances have been made,
the process of metastasis in the bone marrow is still not fully
understood. Therefore, the purpose of this work was to study new
chemo attractors in bone metastasis and to identify factors that
increase the responsiveness of the SDF-1-CXCR4 axis, using as
model human NB cells.
Methods: Interactions between SDF-1-CXCR4 and bradykinin and
effects of these compounds on receptor expression and activity
levels, proliferation, chemotaxis were studied in CHP-100, CHP134 and IMR-32 human neuroblastoma cell lines using flow
cytometry, and imaging together with standard molecular biology
and protein biochemistry methods. Transplantation of NB cells into
nude mice was performed for assaying the capacities of tumor cells
in homing to the bone marrow.
Results: Our results revealed that BK is a priming agent for
cancer cells in response to a SDF-1 gradient, i.e., BK induced
chemotaxis of NB cells to low doses of SDF-1 in vitro. In addition,
BK increased 1) adhesion of NB cells to fibronectin; 2) MAPK
p42/44 phosphorylation; 3) SDF-1-induced intracellular calcium
mobilization. Furthermore, 48 h after tumor cell injection into
immunodeficient mice, we found that following BK-pretreatment
more NB cells were present in the bone marrow cavities when
in control experiments with untreated cells, as evaluated by
detection of human specific α-satellite DNA using real-time PCR.
High expression levels of the kinin system in the bone marrow of
irradiated mice suggest the importance of BK in metastasis to the
bone marrow.
Conclusions: Our study provides novel roles for the kinin system in
metastasis of NB cells to the bone marrow. Intervention in the kinin
system may improve current and future therapies for cancer.
167/B46
Detection and Evaluation of Cellular Phenomena in
the Breast Cancer Cell Line Resistance to Cisplatin
Jelena Markovic1, Bojana Krunic2, Nadezda Apostolova2,
Sergio Bañuls1, Federico V. Pallardo3
1
Core research facility, University of Valencia, Valencia, Spain,
2
Pharmacology, University of Valencia, Valencia, Spain,
3
Physiology, University of Valencia, Valencia, Spain
Cisplatin is a cytostatic drug that is used in the treatment of many
different types of cancer. Recently, several clinical studies have
been initiated introducing cisplatin in the treatment of the triple
negative and Brca1 positive breast cancer with promising results.
However, in some cases, the resistance to therapy has been
detected.
In this study we have evaluated the cellular response involved in
the resistance to cisplatin using the mammary adenocarcinoma cell
line, MCF7, and its counterpart overexpressing human oncogene
Bcl-2.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Saturday,
18 May
Sunday,
19 May
166/B45
Characterization of C22:0 and Saturated Very
Long Chain Fatty Acids (C24:0; C26:0) – Induced
Cell Death by Microscopical and Flow Cytometric
Methods on Human Neuronal Cells (SK-N-BE)
Monday,
20 May
165/B44
152
ISAC 2013 Program and Abstracts
169/B48
Measure of Cell Cycle with the Tali Image Instrument
Using a Broad Range of Dyes
Telma Lopes, Laetitia Roh, Miguel Garcia
École Polytechnique Fédérale De Lausanne, Lausanne,
Switzerland
Poster Session
Abstracts
The most common approach for determining the cell cycle
stages is based on cellular DNA content. The information of the
cell cycleprovides countless information about a consequence
of stimuli, like a drug treatment or a transfection with a gene of
interest. This analysis is based on the ability to stain the cellular
DNA in a stoichiometric manner, with means that the amount
of fluorescence is directly proportional to the amount of DNA
Speaker/Author
Index
ISAC 2013 Program and Abstracts
170/B49
Oral Session
Abstracts
We optimized components in the improved click reaction
conditions which best preserved the R-PE fluorescence while
obtaining a bright EdU signal. By first labeling live cells for surface
Commercial
Tutorials &
Exhibits
The easy-to- use and popular Click-iT® EdU (ethynyl-deoxyuridine)
introduced in 2007 for measuring cell proliferation using click
chemistry, due to the presence of copper and reactive oxygen
species (ROS) mediated damage to fluorescent proteins prevents
the simultaneous detection of EdU, GFP and R-Phycoerythrin (R-PE)
fluorescence. We present here chemical modifications to the click
reaction resulting in “copper safe” catalysis of the click reaction,
whereby flow cytometry measurements of cell proliferation
using Click-iT® EdU are demonstrated to be compatible with
immunophenotype panel using R-PE conjugates and GFP
expressing proteins. R-PE is known to be quenched by exposure
to free radicals. The basis of the click chemistry improvement is
the use of a copper (I) ligand combined with a modified dye azide
detection reagent. Together, the copper ion is sequestered with
the ligand and prevented from quenching GFP fluorescence but
still remains available to catalyze the click reaction. Ligands with
excessively high affinity for copper protect GFP fluorescence, but
result in diminished or no EdU signal. The modified dye azide with
an intrinsic affinity for copper reduces copper-related ROS damage
and facilitates the click reaction.
Poster
Session
Carolyn DeMarco1, Jolene A. Bradford2, Scott Clarke1, Ruth
Deveny1, Kyle Gee1, Scott Grecian1, Upinder Singh1
1
Life Technologies, Eugene, OR, United States, 2 Life
Technologies
Wednesday,
22 May
Improved Click Chemistry Demonstrating Edu Cell
Proliferation with Gfp Expressing Cells and R-Pe
Based Immunophenotyping
Mitochondria are critical cellular organelles that play a fundamental
role in cellular metabolism and oxidative stress and are well
known to trigger multiple cell death pathways including apoptosis
and autophagy. The study of sequence of mitochondrial events
as it relates to both autophagic and apoptosis events can provide
critical insights into mechanism of cellular homeostasis and stress
and death. Availability of rapid and simplified cytometric testing
methods for evaluating mitochondrial changes, autophagy, and
apoptosis can greatly enhance our understanding of mechanism
of compound action. In this study, we investigated a series of
compounds to evaluate apoptotic and autophagic effects in context
of mitochondrial changes using plate-based assays on EMD
Millipore’s easyCyte 8HT system. Studies utilized multiplexed
FlowCellect assays for mitochondrial membrane potential changes
and apoptosis/cell death markers and the information obtained was
compared with results from anautophagic LC-3 based antibody
assay. Dose response studies with Niclosamide, an anti-helmintic
drug was shown to cause rapid mitochondrial depolarization in
short durations of 4 hrs of treatment of Jurkat cells. No apoptotic
or cell death impacts were observed in parallel at low doses,
however interestingly autophagic changes were observed to rise
in concert and almost identicallywith mitochondrial potential
changes. Increased time with niclosamidecaused increase in
mitochondrial, apoptotic and cell death response. Compounds
such as staurosporine and gambogic acid demonstrated significant
mitochondrial depolarization and apotosis or cell death; no
autophagic impacts were observed under the dose range
tested. The assays thus demonstrate great power in being able
to distinguish between potency of compounds and conditions
that result in autophagy and apoptosis and the sensitivity of
mitochondrial changes in these processes. The simplicity of the
assays described coupled with the ease of use of plate based
microcapillary cytometry can allow researchers to obtain a more
comprehensive insight into how compounds modulate apoptotic
and autophagic processes and their relationship to mitochondrial
dysfunction.
Tuesday,
21 May
168/B47
Katherine Gillis, Julie Clor, Kamala Tyagarajan
EMD Millipore, Hayward, CA, United States
Monday,
20 May
It is of great importance to strive to unravel the mechanisms of the
resistance to the neoplastic drugs and understand how to overcome
it in order to create new successful treatment protocols.
Investigating Mitochondrial Changes during
Autophagy and Apoptosis Using Microcapillary
Cytometry
Sunday,
19 May
The increased resistance that MCF7 Bcl2 cells demonstrate can
be attributed to the protective effect that this oncogene has in
the process of apoptosis by attenuating the cellular stress at
different levels. In our cell model we demonstrate that autophagy
contributes, at least in part, to cell survival and its inhibition could
be the way to prevent the induction of the resistance and thus
increase the efficiency of the cisplatin treatment.
Saturday,
18 May
Our results show that cisplatin produces different effects in the two
cell lines studied. The MCF7 WT vs. MCF7 Bcl2 show increased
apoptosis, by both techniques we performed, which points out the
effect of Bcl2 in the resistance to the induction of the cell death.
The inhibition of basal autophagy by Bcl2 was also confirmed in
our model. When the treatment with cisplatin was accompanied
by the inhibition of autophagy with 3MA, we have observed the
increase of the apoptosis level. The cell cycle also shows different
profile depending on the presence of Bcl2: the MCF7 WT cells
show the increase of the cell death while the MCF7 Bcl2 cells are
characterized by the G0/G1 arrest. We have observed, by confocal
microscopy, the increased level of GSH and its nuclear distribution
in the cells surviving the treatment, as well as mitochondrial
reorganization and aggregation around the nucleus, especially
striking in MCF7 Bcl2 cells.
Special
Lectures
markers using R-PE conjugates prior to fix/perm and click chemistry
labeling, cultured Jurkat cells were pulsed with EdU, labeled using
standard work flow, demonstrating the improved detection.In
addition BacMam transduced cells expressing GFP were firsttreated
with cell cycle inhibitors then pulsed with EdU and used to
demonstrate GFP and click chemistry compatibility. The use of the
described modified click reaction is an enabling improvement over
originally described copper based click reactions and will further
enhance the utility of EdU based cell proliferation applications in
flow cytometry.
Congress
Overview
Amnis Image Stream IS 100 was used to simultaneously detect in
the same sample: late apoptosis, by the imaging and analysis of
nuclear morphology, autophagy, by LC3 intensity and distribution,
and cell cycle analysis by the intensity of PI staining. Flow
cytometry studies were performed to assess apoptosis progression,
using Annexin V-488/PI staining, as well as alteration of oxidative
environment, evaluated by dihydrorhodamine 123. Mitochondrial
morphology and dynamics (Mitotracker green staining), and
glutathione distribution (GSH staining by CMFDA), were evaluated
by means of confocal microscopy.
153
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
The image-based cytometry has the advantage of allowing the
visualization and the analysis of thousands of events in seconds.
The Tali Image Based Cytometer from Life TechnologiesTM is a
3-channel (bright field, green and red fluorescence) benchtop
cytometer that is capable of performing a range of quantitative
cell-based suspension assays, including cell viability, GFP and RFP
expression, apoptosis or cell cycle, using only 25μL of sample.
We tested the ability of the Tali to measure cell cyclein fixed cells
using conventional protocols. The bright field and red fluorescence
images were capturedand the data presented in two histograms,
one for doublets exclusion andother for the cell cycle. The files
generated can be exported as flow cytometry standard (.fcs) to be
used in specific software to analyze and calculate percentages and
CV’s of the different phases. A side-by-side comparison between
the Tali-instrument and traditional flow cytometry demonstrate that
the two methods provide comparable data.Though flow cytometry
provides better coefficients of variation.
Many different dyes are available on the market to determine DNA
content. Each has different excitation and emission wavelengths
that allow the combination of cell cycle analysis with many other
different fluorochromesto definesubpopulations.
Methods: Peak-time analysis: A new signal processing approach
was evaluated to detect the fluorescence decay using nonmodulated excitation in flow cytometry. The time shift between
a fluorescence and scattering waveform was measured by simply
observing the different ‘peak-times’ of correlated Gaussian-like
pulses. Shifts occurred owing to the fluorescence lifetime of any
fluorophore tagged cell or microsphere.
Phase-filtered cell sorting: An analog hardware modification to a
cell sorter was evaluated to implement fluorescence lifetime based
sorting. Mammalian cells were sorted by implementing phasefiltering on a frequency-domain adapted FACSVantage.
Cell phasor cytometry: Phasor plots are a new analysis method
for multi-exponential fluorescence decay measurements.
The convenience and clarity of a phasor plot will indicate
heterogeneous fluorescence lifetime expression. Here we
incorporate the phasor plot into flow cytometry for high throughput
lifetime measurements.
Results: Peak-time measurements: Figure 1 presents peak-time
results using four different waveform fitting approaches. From
simulated delays, we resolved all the peak-times with varying
accuracy.
We tested a broader range of dyes that can be used on the Tali
(red fluorescence) and also on a complex multicolor experiment.
Vybrant DyeCycle Orange, Ruby, Sytox Orange, Sytox Advanced,
Draq5 and To-Pro 1 can be measured on the Tali and also with flow
cytometry. Although theexcitation laserswere notthe same forthese
differentexcitationsdyesonvarious instruments usedthe results
betweenTaliand the differentcytometers(Cyan-ADP and LSRII)are
similar.
The Tali Image Cytometer seems to be a quick, easy and affordable
instrument to have for a fast screening of cell cycle before
advancing into a more complex multicolor experience in flow
cytometry.
Cell Sorting and Selection
(B50 – B57)
171/B50
Advances in Fluorescence Decay Analysis: New
Techniques for Use in Cytometers of All Shapes and
Sizes
Ruofan Cao1, Mark A. Naivar2, Jessica Houston1
chemical engineering, new mexico state university, Las
Cruces, NM, United States, 2DarklingX, Los Alamos, NM,
United States
1
Background: Intracellular fluorescence decay measurements
are historically captured using fluorescence lifetime imaging
microscopy (FLIM). With FLIM’s static imaging and long integration
times, multiple fluorescence lifetimes can be obtained at a high
resolution and with optimum signal-to-noise. Because of the
dynamic nature of cytometry, decay kinetic approaches have
evolved slowly and remain non-commercial and less robust. For
several years our laboratory has been developing fluorescence
lifetime detection systems for flow cytometry to provide
cytometrists a concentration-independent means of quantification.
In this contribution we highlight three new findings centered on
approaches that will allow simple and straightforward integration of
fluorescence lifetime detection into modern flow cytometers.
Figure 1. Phase-filtered cell sorting: Sorting to a ---% purity was
accomplished using phase filtering. Figure 2 showes the separation
of fluorescence microspheres having independent fluorescence
lifetimes.
Speaker/Author
Index
Poster Session
Abstracts
within the cell. Flow cytometry has been the method of choice for
analyzing cell cycle and it still stands as the gold standard.
154
ISAC 2013 Program and Abstracts
Congress
Overview
Special
Lectures
Methods: Five commercial HCC cell lines(HepG2, HUH7, Hep3B,
PLC/PRF/5 and SNU475) and one primary cell line obtained in our
laboratory (HCC1) were single cell sorted in positive and negative
fractions for the following markers: EpCAM, CD133, CD44, CD56
and CD90. Cells were then cultured for one month in adhesion
(on collagen-coated plates in triplicate) before colony counting.
Tumor cells with specific CSC-like phenotypes, previously showing
increased ability of colony formation in adhesion, were tested for
sphere formation in suspension using ultra-low attachment plates.
The table below reports the a specific marker that identified a more
clonogenic cell population
HepG
2
0.45
0.4
PLC
0.35
SNU4
0.3
Hep3
B
0.2
0.15
0.1
0.05
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Figure 3
Single Cell Sorting for Cancer Stem Cells Enrichment
in Hepatocellular Carcinoma
Pos/Neg
6,3
0
Pos/Neg
3,1
17,7
Pos/Neg
0
1
sphere
adhesion
12,5
32,1
2,1
8,9
28,1
27,1
2,1
2,1
4,2
7,3
12,5
4,2
2,1
34,4
1
7
sphere
adhesion
sphere
adhesion
14,6
1
0
8
4,2
20,8
0
25
28,1
0
10,4
1
0
13,5
18,4
8,3
22,6
0
22,9
1
0
20,8
0
27,1
0
12,3
4,2
10,5
0
20
14,6
25
0
31,6
sphere
adhesion
0
20.4
0
9.1
0
0
0
0
0
0
0
0
0
0
0
0
sphere
0
0
-
-
-
-
-
-
-
-
Conclusions: In all cell lines, we identified phenotipically
different clonogenic cells, able to generate colonies in adhesion.
Furthermore, two cell lines - HCC1 and HepG2 - were also able to
form spheres in an anchorage-free manner. Interestingly, EpCAM+
and CD56+ cells showed similar clonogenic capacity, suggesting a
potential role of these markers in the identification of CSCs in HCC.
173/B52
Comparison of Proliferation Markers CD71, Ki-67,
and Pyronin Y in Combination with Hoechst 33342
Nicole Hanson1,2, Jessie Brown1,3, Peter Lopez2, Markus
Schober1
1
Ronald O. Perelman Department of Dermatology, New York
University Langone Medical Center, New York, NY, United
States, 2Office of Collaborative Science, New York University
Langone Medical Center, New York, NY, United States, 3Sackler
Institute, New York University Langone Medical Center, New
York, NY, United States
Cellular proliferation is fundamental for tissue function and is
tightly regulated.Actively proliferating cells cycle continuously
through the G1, S, G2 and M phases of the cell cycle, though some
cells arrest at defined checkpoints due to developmental signals, or
in response to cell intrinsic and extrinsic stress situationslike DNA
damage orrestricted nutrient supply. Adult stem cells, for example,
are commonly maintained in a slow-cycling, “quiescent” state that
is governed by developmental signals from the stem cell niche.
The quiescent behaviorof stem cells prevents replicative stress and
DNA damage, and supports self-renewalwhile sustainingthe stem
cells’long-term regenerative potential.
Similar to normal tissues, cancers are also heterogeneous and
comprised of rapidly proliferating and growth arrested tumor cells
with defined tumor initiating and growth promoting potential.
Understanding the proliferative differences between adult tissue
stem cells and their progeny, as well as cancer stem cells and their
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Pos/Neg
6,3
11,5
Poster Session
Abstracts
Background: Differently from other tumors, the cancer stem cell
(CSC) phenotype in hepatocellular carcinoma (HCC) has not been
unequivocally identified, mainly due to the rarity of CSCs within
the tumor bulk. Single-cell colony and sphere formation assays
have been proposed to assess CSC tumorigenic potential. Usually,
limiting dilution techniques are used for this purpose, although
the formation of cell aggregates may affect the results. Therefore,
approaches based on flow cytometry cell sorting are now being
developed. The aim of this work was to evaluate the clonogenic
potential (i.e. the capability of generating colonies in adhesion and
/or spheres in suspension) of phenotypically defined CSC from HCC
by means of single cell sorting.
Pos/Neg
56,5 47,4
Oral Session
Abstracts
Elena Trombetta1, Federico Colombo1, Silvia Mazzucchelli1,
Alessandra Cattaneo1, Daniele Prati2, Paolo Rebulla1, Laura
Porretti1
1
IRCCS Fondazione Ca' Granda Policlinico di Milano, Milan,
Italy, 2Ospedale "A. Manzoni" Lecco, Lecco, Italy
adhesion
Commercial
Tutorials &
Exhibits
172/B51
CD90
Poster
Session
Conclusions: There are numerous possibilities for adopting
fluorescence lifetime-based approaches into flow cytometry.
Coupled with new data acquisition systems, modern lasers and
detectors, and novel signal processing methods, the fluorescence
lifetime can, in a straightforward manner, be implemented. Our
work aims to demonstrate how time-dependent parameters might
be adopted in cytometry, and in this work we propose a few, of
many, ways in which fluorescence decay times can be exploited.
CD56
Wednesday,
22 May
0
CD44
Tuesday,
21 May
0.25
CD133
Monday,
20 May
HCC1
0.5
EpCAM
Sunday,
19 May
Figure 2. Phasor plot cytometer: We successfully demonstrated
phasor plotting with real cells and microsphers. Figure 3. is an
example phasor plot representing the single exponential decay
of a fluorescent microsphere population captured using a multifrequency domain flow cytometer.
Saturday,
18 May
Results: HUH7 generated colonies in adhesion with all sorted
markers but without statistically significant differences. Despite
all cell lines were able to generate colonies in adhesion, only the
primary HCC1 and the hepatoblastoma HepG2 cell lines generated
spheres with overlapping results for EpCAM and CD56. The table
below summarizes the main results. Bold characters indicate
statistically significant differences (p<0.05).
155
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
progenyis critical for the development of effective treatment of
cancer patients, and novel approaches in regenerative medicine.
Although the proliferative behavior of individual cells can
be characterized by flow cytometry based on their relative
incorporation of nucleotide analogs (BrdU), their expression
of proliferation antigens (Ki67), or the senescence marker (SAβ-Gal), and their respective DNA content (PI), their detection
requires fixation and permeabilization procedures that limit
subsequent functional studies.In addition, whole organ and tumor
cell suspensions are complex mixtures that generally depend on
multiple selection markers that distinguish between specific cell
types.
The objective of our study is to directly compare these well
established methods withalternative assays that measure the DNA
content with Hoechst 33342, and distinguish between growth
arrested G0 and actively cycling G1 states based on Pyronin Y and
CD71 expression in unfixed, heterogeneous cell suspensions.We
induced cellular arrest by serum starvation at different time points
and increased quiescence was evident by the decrease in CD71
as well as a reduced number of cells in the G2/M stage of the cell
cycle.
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Method: A piezoelectric transducer is bonded underneath a
microfluidic channel (Fig. 1) to induce an acoustic standing wave
across the channel upstream of a magnet, which is incorporated
near abifurcation point to extract magnetically labeled cells. When
the driving frequency has a wavelength twice of the channel
width, the cells are focused along the central streamlines.4Thus,
by acoustically constraining the initial lateral positions of the
cells, the downstreammagnetic force applied on the labeled cells
becomes uniform, thereby removinginhomogeneities in transit
timesexperienced by the labeled cells if they were dispersed in
different streamlines across the channel. Such inhomogeneities
result in a necessary compromise between sample throughput and
sorting purity.
Our comparative studies revealed that antibodies detecting the
transferrin receptor CD71 in combination with the DNA dye
Hoechst 33342 are the most favorable and versatile markers to
distinguish quiescent from actively proliferative cells in normal
keratinocytes and squamous cell carcinoma cells due to their
compatibility with broad panel of fluorescent proteins and dyes.
174/B53
Speaker/Author
Index
10x. Furthermore, to achieve even higher throughput, AeMACS
is also compatible with parallel separation channels or multiple
acoustically focused streams.3
High Throughput Chip-Based Cellular Sorting via
Acoustically Enhanced Magnetophoresis
Lu Gao 1,2, C. Wyatt Shields IV 2,3, David M. Murdoch 4,
Benjamin B. Yellen1,2, Gabriel P. Lopez3,5
1
Department of Mechanical Engineering and Materials Science,
Duke University, Durham, NC, United States, 2IRG-1, Research
Triangle Material Research Science and Engineering Center,
Durham, NC, United States, 3Department of Biomedical
Engineering, Duke University, Durham, NC, United States,
4
School of Medicine, Duke University, Durham, NC, United
States, 5Research Triangle Material Research Science and
Engineering Center, Durham, NC, United States
Background: Techniques to rapidly sort specific types of cells
from biological samples are of interest in scientific research,
biotechnology industry and medicine.1Comparedto techniques such
as centrifugation, fluorescence activated cell sorting (FACS) and
electrophoresis, magnetism-based cellular sorting techniques such
as magnetically activated cell sorting (MACS) and magnetophoresis
(e.g., H-Filters2) extract magnetically labeled cells from complex
samplesand have advantages including: capability to isolate rare
cells (e.g., immune cells or circulating tumor cells), low cost,
electrochemical stability and wide availability of biofunctionalized
magnetic particles. However, these techniques and their
corresponding devices suffer from several shortcomings, including
(i)thenecessity ofbatchwise (e.g., serial trap / release)operation, or(ii)
aconflict between throughput and separation purity in continuous
operations caused by the highly nonlinear dependence of magnetic
forcing on distance between cells and magnets.
To circumvent these shortcomings, while exploiting all
the conveniences ofthe magnetism-based methods, we
developedthechip-basedmethod, acoustically enhanced
magnetically activated cell sorting (AeMACS).Preliminary tests
performed on prototype AeMACSdevicesshow that, compared to
the conventional chip-based continuous methods (e.g., H-Filters
that typically run at ~10μL/min2), throughput is increased by
156
Figure 1. Schematic depictionof an AeMACS device and its
operation.
Results: Lymphocytes (CD3+) were labeled with magnetic
nanoparticles. Fig. 2a shows thatwhen no fieldsare applied, the
cells entered both outlets; b and c show that when only the acoustic
field is applied, the cells were focused and enter the drain outlet
since the bifurcation point is offset from the focusing axis; d shows
that once the magnetic field is also applied, the labeledcells were
extractedtoward the target outlet.
Figure 2. Preliminary results for a flow rate of 100μL/min.
Conclusion: We have demonstrated the concept of the AeMACS
technique. This technique combines the high efficiency of acoustic
focusing with thepower of magnetic separation and the accessibility
of magnetic labeling reagents. In ongoing studies, we are
optimizing the design of the device and performing flow cytometry
to evaluate cell sorting efficiency.
References:
1. A. Asgar, et al. Med Biol Eng Comput 2010 (18): 999 - 1014
2. J. J. Lai et al. Lab Chip 2009 (9): 1997–2002
3. M. E. Piyasena, et al. Anal Chem2012(84): 1831−1839
4. T. Laurell, et al. Chem Soc Rev 2007 (36): 492–506
ISAC 2013 Program and Abstracts
Microvesicle Detection and Cell Sorting
Background: In order to sort cells with high purity, droplet
generating cell sorters typically abort cells that are too close
to another unwanted cell electronically and thereby reduce
contamination of the sorted populations. In samples of adherent
cells, or antigen presenting cells such as monocytes and dendritic
cells or those that have been processed in a manner that increases
cell to cell interaction, this can often lead to poor recovery. In
a well dispersed sample, the distribution of cells is random and
follows the statistics of a Poisson distribution. As noted by Lindmo
(1981) by measuring the time interval between events it is possible
to evaluate the deviation from the ideal distribution. We describe a
method to assess sample behavior using a simple dispersion index
and introduce the concept of “entrainment factor”, and demonstrate
using different samples the utility of this tool in distinguishing
performance issues arising from sample behavior from those
intrinsic to the sorter.
Poster
Session
Methods: Particle arrival time was measured with a high degree of
accuracy using intrinsic time stamps in firmware with a resolution
of from 1/16th of a drop (1.5625 microseconds at 40 KHz, BD
Influx™), to 0.1 microseconds (BD FACSAria™ III), to 17.625
nanoseconds (48 bit time stamp, BD FACSJazz™) and including that
information in the event frame of each processed event as a time
stamp and in the case of the Influx and FACSJazz additionally as the
distance to the previous event in time. The data were binned and
fitted as a Poisson exponential distribution, and the probability of
another event occurring within time bins related to drop boundaries
was obtained based on traditional Poisson estimates (Pinkle and
Stovel, 1985). A simple ratio of observed/expected for different
relevant time spans) provided an “entrainment factor” metric such
that the result would be 1.0 for true Poisson distributions and
significantly greater for those particles that entrain in a non-random
manner.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Results: The behaviors of different sample/cell types, and of
subpopulations within heterogeneous samples such as PBMCs,
show entrainment behaviors in flow that range from true Poisson
distributions to non-random entrainment significantly affecting
sorting performance and reducing sort yield.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Joe Trotter, Sujata Iyer
R&D, BD Biosciences, San Jose, CA, United States
Wednesday,
22 May
The absorption of light by mammalian tissues in wavelengths
shorter than 750 nm results in poor excitation and emission
efficiencies for detection corresponding fluorescent dyes. For
these reasons, and to decrease autofluorescence, in vivo imaging
technologies use near-infrared emitting dyes. However, little has
been done to further characterize these fluorescent molecules
by flow cytometry because of the low excitation and detection
efficiencies for these fluorochromes using the commonly available
lasers and detectors in conventional flow cytometers. However,
in this presentation we show that flow cytometry detection can
be good enough to allow a combination of these two modalities
Assessing Sample Behavior in the Context of Sorter
Performance Using a Dispersion Index
Tuesday,
21 May
Marty Bigos1, David Parks2, Tobi Schmidt3
Center for Molecular and Genetic Medicine, Stanford Univ,
Stanford, CA, United States, 2Stanford Univ, 3Pediatrics,
Stanford University, Stanford, CA, United States
1
177/B56
Monday,
20 May
Use of Near-IR Detection in Flow Cytometry
Finally, we used this new flow cytomtery analysis approach in
conjunction with whole body fluorescence imaging to track and
quantify human immune cells trafficking into breast cancer tumors
in vivo. Human derived cytokine-induced killer (CIK) cells labeled
with IRDye800cw were injected into SCID mice bearing GFP and
luciferase positive MDA-MB-231 tumors. Whole body imaging
was done to follow the trafficking of CIK cells to the tumor. Flow
cytometry analysis was used to assess intensity of cell labeling
before in vivo injection and again after tumor isolation to quantify
the number of CIK cells present at the tumor site. We report these
data here and conclude that the Becton Dickinson Influx can be
used for the detection of near-infrared fluorochromes commonly
used for in vivo studies.
Sunday,
19 May
176/B55
Using a conventional flow cytometer, the Becton Dickinson Influx,
we were able to detect and quantify cells stained with Indocyanine
Green or IRDye 800cw (Li-Cor, Lincoln, NB). Additionally, we
worked to enhance the detection sensitivity of these fluorochromes
by testing two types of PMTs with reported greater sensitivity in the
wavelengths emitted by these dyes in comparison to the standard
red-sensitive Hamamatsu 3896s.
Saturday,
18 May
There is great interest in both medical and scientific communities
in submicron cell-derived particles, termed microparticles or
microvesicles. A great difficulty in this field, however, has been
the optimization and standardization of techniques to measure
these small particles. Although competing techniques have been
developed, flow cytometry remains the dominant approach.
The hurdle in analysis has always been the ability to accurately
measure the size characteristics of small particles; especially when
only considering scatter properties. Instrumentation was designed
around whole blood analysis and, therefore, cellular measurements
above 3um. Particles detected below the 3um “threshold” were
considered to be debris. Due to advances in microscopy and the
ability to identify the existence of less than 1um cellular particles,
flow cytometry instrumentation has been developed to have the
ability to identify populations from 400nm to 1um. However, the
accuracy of these measurements and the validity of the results are
frequently questioned. Therefore, we propose a hardware upgrade
that will not only improve accuracy, but will allow for validation
of the procedure by recovery of the microparticles In this study, we
present the results of our independent testing of the new Beckman
Coulter MoFlo Astrios 1.0. A new forward scatter detection system
has been designed for the MoFlo™ Astrios to measure small and
large particles simultaneously using a two-parameter head-on
PMT-based optical assembly. The noise and resolution of the
design allows for FSC trigger of 0.2 ìm and SSC trigger at 0.1 ìm
particles while simultaneously visualizing particles up to 30 ìm.
The additional resolution on both the small and large particle
forward scatter greatly enhances the scientist’s ability to sort and
gate on scatter as well as, providing tools for expanded research
and diagnostics.1 Many cells, including platelets, endothelial
cells, leukocytes, and erythrocytes, shed fragments of their plasma
membranes into the circulation. There is increasing evidence that
these submicron fragments, termed microparticles, have important
physiological roles. Although many techniques have been derived
for the identification and characterization of microparticles, flow
cytometry is still deemed to be the most accurate and reproducible.
With the addition of the two-parameter head-on PMT-based
optical assembly, the ability to identify and recover microparticles
(<200nm-30um) for further analysis has been simplified and
techniques easier to validate. The MoFlo Astrios 1.0 sorting system
allows for advanced applications with microparticles and will
alleviate some of the roadblocks associated with this research area.
MoFlo Astrios Enhanced Forward Scatter: 0.2 ìm to 30 ìm Dynamic
FSC Detection Dr. Carley Ross.
to significantly expand the scope and quality of data that can be
derived from in vivo imaging alone.
Special
Lectures
Vasilis Toxavidis1, John Tigges2, Kaitlin Groglio3
1
BIDMC, BOSTON, MA, United States, 2Beth Israel Deaconess
Medical Center, 3Flow Cytometry, BIDMC, BOSTON, MA,
United States
Congress
Overview
175/B54
Conclusion: Our metric serves as an effective means to generally
describe how “Poisson” a population actually is during a sort,
157
Congress
Overview
Special
Lectures
thereby aiding in improving sample preparation, and sheds light on
why a classified population may be exhibiting poor performance
contributing to a disappointing sort yield.
generating a standardized methodology for studying MPs by flow
cytometry. However, due to historic reasons and the complexity of
the field, which protocol is optimal is still under debate.
Lindmo T, Fundingsrud, K (1981): Cytometry. 1981 Nov;2(3):151154.
Pinkle D, Stovel R (1985): (M.A. Van Dilla, P. N. Dean, O.D.
Laerum, M. R. Melamed, eds), 3, 77-128, Academic Press, London.
Here we demonstrate an instrument setup protocol on the BD
FACSVerse flow cytometer to analyze platelet-derived MPs (PMPs).
First, a set of SPHERO™Nano Fluorescent Particles (0.22, 0.45,
0.80, and 1.33µm) was used to set up the instrument, as well as the
BioParticles fluorescent bacteria (Staphylococcus aureus). Second,
PMP samples from healthy donors’ whole blood, which were
isolated by two-step centrifugation followed by staining with CD31
and CD41a fluorescent conjugates, were acquired on the flow
cytometer using established instrument settings. A standard curve
of serial diluted sample was also established to demonstrate the
detection capability of the instrument.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
178/B57
Performance Study of the Closed Piezo-Based Cell
Sorter (CyFlow® Sorter)
Juliane Klose, Danny Köhler, Volker Ost
Partec GmbH, Münster, Münster, Germany
Sorting of cells is of major relevance for biomedical applications
including cellular cloning steps or the isolation of rare cell
populations. Whereas conventional cell sorters often include a high
financial investment and sophisticated operation requirements the
Partec cell sorter is easy in handling and can be operated by any
worker in the lab.
Sorted cells from conventional cell sorters often suffer from
mechanical stress. Besides that, a contamination of both the
sample and the environment by creation of aerosols is an issue that
should not be underestimated.
The Partec CyFlow® Sorter combines in a unique way user comfort
with superior performance characteristics and can be included
in both the Partec flow cytometers CyFlow® space and CyFlow®
Cube. The Partec sorter guarantees lowest possible mechanical
impact on the cells and hence, allows for non-destructive cell
sorting of fragile or electrically delicate cells (like cardio myocytes).
The virtually aerosol-free condition of the cell sorter means a highly
reduced risk of contaminations.
The Partec CyFlow® Sorter has been successfully employed manyfold for sorting of various cells types with very distinct cellular
properties including bacteria, stem cells, cultured cells, protoplasts,
pollen, cardio myocytes and many others. A novel procedure of
concentrating the cells directly during the sorting process minimizes
the dilution of the cells and yields a highly enriched suspension of
sorted cells.
Examples of such experiments are summarized in the actual
scientific study. We present data of sorting results for various
cell types and based on these data we are able to classify the
performance of the sorter as given by sorting rate and sorting purity
with values up to 300 cells / sec and > 98%, resp.
Cell-Derived Microvesicles
(B58 – B62)
179/B58
Study of Platelet-Derived Microparticles Using the
BD FACSVerse™ Flow Cytometer
Liping Yu, Yibing Wang
BD Biosciences, San Jose, CA, United States
Microparticles (MPs) are small membrane fragments released
by many types of cells including, but not limited to, platelets,
leucocytes, and endothelial cells. MPs can contain both surface
markers and intracellular contents (eg, protein and mRNA) of the
cells they originated from. The presence of MPs in circulating
blood has become of increasing interest to researchers as a valued
biomarker for studying certain disease states, such as in hemostasis
and thrombosis, cancer, and inflammatory diseases. Flow cytometry
is a preferred method for studying MPs because of its ability
to perform multicolor analysis for looking at several markers
simultaneously and to analyze a large amount of different sized
particles in a short period of time. Many efforts have been put into
158
Our data confirms that scattered light detection of a given size of
polystyrene beads on a flow cytometer cannot be used to measure
MP size, but can be used to set up optimal instrument settings
for detecting MP populations. Also, the approach of using side
scattering (SSC) in combination with fluorescence signals on the BD
FACSVerse enables resolution of PMPs from residual platelets and
background noise.
180/B59
Importance of Quality Control to Follow-Up and
Compare Instrument Performance for Microparticle
Analysis by Flow Cytometry: Correlation to
Microparticle Count
Nicolas Bailly1, Philippe Poncelet2, Bérangère Devalet3,
Stéphane Robert4, Romaric Lacroix4, Jean-Michel Dogné5,
Françoise Dignat-Georges4, Bernard Chatelain1, François
Mullier1,5
1
Hematology Laboratory, CHU Mont-Godinne, Yvoir, Belgium,
2
BioCytex, Marseille, France, 3Hematology, CHU MontGodinne, Yvoir, Belgium, 4NSERM UMR-S608 and Faculté de
Pharmacie, Université de la Méditerranée, Marseille, France,
5
Pharmacy Department & Namur Thrombosis and Hemostasis
Center, University Of Namur, Namur, Belgium
Background: Microparticles(MPs) have high potential as diagnostic
biomarkers. Standardization of their analysis by flow cytometry
(FCM) is limited by size-related issues.
Aims: To apply a simple formula describing instruments’ scatter
resolutionbased on submicron beads [Megamix (Mgx)] for
instrument qualification, day-to-day monitoring, optimization trials
and inter-platform comparisons.
To illustrate the effect of time-related drifts ofan instrument’s scatter
resolution on platelet microparticle (PMP) counts.
Methods: Performance levels were compared on 30 flow
cytometers(FCMrs) of different types and brands using a separation
index (SI) based on the forward scatter (FSC) or side-scatter (SSC)
distributions of Mgx and calculated as follows: SI=(Median(Md)
0.9µm – Md 0.5µm)/(Standard Deviation (SD) 0.9µm+SD 0.5µm).
Using one particular instrument (our FACS-ARIA I) showing low
and fluctuating SI values on both FSC and SSC, the absolute PMP
count of a Platelet Free Plasma (PFP) sample frozen as multiple
aliquots was determined over-time and correlated to SI values.
This instrumentrequiring the use of a combined threshold on both
parameters to get rid of background, the product {SSC SI x FSC SI)
was also calculated and compared to PMP counts.
Results: i) Becton-Dickinson FACSCantoII, AriaI, FACSVerse and
LSRII, showed significant inter-instrument variability on FSC with
highly variable SI(maximum SI: 21) but more homogeneous SI
on SSC (maximum SI: 25) ii) Beckman-Coulter FC500, Navios
and Gallios showed rather homogeneous FSC resolution with
a maximum SI of 35 iii) Accuri C6, Apogee A50, LSR-Fortessa,
showed encouraging resolution values(SI ≥10) iv) Monitoring
ISAC 2013 Program and Abstracts
Methods: Polystyrene counting beads greater than 1 µm in diameter
were enumerated and normalized over 30 seconds of collection
time. The side scatter threshold was varied to the level of detection
of 1 µm to 200 nm beads. Similar analysis was performed using
dual trigger for FSC and SSC or SSC and fluorescence.
Results: Using a single SSC threshold, lowering the threshold
reduced the number of counting beads detected. Interpretation: On
instruments lacking a built-in doublet detect and abort mechanism,
large beads with low threshold will yield a higher count of
unstained MPs in the scatter channel used for the threshold. These
may appear as noise or unstained MPs. As the scatter threshold is
lowered on instruments with a built in doublet detect and abort
function, the larger beads will start aborting as they will generate
a footprint similar to coincident events. Solution: Stratedigm
designed a Laser Trimming Technology to disable the doublet
detect and abort function. This system contains an optional built in
doublet detect/abort capability that can correctly account for large
particles.
Saturday,
18 May
Keywords: Platelet, Microvesicles, Microparticles, Flow cytometry,
separation index, quality control
Thus, we tested the relationship of various scatter triggering
thresholds on the enumeration of counting beads.
Special
Lectures
Conclusions: Thus, the SI value of Mgx submicron beads is a useful
quality control parameter for MP analysis. This study of scatter
resolution on multiple FCMrs supports some modifications to the
current ISTH protocol, using SSC (rather than FSC) to define the MP
gate on instruments which measure FSC at low angle.Monitoring
and maintenance of instrument performance is important since
PMP counts may be influenced by instruments’ scatter resolution.
Congress
Overview
FSC SI showed time-related drifts and allowed raising the need
for maintenance on some FCMrs v) Optics cleaning (FC500) and
optimization of the optical design (Apogee instruments and LSRFortessa) led to improved FSC resolution.vi) In the particular case of
an instrument showing low and fluctuating SI values and requiring
a combined FSC and SSC thresholding strategy, PMP counts where
influenced by both SI and best correlated withSSC SI x FSC SI
(Figure 1).
Sunday,
19 May
Monday,
20 May
Conclusion: Investigators should be aware of and take steps to
ensure that the flow cytometer is counting all of the sub-micron
particles and larger sized counting beads accurately.In addition,
it is critically important for all publications on MP studies by flow
cytometry to include the instrument, gating strategy and triggers
used. This abstract does not reflect EPA policy.
182/B61
Figure 1. Evolution of the PMP concentration according to the SI
181/B60
Accurate Detection of Counting Beads for Cell-Derived
Microparticle Enumeration by Flow Cytometry
Oral Session
Abstracts
The purpose of this study was to identify and quantify by flow
cytometry circulating EMPs in plasma of rats treated with SK&F
95654 (PDE-3 inhibitor) and methoxamine (alpha 1-adrenocepter
agonist). In this time-course study, platelet-poor plasma (PPP)
was collected from male Sprague Dawley rats (6/time point) at 4,
8, 16, 24, 48 hours and at Day 30 after a single dose. Detection
of EMPs in PPP was accomplished using markers specific for
endothelial cells (CD31, CD106, CD146, CD54, Flk-1, vWF).
Additional exclusion markers (CD45 and CD42d) and the apoptotic
marker Annexin V were included to further characterize the
origin of microparticles. All stained PPP samples were processed
concurrently with microbeads to set a baseline for size and EMPs
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Commercial
Tutorials &
Exhibits
Microparticles (MPs) are sub-micron sized fragments that are
shed by cells undergoing activation or apoptosis. Enumeration
of biological MPs is critical to the study of their role in disease
processes such as thrombosis or cancer. Typically, enumeration
of cells is performed by diluting a sample of cells with a known
concentration of fluorescent counting beads to the unknown
particles being counted. We found that the size characteristics of
the beads and the electronics of the machines are very important
for this assay to work correctly. The beads are gated by either size
or fluorescence and a given number of beads aids in the calculation
of a volume of cells analyzed. This formula may be compromised,
however, if the scatter threshold for detection of the MPs is set so
low that the bead signal is frequently aborted due to errant scatter
signal at such low thresholds. We found that on some instruments,
a constant amount of counting beads took longer and longer to
count after the scatter threshold was dropped to include the MPs.
The outcome was an incorrectly increased number of MPs counted,
since the counting beads were being frequently aborted or just not
counted, thus increasing the counting time for MP enumeration.
Drug Induced Vascular Injury (DIVI) is caused by many types of
drugs in development including PDE inhibitors, dopamine agonists,
and endothelin receptor antagonists. DIVI can present differently
in various preclinical species (mesenteric arteries affected in the
rat and coronary arteries in dog) without corresponding findings
in humans. Many potential drugs are terminated in development
because of DIVI andthe lack of translatable peripheral blood
biomarkers.To address the need for biomarkers, endothelial
microparticles are being investigated as biomarkers for drug
induced vascular injury. Endothelial microparticles (EMPs) are
small vesicles (0.1-1μm) that are released into circulating blood
from activated, injured, or apoptotic endothelial cells and are found
at elevated levels in a number of diseases associated with vascular /
endothelial dysfunction. EMPs retain markers, or surface receptors,
from the cell of origin such as FLK-1, CD144, CD146, CD105
and CD106 and can be identified by specific antibodies via flow
cytometry. Because expression of surface receptors can change
during injury, inflammation, or activation, the phenotype of the
EMPs may also correspond to the overall physiological status of the
cell of origin.
Poster
Session
Nancy Fisher1, Micah Mooberry2, Shervin Javardi3, Robert
Zucker4, Nigel Key2
1
Microbiology and Immunology, University of North Carolina,
Chapel Hill, NC, United States, 2Medicine, University of North
Carolina, Chapel Hill, NC, United States, 3Stratedigm, Inc.,
San Jose, CA, United States, 4NHEERL Toxicology Assessment
Division, US EPA, Research Triangle Park, NC, United States
Sharon Sokolowski, Amy Shen, Leslie Obert, Todd Wisialowski,
Michael Lawton, Paul Nugent, Terri Swanson, Susan Portugal,
Catherine Rief, Bradley Enerson
Pfizer Inc., Groton, CT, United States
Wednesday,
22 May
Tuesday,
21 May
Identification and Analysis of Circulating Endothelial
Microparticles as a Potential Biomarker of DrugInduced Vascular Injury
159
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
were identified based on size and labeling with specific markers.
TruCount beads were utilized to provide absolute microparticle
counts. Significant increases in EMPs counts were observed
after administration of SK&F 95654 (FLK-1, CD31FLK-1) and
methoxamine whichcorrelated with histopathology findings. The
present study suggests that EMPs potentially represent a novel
biomarker for DIVI observed in preclinical species.
183/B62
Flow and Imaging Cytometry Characterization of
Microparticles: Analysis and Sorting on the Basis of
Size and Fluorescence
Natasha Barteneva1, Elizaveta Fasler-Kan2, Larry Duckett3,
Ivan Vorobjev1,4
1
PCIMM, Boston Childrens Hospital, Boston, MA, United
States, 2Biomedicine, University of Basel, Basel, Switzerland,
3
Special Order Instruments, BD Biosciences Inc, San Jose,
CA, United States, 4A.N.Belozersky Institute, Moscow State
University, Moscow, Russia
Microparticles (MPs) are a heterogeneous population of membranedelimitated vesicles released by the different cells and retaining
certain antigens of their cell’s of origin. Reported sizes of MPs
vary among publications, ranging from 50 nm to 1000-2000 nm.
Isolation of MPs typically involves a combination of centrifugation
and size-based filtration. Isolated MPs are further characterized
using light-scattering methods, flow cytometry, flow imaging
cytometry, electron microscopy, Western blotting, proteomics etc.
We have used Special Order (SORP) FACSAria instrument equipped
with 5 lasers, photomultiplier on forward scatter channel and digital
focusing (red laser) and imaging cytometer Imagestream 100 for
characterization and sorting of MPs of different origin(erythrocytes
and endothelial cells). A set of size beads ranging from 190 nm
to 1 ì were used as controls. The size distribution of MPs fractions
was confirmed by light-scattering method using Nanosight 500.
We were able to purify subpopulations of MPs of different size
(starting from 190 nm) labeled with glycolipid dye (calcein) and
specific antibodies conjugated with Alexa-647. The FACS-based
MPs sorting is currently the only method allowing to separate
specific subpopulations of MPs based simultaneously on their size
and immunophenotype. This approach can be critical for analysis
of MPs function, and also may be applied for sorting of viruses and
similar particles of different size.
Results: the interaction of AO with double strand and single
strand DNA are resulted to emit green and red color, respectively.
So, we analyzed DNA fragmentation in fl1 and fl3 channels by
FACSCalibur. Flow cytometry Data was stored in “flow repository”
with Repository ID (FR-FCM-ZZ2B). Moreover, different phases of
cell cycle determine using AO.
AO is taken up by both viable and non-viable cells and EB are
taken up only by non-viable cells so AO and EB can show live
cells (green), necrotic cells(red) , late apoptotic (orange)and early
apoptotic cells (green cells with fragmented nucleus) in florescent
microscope.
Conclusion: the determination of Cell cycle, apoptosis and
discrimination of ssDNA and dsDNA can be done as easy as
possible by acridin orange. On the other hand, it could be use in
sperm chromatin Structure assay (SCSA). Therefore AO can be used
in research and clinical diagnosis in cost effective manner.
Key words: Acridine Orange (AO), cell cycle, apoptosis, DNA
fragmentation, SCSA
185/B64
Simultaneously Restorations of Foxp3+Treg, Type
1-Like Treg and B Cells by Anti-TNF Therapy for IBD
Zhe Li1,2, Severine Vermeire2, Dominique Bullens1, Marc
Ferrante2, Kristel Van Steen3, Maja Noman2, Paul Rutgeerts2,
Jan Ceuppens1, Gert Van Assche2
1
Laboratory of Clinical Immunology, Catholic University of
Leuven, Leuven, Belgium, 2Division of Gastroenterology,
Catholic University of Leuven, Leuven, Belgium, 3Dept of
Bioinformatics-Statistical Genetics, Universite de Liege, Liège,
Belgium
Background: Infliximab (IFX) therapy increases circulating Foxp3 (+)
T cells in patients (pts) with Crohn’s disease (CD), ulcerative colitis
(UC), rheumatoid arthritis (RA), psoriasis and Behçet’s disease.
Co-expression of CD45RA & Foxp3 distinguishes resting & active
Treg (rTreg & aTreg) from Foxp3 (+) effector T cells (Teff). IFX also
up-regulates blood total memory and pre-switch memory B cells in
RA. In IBD, IgM (+) memory B cells are decreased. CD19(+) B cells
in the inflamed intestinal mucosa predicts long lasting remission
to IFX in CD. IL-10/IFNg producing Tr1-like cells (Tr1L) have been
characterized in human blood. Genetically modified B cells induce
Tr1L in vivo. Recently resting B cells have a role in expanding
Foxp3+Treg.
Clinical Trials (B63 – B65)
We aimed to investigate the kinetics of these cells in pts with IBD
during IFX therapy.
184/B63
Methods: Blood was taken from healthy controls (HC, N=37) and
pts with IBD (70 CD, 39 UC) before and during therapy (5mg/kg IV
0-2-6 and q8 wks). The 3 subsets of Foxp3 T cells, Tr1L and B cells
were assessed by flow cytometry after staining for CD4, CD45RA,
Foxp3, CD25, CD127 and CD19. Assessment of symptoms,
endoscopic healing & histological improvement was used to
distinguish responders (RS) from non-responders (NRS) at 4 to 12
weeks after start of therapy. Serum CRP was collected to monitor
biological response.
The Applications of Acridine Orange in Cytometry
Fazel Samani1, Pardis Khosravani2, Ehsan Jan Zamin2, Marzieh
Ebrahimi2
1
Stem Cells and Developmental Biology Group of Cell Science
Research Center, Royan Institute for Stem, Tehran, Iran, 2Stem
Cells and Developmental Biology Group of Cell Science
Research Center, Royan Institute for Stem, tehran, Iran
Introduction: Acridine Orange (AO) is a dye that interacts
with DNA (green fluorescence) and RNA (red fluorescence) by
intercalation or electrostatic attractions. However AO has wide
applications in biology, in this study AO were used to study DNA
fragmentation, cell cycle analysis and apoptosis detection.
Method: 1x106 cord blood cells were stained by 6 μg/ml AO for
cell cycle analysis and DNA fragmentation, 10 μl/ml AO/Ethidium
bromide (EB) was used for apoptosis detection.FCM samples were
acquired by FACSCalibur, analyzed by CellQuest software and
images were taken by IX71 Olympus fluorescent microscope.
160
Results:
1. Pts with active IBD before therapy had low circulating
rTreg(0.43±0.080, p<0.001), aTreg(0.62±0.12, p<0.001),
Foxp3Teff(2.38±0.27, p=0.002) , Tr1L(4.79±0.68, p<0.001) and
B cells (0.17±0.02, p=0.002) (N=25), compared with HC(1.47
±0.16),(2.40±0.17),(3.75±0.34), (16.82±1.7) and (0.27±0.02)
(N=37) (10e6/L blood mean±SEM for Tr1L, 10e9/L for others).
ISAC 2013 Program and Abstracts
In addition, a database of all major instruments used in
flow cytometry has been developed. This database includes
manufacturers, models, and default laser and filter sets used in all
instruments.
Results: We have successfully established a growing database of
more than 50,000 reagents used in flow cytometry. This database
provides a standard ontology for targets, fluorophores, target
species, regulatory status, clone, and several other criteria. This
common ontology provides the basis to establish the PanelXML.
InstrumentXML was built utilizing instrument manufacturer,
model, laser types, wavelengths and detection ranges. These
InstrumentXML can be shared individually or as part of the
PanelXML.
Poster
Session
Conclusions: The MIFlowCyt standard requires specific information
regarding the instrument and reagents used in the flow cytometry
experiment. We propose that our developed InstrumentXML
and PanelXML provide a starting point of a set criteria for these
components of the MIFlowCyt standard. In order to encourage
adoption, we have also developed web based and Java based
applications with user interfaces that assist in building and sharing
InstrumentXML and PanelXML. In order to integrate with resources
available, we have worked with FlowRepository to accept our
InstrumentXML and PanelXML. InstrumentXML can be shared
with other scientists in order to provide the specific configuration
a flow cytometry experiment was run on. InstrumentXML can
also be embedded into PanelXML to provide a complete list of
not only the exact reagents used an experiment, but the specific
channels each reagent was detected with. We suggest that our
developed InstrumentXML and PanelXML will make it easier to
share instrument and reagent information with fellow scientists.
This will allow quicker development of new reagent panels or the
elaboration on existing reagent panels.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Results and Conclusions: A description of the qualification plan
and an example of results from a 12-color T regulatory cell flow
cytometry assay qualification are presented. The T regulatory
cell assay met qualification performance targets. The goal of
this project—development of an assay qualification strategy to
ensure that flow cytometry panels are scientifically sound, robust,
reproducible, and deliver the results intended for their laboratory
purpose—was achieved.
Material and Methods: A common ontology for reagents used in
flow cytometry was created utilizing the antibody portfolios of
13 major reagent suppliers (BD Biosciences, Life Technologies,
BioLegend, eBioscience, Beckman Coulter, Abcam, Stemcell
Technologies, Novus Biologicals, Exbio, Sony Biotechnology,
Leinco Technologies, Miltenyi Biotech and Cedarlane.)
Wednesday,
22 May
Methods: A flow cytometry assay qualification plan was designed
incorporating four experiments to measure intra-assay precision,
inter-assay precision, intermediate precision, and sample stability.
Animplementation of this qualification plan was performed on
multi-color flow cytometry panelsto be used for clinical research.
Peripheral blood mononuclear cells from one normal donor were
used for the qualifications. A performance target was set at <
20% CV among cell population measurements whosefrequency
was>10% positive.
Introduction: TheMIFlowCyt Standard (Minimum Information
about a Flow Cytometry Experiment) is anISAC (International
Society for the Advancement of Cytometry) recommend data
presentation standard for flow cytometry data. Submitted in
2008, the MIFlowCyt standard outlines the minimum information
about a flow cytometry experiment that should be published with
every experiment employing flow cytometry as a method. The
MIFlowCyt standard can be difficult to follow. We present here
primary standards for the instrument and reagent components of
the MIFlowCyt standard, which make it easier to share and transfer
these elements of the MIFlowCyt standard.
Tuesday,
21 May
Background: When a formal assay validation is not needed but
a measure of assay integrity and robustness is, a qualification
wouldbe appropriate. Method qualification characterizes and
documents assay performance to ensure the assay is fit for its
intended purpose. Multi-color flow cytometry panels used for
cellular characterization and comparability studies, that is, nonQC applications, are good candidates for assay qualification. The
benefit of qualification is that it is typically less expensive, less
labor/resource intense and takes less time than an assay validation.
Given the complexity of developing multi-color flow cytometry
panels and the need for multiple controls (compensation, gating,
biological), processes that help reducetime and resources between
panel conception and use are beneficial.
Nicholas Ostrout1,2, Jasper Katz1,2, Mike Stadnisky2, John
Quinn2, Adam Treister3
1
Fluorish, LLC, Ashland, OR, United States, 2FlowJo, LLC,
Ashland, OR, United States, 3Tree Star, Inc, Ashland, OR,
United States
Monday,
20 May
Juliane Hill1, Erica Dearstyne1, Travis Beckett1, Nikole Purdue2,
Stephen Apone1
1
Analytical Sciences, Dendreon, Seattle, WA, United States,
2
Clinical Immunology, Dendreon, Seattle, WA, United States
Creating Standards for InstrumentXML and PanelXML
Sunday,
19 May
An Approach to Qualifying Flow Cytometry Panels
187/B66
Saturday,
18 May
186/B65
Computation and informatics
(B66 – B72)
Special
Lectures
Conclusions: Circulating Foxp3T cells, Tr1L and B cells are
decreased in active IBD and an increase in these cells (except for
Foxp3Teff) correlates with biological response to IFX and/or with the
clinical response to IFX. The positive correlation between B cells
and Foxp3Treg subsets or Tr1L suggests that there might be a cross
talk between B cells and Tregs.
Congress
Overview
2. Compared with baseline before therapy, change of these cells
after IFX treatment was seen in rTreg(RS: 1.57±0.21, p<0.001;
NRS: 1.14±0.24, p<0.001), aTreg(RS: 2.70±0.26 , p<0.001;
NRS: 1.48±0.33, p=0.0057), Foxp3+Teff (RS: 3.19±0.24,
p=0.09; NRS: 3.02±0.41, p= 0.25), Tr1L(RS: 26.09±2.21,
p<0.001; NRS: 8.92±1.00, p=0.013) and B cells(RS: 0.25±0.03,
p=0.035;NRS:0.14±0.01, p=0.31) (N=59 in RS, 15 in NRS).
3. Significant differences between RS and NRS were seen only for
aTreg, Tr1L and B cells (p=0.0067, <0.001, <0.001), but not in
rTreg and Foxp3Teff (p=0.32, 0.72).
4. CRP negatively correlated with rTreg, aTreg, Tr1L (as %of CD4T
cells) and B cells (absolute number)( p=0.0011, r=- 0.32),(
p<0.001, r=- 0.40),( p<0.001, r=- 0.39) and( p=0.044, r=- 0.21).
5. B cells positively correlated with rTreg, aTreg and Tr1L (absolute
number)(p=0.002, r=0.31), (p<0.001, r=0.49) and (p<0.001,
r=0.37).
Speaker/Author
Index
ISAC 2013 Program and Abstracts
161
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
188/B67
Variance-Stabilized Meta-Clustering in Flow
Cytometry
Ariful Azad1, Bartek Rajwa2, Alex Pothen1
Computer Science, Purdue University, West Lafayette, IN,
United States, 2Bindley Bioscience Center, Purdue University,
West Lafayette, IN, United States
1
Clinical immunology studies with large numbers of samples require
the data to be summarized by a few high-dimensional templates,
each characterizing the samples from a particular class. Here, a
template is a collection of homogeneous meta-populations, more
generally known as meta-clusters, formed by combining cell
populations expressing similar phenotypes. Previous studies (Hahne
et al. 2009, for example) shifted the distribution of the populations
to ensure homogeneity in meta-populations, but this can hide
useful biological signals. We show by using an analysis of variance
(ANOVA) model, that homogeneity of meta-populations can be
achieved by stabilizing variance across populations.
We achieved the variance stabilization within the ANOVA model
by applying an inverse sine hyperbolic (asinh) transformation with
different normalizing factors (cofactors) for each fluorescence
channel. We then compute high-dimensional meta-populationsin
the transformed marker space by a hierarchical meta-clustering
algorithm, flowMatch, which combines combinatorial techniques
with a UPGMA-like tree construction method in phylogenetics.
We can assess the homogeneity of a meta-population statistically
by testing a null hypothesis that the mean fluorescence intensities
(MFIs) of the populations in a meta-population are equal in the
presence of fixed within-population noise. However, this test has
a high probability of making a Type I error, because the number
of cells in each population is large. To address this limitation, we
obtain the signal-to-noise ratio (SNR) of the meta-populations by
computing the ratio of between-population to within-population
variance. A meta-cluster is homogeneous when the confidence
interval of its SNR is below a given threshold.
The SNR-based ANOVA model can, therefore, statistically assess
the homogeneity of meta-populations without shifting them in the
marker space and can detect potential outlying populations. The
variance stabilization can also be used with other meta-clustering
algorithms such as FLAME and flowClust.
Gating the Gate Makers: How to Decide Whose
Gates Are Bright, and Whose Are Dim in a HighThroughput Manner
John Quinn , Jay Almarode , Mike Stadnisky , Ian Taylor ,
Adam Treister3
1
Tree Star, Inc., Ashland, OR, United States, 2FlowJo, LLC,
Ashland, OR, United States, 3Tree Star, Inc.
2
Background: Flow cytometric analysis depends largely on gating,
and regardless of whether gates are created by algorithms or
human experts their quality and consistency are difficult to assess.
162
This initial study has allowed us to develop the FlowDx platform.
The platform provides the means through a simple GUI interface
for a user to select any number of populations and any number of
gating results and calculate one or more statistics on each. The
embedded statistics are F-measure, the Davies-Bouldin score,
and the Match Ratio, another product of this work. One of the
advantages to this platform is that the gate generators can be a
heterogeneous mixture of humans and algorithms, and that results
themselves can be consequently gated to analyze the analyzers.
Conclusions: This experiment provided quantitative measurements
that the laboratory staff were producing work acceptable to the
laboratory standard, and that the lab manager was a proper lead.
It also demonstrates the utility of applying software evaluation of
gating in either a QA, training assessment, or validation role in a
typical cytometric workflow.
190/B69
Generative Modeling of F-Actin in Cells to
Understand Drug-Induced Cytoskeletal Changes
Jason Kinser 1, Tommy Turbyville 2, Karlyne Reilly 3, John
Beutler2, Stephen J. Lockett2
1
School of Physics and Computational Sciences, George Mason
University, Fairfax, VA, United States, 2Optical Microscopy and
Analysis Laboratory, Frederick National Laboratory, Frederick,
MD, United States, 3Mouse Cancer Genetics Program,
Frederick National Laboratory, Frederick, MD, United States
189/B68
1
Methods: We set up a study at a laboratory that received a
constant stream of data files, distributed the analysis load between
technicians, and used templates as the starting point for gating.
One data set consisting of 10 FCS files was given to a set of five
technicians including our presumed ‘gold-standard’, the laboratory
manager. The set was given to them five times on five different
days without the analysts’ knowledge that they were receiving the
same set. The processed data in the form of FlowJo workspaces
was uploaded to a repository from which we applied our statistical
platform, FlowDx. We used FlowDx across all 5 time points and
18 populations to calculate the Match Ratio for each technician
to measure individual consistency, the Match Ratio (a statistic that
scores event assignment to gates using the ensemble gating results
as a standard) of all participants versus each other to measure
accuracy versus the consensus, the F-measure of each technician
versus the lab manager to gauge of accuracy versus the standard,
and the Davies-Bouldin statistic to test the assumption that the lab
manager makes the best gates.
Results: Our analysis revealed that the lab manager did make
the best gates statistically, and that the technicians were largely
consistent both with themselves over time and with their coworkers,
including the lab manager. We did notice that one technician
performed more poorly than the rest and ran a series of comparative
statistical tests to establish an equivalence measure in gating terms
that these scores equated to.
We constructed variance-stabilized templates on two data sets: (a)
healthy data containing samples from five healthy subjects with
five replicates at four different time points, and (b) lymphoma data
containing 30 randomly selected lymph-node biopsies from patients
with diffuse large B-cell lymphoma. In both data sets, we show that
variances can be stabilized by optimizing cofactors of the asinh
transformation on each channel and that statistically homogeneous
meta-populations can be obtained on the transformed data. In the
healthy data we observe that the SNR of a meta-cluster increases
when we go up in the hierarchy of samples (from replicates to time
points to subjects). Such studies can obtain a threshold on SNR
in order to detect true biological signals at the meta-population
level. In the lymphoma data, different classes of samples were
automatically separated into distinct meta-populations.
1
High-throughput facilities, particularly clinical operations and
CROs, must employ either multiple technicians or use algorithms
to process large numbers of data files daily. Tools are needed
to examine and quantify technician consistency and algorithm
accuracy. In this work we partnered with Harvard to develop and
demonstrate a suite of statistical methods to evaluate the quality of
the gating.
1
Many reagents, including drugs, cause cytoskeletal changes in cells
that can be quantified from optical microscope images. However,
it remains challenging to understand the molecular mechanisms
underlying such changes based on quantitative imaging in
combination with results from in vitro biochemical assays.
Generative modeling is a crucial tool for overcoming this challenge,
because it gives interpretation of image-based measurements in
terms of biophysical changes to the cytoskeleton. The modeling
builds simulated images based on known or hypothesized
ISAC 2013 Program and Abstracts
In recent years, efforts have been made to improve analysis of flow
cytometry data by applying multivariate methods, thus taking into
account the joint structure of a cell population across multiple
dyes. However, this does not eliminate the need for compensation,
and indeed, it is not known the extent to which compensation
in fact distorts the data. Additionally, in the absence of prior
knowledge about the cell type make-up of a patient, unsupervised
learning methods such as clustering are employed. Unfortunately,
a fundamental statistical limitation of any unsupervised learning
method is that its success in describing the structure of the data
cannot be directly evaluated.
Here, we propose new methods that simultaneously mitigate these
two limitations. On the technical side, we use an approach taken
from multi-spectral imaging, which captures images at separated (3
to 7) wavelengths. In the context of flow cytometry we used optical
bandpass filter (BP) to divide the emission spectrum into six equal
parts in order to get a fingerprint of each fluorophore combination,
and the need for compensation is thus eliminated. Furthermore,
the analysis problem is transformed from an unsupervised learning
problem into an analogue of a two-sample problem by using FMO
controls. This means we can determine the cell type make-up of a
patient in an assumption-free manner, and due to the undistorted
signal, start characterizing biological variation that may occur in
particular cell types.
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
In a common flow cytometer a combination of optical filters and
dichroics is used in order to detect a specific fluorescence emission
range of a single dye. The spillover in a multicolour experiment
needs to be compensated and careful analysed by sequential gating
strategy. As a consequence, limitations arise in the sensitivity and
resolution of partially overlapping fluorescence signals.
Oral Session
Abstracts
Methods: The CytometryML schemas are written in the XML
Schema Definition (XSD1.1) language and validated to demonstrate
adherence to the XML Schema Definition language, XSD. Objectoriented methodology was employed to create the CytometryML
schemas. Their content was tested by translating specific XSD
elements into XML and filling in the values of the objects contained
therein. The attribute based syntax description of relationships in the
Resource Description Framework (RDF) has been replaced by an
Kristen Feher1, Toralf Kaiser2
1
University of Potsdam, Potsdam-Golm, Germany, 2DRFZ,
Berlin, Germany
Commercial
Tutorials &
Exhibits
Introduction: The development of cytometry standards is
complicated by the fact that much of their information space is
relevant to other disciplines: medical informatics, specifically
that pertaining to pathology, and biological science in general.
Presently, all three groups have their own standards. Another
complication is that both the objects and their relationships need
to be described. CytometryML, the cytometry markup language, is
an attempt to create a continuum of interoperable XML standards
for flow and image cytometry that can be used by all three groups.
Wherever possible, CytometryML is based on existing standards,
specifically those of the International Society for Advancement of
Cytometry, ISAC, Digital Imaging and Communication in Medicine,
DICOM, and International Digital Publishing Forum, IDPF.
Automated Flow Cytometry Data Analysis
Poster
Session
2
192/B71
Wednesday,
22 May
Robert Leif , Stephanie Leif
1
R&D, Newport Instruments, San Diego, CA, United States,
2
President, Newport Instruments, San Diego, CA, United States
1
Keywords: CytometryML, Cytometry, DICOM, EPUB, FCS, Instance,
Series, Schema, XML, RDF
Tuesday,
21 May
A Shared Standard for Cytometry and Pathology
Conclusions: This DICOM based design together with the use
of an EPUB container of CytometryML could serve as a reliable
efficient means for the transmission of research and medical data,
an extension of the pathology part of DICOM and as a prototype of
an XML version of DICOM. The present implementation of a simple
version of RDF in XSD could be extended to provide XSD with full
RDF capabilities.
Monday,
20 May
191/B70
Sunday,
19 May
Funded by NCI Contract No. HHSN261200800001E and
technology enhancement funds from SAIC Inc.
Results: An XML based system that includes the DICOM specified
separation of series and instances and includes relationships has
been created. The EPUB container file design is consistent with
client-server architecture of DICOM and with addition of binary
containing data files can be used as an ISAC ACS container file. The
use of data structures based upon elements to describe relationships
permitsbidirectional and multiple relationships between two
objects to be expressed. Very preliminary data indicates that the
CytometryML XML data elements can be used with XHTML5,
which would permit the creation of a medical informatics system
that has access to the full power of the Internet.
Saturday,
18 May
At this juncture, simulations and actual images are visually
similar and the current task is determining metrics for quantitative
comparison. With this tool in place, we will be able to quantify
F-actin alterations in cells in response to reagents in terms of
changes to number of fibers, spatial distribution of fibers in the cell,
physical properties of fibers and which F-actin structures were most
affected by a test reagent.
XSD element based implementation. The ISAC Archival Cytometry
Standard concept of a zipped data container file was further refined
to be an IDPF EPUB (electronic publication) file. Since the ToC
XML page locations are already present in the EPUB container, this
redundant information was minimized in the Relations schema,
which replaced the Table of Contents (ToC) schema of the Archival
Cytometry Standard (ACS). The Relations schema includes a
modified and extended version of the ToC RDF capabilities.
Special
Lectures
Although F-actin forms at least 15 distinct structures, we modeled
two structures: lamella and lamellipodia. In addition, modeling was
performed on cells grown individually on fibronectin micropatterns
in order to subsequently facilitate high throughput analysis. For
lamella, actin fibers were initially simulated as randomly positioned
rods that formed cross-links at their intersections. The model was
mathematically represented as a Hamiltonian energy equation.
The optimization minimized the total energy by moving the rods,
allowing cross-links to break under high fiber stress and adjusting
coefficients for each energy term. Two energies represented
biophysical properties of fibers: stretch and bend. Two further
energies were derived from images: coherence and flow, which
required that simulated fibers collect in regions of the image where
actual actin was most dense and be oriented in a similar direction.
Agreement between a simulated and actual image was further
optimized by adjusting the number of rods and implementing
features associated with optical imaging. Simulation of lamellipodia
F-actin was similar in principle but was restricted to a user-defined
distance from the edge of the cell. In addition, two non-interacting
layers of lamellipodia were simulated, a lower layer juxtaposed to
the glass substrate and an upper layer approximately 0.5 micron
above.
Congress
Overview
biophysical properties of the cytoskeleton, and the simulations are
optimized to approximately match actual images. In this study, we
built models of the lamella and lamellipodia F-actin cytoskeleton
in individual cells. This modeling can be applied to interpreting
observed changes to F-actin in response to a test reagent, in terms
of where, when and how F-actin physically changes in the cell.
Such understanding will in turn implicate which actin-associated
proteins are affected by direct or indirect interaction with the
reagent.
163
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Automated Flow Cytometry Data Analysis with the
OpenCyto Framework
John Ramey1, Greg Finak1, Mike Jiang1, Jafar Taghiyar 2,
Stephen DeRosa1, Ryan Brinkman3, Raphael Gottardo4
1
Fred Hutchinson Cancer Research Center, Seattle, WA,
United States, 2British Columbia Cancer Agency, Vancouver,
BC, Canada, 3Terry Fox Laboratories, British Columbia Cancer
Agency, Vancouver, BC, Canada, 4Fred Hutchinson Cancer
Research Center, Seattle, WA, Canada
Background: Advancements in flow cytometry (FCM) technology
have enabled rapid quantification of multidimensional attributes
for millions of individual cells to identify meaningful cellular
subpopulations and to assess cellular heterogeneity. However, the
analysis of the resulting large, high-dimensional data sets typically
involves a time-consuming sequential manual-gating strategy that
is inherently subjective as the gating depends on the analyst. This
subjectivity can yield highly variable gate placement from person
to person if an experiment is not well-controlled or if a marker is
not well-resolved. Alternatively, automated, data-driven pipelines
are necessary to expedite the gating of FCM data and to remove the
subjectivity intrinsic to manual gating. This is particularly important
in clinical trials where assays must be extremely well controlled in
order to generate data that is comparable over time.
Methods: We have developed the OpenCyto framework, a
collection of well-integrated open-source R packages that delivers
robust, reproducible, and data-driven gating in an automated
pipeline by incorporating expert-elicited and data-driven prior
knowledge within a Bayesian model. OpenCyto promotes relatively
fast and exhaustive gating that is interpretable in the context
of standard hierarchical, two-dimensional projections of cell
populations, which analysts are used to seeing. Our automated
gating approach allows gating thresholds to be fine-tuned to
optimize detection of informative cell populations in order to
discriminate between subject cohorts based on objective external
criteria such as vaccination status. We also utilize the LabKey
software to provide an easy-to-use web interface to visualize and to
analyze FCM data with our automated pipeline.
Results : We demonstrate that OpenCyto can recapitulate
manual-gating efforts obtained on ICS data sets from the Human
Immunology Project Consortium and the HIV Vaccine Trials
Network. We calculated the coefficients of variation of cellular
population proportions across the samples and found that the
variability from OpenCyto is well within the range of that of manual
gating, even for rare cellular subpopulations. Furthermore, using
the gates constructed by OpenCyto, we were able to discriminate
accurately the vaccination status of the subject cohorts as well as to
identify the antigen-specific T-cells responding to the vaccine.
Conclusions: OpenCyto provides automated, data-driven gating
of high-dimensional FCM data sets quickly, removing the timeconsuming task of manual gating. By incorporating expert-elicited
and data-driven prior knowledge, OpenCyto attains accurate
gating of cell populations, including rare populations, while
controlling variability relative to manual gating, thereby overcoming
the subjectivity in manual gating. Finally, using OpenCyto we
can reproducibly identify associated biomarkers to distinguish
vaccination status within a cohort in clinical trial data.
Poster Session
Abstracts
Oral Session
Abstracts
193/B72
Cytometry in Resource-Poor Settings
(B73)
194/B73
Reliable and Accurate CD4 T Cell Count and CD4
Percent of the New Portable Flow Cytometer Cyflow
Minipoc
Milena Nasi1, Sara De Biasi1, Elena Bianchini1, Lara Gibellini1,
Marcello Pinti2, Tiziana Scacchetti3, Tommaso Trenti3, Vanni
Borghi4, Cristina Mussini4, Andrea Cossarizza1
1
Department of Surgery, Medicine, Dentistry and Morphological
Sciences, University of Modena and Reggio Emilia, Modena,
Italy, 2Department of Life Sciences, University of Modena
and Reggio Emilia, Modena, Italy, 3Department of Clinical
Pathology, NOCSAE Baggiovara, Modena, Italy, 4Infectious
Diseases Clinics, Azienda Ospedaliero-Universitaria
Policlinico di Modena, Modena, Italy
Background: Human immunodeficiency virus (HIV) infection
is increasing at an alarming rate worldwide, and the burden is
heaviest in sub-Saharan Africa. Since the virus kills, directly or
indirectly, CD4+ cells, the accurate, reliable, and affordable
CD4 T cells count is essential in determining disease stage and
progression. It is also crucial in determining when antiretroviral
(ARV) therapy has to start, and in its monitoring. Flow cytometry is
clearly the “gold standard” for CD4 T cell count, but this technique
is expensive and requires sophisticated equipment and trained
personnel. In addition, the lack of ready access to technical support
and quality assurance programs limits the use of flow cytometry
techniques in resource-constrained countries. Instruments are now
available that can solve these problems. We have tested the new
portable flow cytometer for CD4 T cell count percentage, named
CyFlow MiniPOC (Partec), and analysed its sensitivity, carry-over
contamination and repeatability. Its accuracy has been compared
analysing the same blood samples with 2 other systems: CyFlow
Counter (Partec) and Cytomic FC 500 (Beckman Coulter).
Methods: Venous blood from 59 adult HIV-1 infected patients (age
25-58 years; sex: 43 males; CD4 count range: 34-1, 115 cells/ul;
CD4% range: 3.1-48.0%) was collected in EDTA blood, stained
with the Partec miniPOC CD4% count kit - dry kit, and analysed
within a maximum of 2 hours. CD4 T cell count and percentage
were determined by the CyFlow MiniPOC instrument, equipped
with a 30 mW, 532 nm laser and three optical parameters for
detection of side scatter (SSC), orange and red fluorescence. CD4
T cell count and percentages were measured in parallel by CyFlow
Counter and by a dual platform system based upon Cytomic FC 500
(Cytostat tetrachrome kit for mAbs) and Coulter HMX (for absolute
cell count). All measures were performed in triplicate.
Results: The accuracy of CyFlow MiniPOC against Cytomic FC
500 showed a correlation coefficient of 0.98 and 0.97 for CD4 T
cell count and percentage, respectively (linear regression analysis).
The accuracy of CyFlow MiniPOC against CyFlow Counter showed
a correlation coefficient of 0.99 for both CD4 T cell count and
percentage. CyFlow MiniPoc showed an excellent repeatability:
CD4 absolute number and percentage were analysed on two
instruments, with a intra-assay precision below +/- 10% deviation.
The sensitivity was linear in the range 0-5,000 CD4 T cells/ul. There
was no effect of carry-over contamination for samples at all CD4
values, regardless of their position in the sequence of analysis.
Speaker/Author
Index
Conclusions: The cost-effective and portable instrument MiniPOC
produces reliable and accurate results that are fully comparable
with highly expensive dual platform systems. Indeed, data perfectly
correlate with those obtained with Cytomic FC 500/Coulter HMX.
Finally, using CyFlow MiniPoc permits to perform in a fast and easy
way a very high number of CD4 T cells counts per day.
164
ISAC 2013 Program and Abstracts
195/B74
Alexey Polshchitsin, Vyacheslav Nekrasov, Andrey Chernyshev,
Valeri Maltsev
Laboratory of Cytometry and Biokinetics, Institute of Chemical
Kinetics and Combustion, SB RAS, Novosibirsk, Russia
Tuesday,
21 May
Wednesday,
22 May
Methods: We developed a polychromatic flow cytometry protocol
to detect intracellular LAMP2 protein in leukocyte subsets.
Key parameters of the assay are: leukocyte subset definitions,
permeabilisation control (via LAMP1/CD107a detection), doublet
discrimination (decreasing a false positive events), high cell number
processed (3mil leukocytes, enabling a rare cell detection). We
employed this protocol in 1) screening of patients with clinical
suspicion of DD 2) LAMP2 deficiency assessment in families with
molecular proof of LAMP2 gene mutation.
Poster
Session
Results: We detected LAMP2 negative granulocytes and monocytes
indicative of LAMP2 deficiency in four male hemizygotes (all
proved to carry specific LAMP2 gene mutation) with clinical
symptoms, one symptomatic female heterozygote (with 13%
LAMP2 deficient leukocytes) and asymptomatic mother of DD
patients (founder with combined X-inactivation and somatic
mosaicism – with 0,06% LAMP2 deficient granulocytes) in two
unrelated families. Our protocol is routinely used to test patients
with cardiomyopathy suggestive of DD and family members of DD
patients. Although LAMP2 plays a nonredundant role in lysosomal
handling of autophagosomes and phagosomes by mediating
membrane and membrane/cytoskeletal interactions, we show that
LAMP2 is not essential for degranulation of cytotoxic vesicles in
T-cell or NK-cells.
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Conclusions: We present a new method of quantitative
immunoassay with the use of light-scattering flow cytometry.
Using the proposed technique it is possible to determine not only
the concentration of the target protein in a sample, but also such
important parameters as, for example, the affinity constant of
antigen-antibody and the number of antibodies on the surface of
one bead. This allows one to control the quality of the regents in the
clinical diagnostics and significantly increases the accuracy of the
analysis.
Background: Danon disease (DD), an X-linked disorder, results
from mutations in the lysosomal associated membrane protein-2
(LAMP2) gene (CD107b) and presents with hypertrophic
cardiomyopathy, skeletal myopathy, and mental retardation.
Although skeletal and cardiac musclesare primarily affected by
LAMP2 deficiency, LAMP2 absence can be readily detected in
leukocytes (neutrophil granulocytes and monocytes are uniformly
LAMP2 positive in healthy donors, lymphocytes are heterogeneous).
We demonstrate a flow cytometry protocol for LAMP2 deficiency
detection in peripheral blood leukocytes, whichallows fast,
minimally invasive and powerful method for diagnostics in affected
males, but also efficient detection of female carriers/heterozygotes
with more variable DD phenotype. Furthermore, we tested LAMP2
deficient individuals for their T-cell and NK-cell degranulation
capacities.
Monday,
20 May
Results: A new spotted spheres agglutination rate kernel was
developed. Based on the Smoluchowski equation we theoretically
predicted and experimentally demonstrated the linear increase
in the ratio of the total number of aggregates to the number of
monomers in time in a wide range of the monomers conversion
degree and the fractal dimension of particle clusters. Using the
slope of the ratio in time we determined the dimerization rate
constants for all experimental sets. Using the DiRect method
of global optimization we achieved the best possible match of
theoretical and experimental data. Thisprocedureallowed us to
determinethe characteristic values of examined system. The values
obtained were in good agreement with data in literature.
Ondrej Pelak1, Jakub Sikora2, Ladislav Krol1, Filip Majer2,
Lenka Dvorakova2, Hana Vlaskova2, Tomas Honzik3, Tomas
Palecek4,5, Milos Kubanek6, Tomas Kalina1
1
Pediatric Hematology and Oncology, Charles University
Prague, 2nd Medical Faculty, Praha 5, Czech Republic,
2
Institute of Inherited Metabolic Disorders, Charles University
in Prague and General University Hospital, First Faculty of
Medicine, Prague, Czech Republic, 3Department of Pediatrics
and Adolescent Medicine, Charles University in Prague and
General University Hospital, First Faculty of Medicine, Prague,
Czech Republic, 42nd Department of Medicine - Department
of Cardiovascular Medicine, Charles University in Prague
and General University Hospital, First Faculty of Medicine,
Prague, Czech Republic, 5International Clinical Research
Center, St. Anne's University Hospital Brno, Brno, Czech
Republic, 6Department of Cardiology, Institute for Clinical and
Experimental Medicine, Prague, Czech Republic
Sunday,
19 May
Methods: The agglutination of the biotin conjugated yellowgreen fluorescent 1 µm beads mixed with streptavidin in different
proportions was studied in the series of the experiments by means
of the scanning flow cytometry, the method for measurement of
angle-resolved intensity light scattering patterns (LSPs) of individual
particles. We measured LSPs and fluorescent signals with the
Scanning Flow Cytometer fabricated by CytoNova Ltd. (Novosibirsk,
Russia, http://cyto.kinetics.nsc.ru/). Using the LSPs and fluorescent
signals we measured the relative fractions of aggregates composed
of different number of monomers.
Flow Cytometry Detection of LAMP2 Protein in
Danon Disease—a Rare X-Linked Cardiomyopathy
Saturday,
18 May
Background: Immunoassay tests based on agglutination of
polymer particles are widely used in biology and medicine for
the determination of low concentrations of antigens or antibodies
in the sample.Most of these tests are based on characterization
of the whole particles ensemble properties with heterogeneity
of population elements in biological and physical features being
omitted.This imposes significant limitations on the reliability of
the results obtained. Implementation of methods of population
individual particles properties study (e.g. scanning flow cytometry)
would allow one to interpret the results without any priori
assumptions about the distribution functions of parameters.
However, these methods have not been sufficiently developed
and are notused in practice. So the purpose of the work is to
create and to verify experimentally the detailed kinetic model of
immunoagglutination process.
196/B75
Special
Lectures
Investigation of Protein-Coated Particle Aggregation
Using Scanning Flow Cytometry
Congress
Overview
Diagnostics (B74 – B83)
Poster Session
Abstracts
ISAC 2013 Program and Abstracts
Speaker/Author
Index
Conclusions: Diagnosis of DD and detection of heterozygotes
with X-inactivation can be readily made using flow cytometry
detection of LAMP2/CD107b in leukocytes. This approach is less
invasive then muscle biopsy, quantitative and more sensitive then
microscopy of blood smears. Presented protocol offers reliable,
reproducible results even at very low frequency of affected cells,
such as in one case of X-inactivation/mosaic. In conclusion, flow
cytometry (followed by molecular genetic assessment) proved to
165
Congress
Overview
be the first choice method for diagnosis of hereditary disorder of
skeletal and cardiac muscle.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
Special
Lectures
Supported by: UNCE 204012, P/302/12/G101, PRVOUK-P24/LF1/3
and RVO-VFN64165/2012
T.K. supported as ISAC Scholar
197/B76
Determining Reference Bead Concentration and
Fluorescence Intensity for Quantitative Flow
Cytometry at 660 nm and 760 nm
Paul DeRose, Adolfas Gaigalas, Lili Wang
NIST, Gaithersburg, MD, United States
Background: The accurate determination of antibodies bound per
cell (ABC) is an important challenge in quantitative flow cytometry
(QFC) and other clinical assays. The comparability and accuracy
of these assays often depend on the accuracy of the ABC values
obtained. In QFC, the fluorescence channels being used in an assay
must be calibrated, typically using reference beads with known
fluorescence intensities, before ABC assignments of samples can
be made. Fluorophore solutions of known concentration are used
to assign fluorescence intensities to these standard beads in units
of equivalent number of reference fluorophores (ERF). To make
this assignment, the concentrations of these reference fluorophore
solutions need to be known with high accuracy.
Methods: HPLC, quantitative NMR and elemental analysis were
used to determine the purity of Nile Red. Gravimetry was then used
to determine the concentration of reference fluorophore solutions
of Nile Red. NIST’s High Accuracy Fluorescence Spectrometer
was used to measure the fluorescence intensities of reference
fluorophore solutions and corresponding bead suspensions. The
concentrations of the beads in suspension were determined using
three independent methods that implemented a flow cytometer, a
coulter counter and a hemocytometer.
Results: The purity of Nile Red used in the reference solutions was
determined to be greater than 99%, producing reference solutions
with concentrations known to better than 1% uncertainty. A calibration
curve of fluorescence intensity versus concentration enabled ERF
assignments of calibration bead suspensions with uncertainties of
about 5% or less. The concentrations of the beads in suspension
were also determined with an uncertainty of less than 5%.
Conclusion: NIST previously demonstrated the ERF assignment
of FITC standard bead suspensions for the calibration of the FC
channel centered at 530 nm using SRM 1932 Fluorescein Solution,
which was certified for concentration. Here we have outline
methods used for the determination of the concentration and ERFbased fluorescence intensity of reference beads for the calibration
of the FC channels centered at 660 nm and 760 nm, using Nile
Red as a reference fluorophore. We foresee this improvement in
calibration accuracy of flow cytometers will lead to improved
accuracy in ABC determinations.
198/B77
Characterization of Two Human CD4+ Lymphocyte
Preparations for Quantitative Flow Cytometry
Lili Wang, Meiyao Wang, Hua-Jun He, Martin Misakian,
Illarion Turko, Kenneth Cole
NIST, Gaithersburg, MD, United States
Background: It’s well known that CD4 expression level is fairly
consistent on normal human T helper cells, and therefore, can
serve as a good biological calibrator for quantification of other
cell surface and intracellular antigens using flow cytometry. In
our previous study of characterizing different normal human T
lymphocyte preparations, including cryopreserved peripheral blood
mononuclear cells (PBMC) and lyophilized Cyto-Trol Control
Cells obtained commercially [Cytometry Part A, vol. 81A: 567-
166
575 (2012)], we observed an approximately 15%-16% lower CD4
expression level from Cyto-Trol cells than that of cryopreserved
PBMC. The underlying reason for the lower CD4 expression level is
currently unknown.
Methods: A multiple reaction monitoring mass spectrometry (MRM
MS) method combined with an isotope-labeled recombinant CD4
peptide as an internal standard developed in house is used to
quantify CD4 receptors on human T cells. Application of isotope
(13C and 15N) labeled CD4 peptide as internal standards overcomes
quantitative errors arising from protein hydrolysis and variations
associated with complexed biological sample processing. The cell
lysates of known numbers of CD4+ cells are supplemented with the
standard peptide followed by trypsin digestion. After separation of
the peptides, mass spectrometry analyses of peptides are performed.
With known amount of CD4 peptide standard added and known
number of CD4+ cells present, CD4 receptor density on CD4+ cell
is therefore obtained. Additionally, scanning electron microscopy
(SEM) is used for probing changes in CD4+ cell membrane structure
and morphology.
Results: The preliminary MRM MS results are larger than those
determined using both flow and mass cytometry methods reported
in our previous study, which rely on the affinity binding between
CD4 receptors and anti-CD4, suggesting that not all CD4 receptors
are accessible for anti-CD4 binding. The SEM images of CD4+ T
cells from cryopreserved PBMC, fresh whole blood and lyophilized
Cyto-Trol reveal that CD4+ cell membrane structure and
morphology are largely altered due to the lyophilization process.
Conclusion: To serve as a biological calibrator for the
transformation of a linear fluorescence intensity scale obtained
with fluorescent microspheres to an antibody bound per cell (ABC)
scale [Cytometry Part A, vol. 73A: 279-288 (2008)], a candidate cell
reference material must have a reproducible and tight ABC value
for CD4 expression. The present investigation and previous study
support that cryopreserved PBMC and Cyto-Trol fulfill reasonably
the requirement of a reference cell material. Depending on specific
flow cytometry applications, users could make good use of these
human lymphocyte preparations for achieving quantitative flow
cytometry measurements.
199/B78
Assessment of Myeloid Nuclear Differentiation
Antigen (MNDA) in Myelodysplastic Syndrome and
Acute Myeloid Leukemia
Kah Teong Soh1, Paul K. Wallace2
Biotechnical and Clinical Laboratory Science, SUNY
University at Buffalo, Buffalo, NY, United States, 2Flow &
Image Cytometry, Roswell Park Cancer Institute, Buffalo, NY,
United States
1
Myelodysplastic syndrome (MDS) is a set of clonal marrow failure
disorders that is difficult to diagnose due to the lack of standard
diagnostic parameters, admixture of normal bone marrow in the
MDS samples and large differential diagnosis of the disease. Left
untreated, MDS patients will experience reduced life expectancy
due to either infectious complications, a consequence of the
neutropenic state, bleeding, or progression to leukemia. Myeloid
nuclear differentiation antigen (MNDA) is a potential marker that
has been demonstrated to help diagnose the disease. This marker
is expressed at high levels in myelomonocytic cells, especially of
the mature granulocyte and monocyte lineages. Previous studies
have shown that MNDA expression is decreased in some familial
and sporadic MDS cases. We set out to validate the diagnostic
capabilities of this marker by examining the staining pattern of
bright monocyte and bright granulocyte using dim lymphocytes as
the threshold for the internal control. MNDA expression on patients
with either known or suspected MDS and AML were compared
with healthy donors. CD45, CD14, CD33 and CD66abce were
used to define each leukocyte subpopulation and the MFI of bright
ISAC 2013 Program and Abstracts
Results: There were significant amount of epithelial cells and
leukocytes in whole saliva demonstrated by light microscopy.
They could be identified by morphology. Among WBCs, there was
variable percentage of CD3 T cells. We anticipated the presence of
other subsets as well.
The extent of gum bleeding could be evaluated by the presence of
RBCs in whole saliva. It did varied significantly among individuals.
The current flow cytometric protocol can provide data on both
intact and lyzed RBC. The absolute number and its ratio could
suggest timing of bleeding.
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Using SYBR Gold, we were able to visualize smaller cellular
elements including oral bacteria and cellular microparticles. The
various morphotypes and differences in nucleic acid content could
potentially provide classification information.
Oral Session
Abstracts
Conclusions: This study is an ongoing project. We hope to provide
a working framework for those interested in salivary cytomics. The
detection of various cellular elements could be a useful diagnostic
tool for dental professionals. Early diagnosis of oral squamous cell
carcinoma, evaluation of gum diseases, and study of caries are
among the examples. In addition, dental professionals may choose
to collect gingival fluid or from tongue to collect site-specific
information. For areas with limited resources, dentists may choose
to establish rapid chair-side testing protocols, or preserve samples
for future longitudinal study at larger facility.
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Methods: Whole saliva samples were collected through natural
draining from volunteers with informed consent. Light microscopy
with Sedi-Stain was utilized to visualize cellular elements, and
those elements were quantitated using hemocytometer. In order
to keep saliva samples for future studies, we used ethanol to
preserve cellular elements. For projects looking at RBCs, Streck
Cell Preservative was used instead to preserve for up to 5 days. The
amount of RBC was quantitated by CD235a staining. Following
proper hydration, the presence of leukocyte (CD45) and T cells
(CD3) were demonstrated. In addition, using SYBR Gold, cellular
elements with nucleic acid were visualized and quantitated by
fluorescence microscopy and flow cytometry.
Poster
Session
Conclusion: Mid-induction peripheral blood sample cannot replace
bone marrow aspirate for detection of MRD by flow cytometryin
paediatric patients of B lineage acute lymphoblastic leukemia.
Background: Oral health is an open window to our overall health.
Examining oral cavity for focal periodontitis for example, provides
an opportunity to evaluate the oral health and general condition.
Saliva, particularly the supernatant, has been tested for molecules
present in plasma and saliva. While sediment after spinning was
discarded sometimes, the potential information could be discovered
particularly from those cellular elements in generally hypotonic
environment. We aimed to establish methodology for studying those
cellular elements in saliva. Among the protocols in development,
there are those designed for rapid chair-side testing, and those for
preserving saliva for longitudinal study. It is likely that they can be
easily adapted to areas with limited resources.
Wednesday,
22 May
Results: Residual leukemic cells were detected in marrow and
blood in eight (34.78%) pairs, with MRD values ranging from 0.032.1% (mean 0.67%) and 0.01-0.83% (mean 0.2%), respectively.
MRD was undetectable in six(26.08%)pairs. Discordance was
noted in nine(39.13%) pairswith MRD detected in marrow and not
in blood, with the MRD values ranging from 0.023-0.6% (mean
0.23%).None of the cases showed MRD in peripheral blood alone.
In other words, MRD was detected in 17/23 (73.91%)mid-induction
bone marrow samples with 9 of these 17 cases not showing MRD
in their peripheral blood sample.
Zhibin Chen1, Fang Yao Stephen Hou2
1
Periodontology, Peking University School of Stom, Beijing,
China, 2Clinical Laboratory Science, Marquette University,
Milwaukee, WI, United States
Tuesday,
21 May
Methods: Twenty three newly diagnosed paediatric B lineage acute
lymphoblastic leukemia patients with presence of <5% blasts on
morphological examination of bone marrow aspirate on day 15
of chemotherapy (Vincristine, L-Asparaginase & Dexamethasone),
were assessed for MRD levels by flow cytometry.EDTAanticoagulated, paired peripheral blood and bone marrow aspirate
samples were collected after informed consent. Lyse-stain-wash
technique was used to process a single 6 colour tube comprising
of Syto13, CD34PE, CD20PerCP, CD10APC, CD19PECy7 and
CD45APCH7. One million events were acquired or complete
acquisition of the tube (whichever came earlier) was carried outon
BDFACS Canto II and analysed by BDFACS Diva software. MRD
was defined by presence of >0.01% leukemic cells.
Salivary Cytomics — A Useful Clinical Diagnostic
Tool for Dental Professionals
Monday,
20 May
Background: There is a strong correlation between minimal residual
disease (MRD) levels and risk of relapse in childhood leukemias.1,2
The assessment of MRD during mid-induction phase (day 1519) is simpler due to lack of hematogones (<0.01%) in bone
marrow, and thus a mid-inductionbone marrow aspirate sample
is commonly used to assess MRD levels for further management
decisions. A peripheral venous blood sample might be used for the
same if proven to yield similar MRD levels, thus avoiding a more
invasive procedure. This study was planned to assess the utility of
mid-induction peripheral blood samplein comparison toa paired
bone marrow aspirate sample for detection of MRD in paediatric
patients of B lineage acute lymphoblastic leukemia using six colour
flowcytometry.
201/B80
Sunday,
19 May
Man Updesh Sachdeva1, Karthik Bommannan B.K. 1, Parveen
Bose1, Neelam Varma1, Deepak Bansal2, R.K. Marwaha2
1
Department of Hematology, Postgraduate Institute of Medical
Education & Research, Chandigarh, India, 2Department of
Paediatrics, Postgraduate Institute of Medical Education &
Research, Chandigarh, India
Coustan-Smith E et al. Immunological detection of minimal residual
disease in children with acute lymphoblastic leukaemia. Lancet
1998, 351:550-4.
Saturday,
18 May
Six Colour- Single Tube Analysis of Minimal Residual
Disease in Paediatric B Lineage Acute Lymphoblastic
Leukemia on Paired Mid-Induction Peripheral Blood
and Bone Marrow Samples: Can Peripheral Blood
Replace Bone Marrow Aspirate Sample?
References: Cave H et al. Clinical significance of minimal residual
disease in childhood acute lymphoblastic leukemia. NEJM 1998,
339:591-8.
Special
Lectures
200/B79
Congress
Overview
and dim MNDA expression was calculated for each population. In
our studies, we were able to detect a population of granulocytes
with diminished MNDA expression in bone marrow samples from
some patients diagnosed with MDS. A MNDA dim population was
not observed in monocytes from these patients. Further clarification
is being performed on this dim granulocyte using eight different
monoclonal antibodies (CD11b, CD11c, CD13, CD16, CD38,
CD64, CD 133 and HLA-DR) to determine the extent of how these
antigens are modulated in this population. Ultimately, the goal of
this research project is to validate the usefulness of this assay to
assist in the identification and classification of MDS in the clinical
setting.
167
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
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Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
202/B81
TransFix®/EDTA Stabilisation of Leukocytes in
Cerebrospinal Fluid for Flow Cytometric Screening of
Patients with Suspected Leukaemic Conditions
Tim Almond1, Daniel Harrison1, Michelle Crawford2, Ulrika
Johansson2
1
Caltag Medsystems Ltd., Buckingham, United Kingdom,
2
Haematology Oncology Diagnostics, Bristol Royal Infirmary,
Bristol, United Kingdom
Leukocytes present in cerebrospinal fluid (CSF) samples, collected
by lumbar puncture, provide an invaluable medium with which
to screen patients with suspected central nervous system (CNS)
localised leukaemia or lymphoma. However, cells within these
samples are typically low in numbers, sparse in concentration
and degrade quickly resulting in the necessity to promptly test
CSF specimens to prevent false negative results. Coupled with
the painful and cumbersome lumbar puncture procedure, it has
been recognised that the stabilisation of CSF is required for patient
screening: to provide accurate results, to allow flexibility when
conducting the flow cytometric analysis and to prevent repeat
sampling. Previous studies have successfully used TransFix to
maintain the immunophenotypic profile so that these cells can be
analysed at a later time point. However, no study has systematically
compared cell yield or immunophenotypic profile for fresh and
TransFix-treated CSF samples.
In this study CSF specimens from 10 patients with suspected CNS
involvement of haematological malignancies were analysed fresh
(2 hours after lumbar puncture) using the flow cytometer. The
same samples were treated with TransFix/EDTA and were tested
identically after at least 72 hours (between 3-5 days) of storage
at 2-8˚C. The absolute number of lymphocytes, monocytes and
neutrophils and immunophenotypic profiles were compared using
a comprehensive monoclonal antibody panel.Data demonstrated
that the cell recovery - measured in absolute counts -of most
lymphocyte subsets observed in TransFix/EDTA treated CSF samples
matched and exceeded that of the fresh sample. In addition, antigen
expression profiles ofreactive and neoplastic cell populations
were maintained for the majority of the 20 antibodies tested.
The diagnostic conclusion would have remained the same for all
patients using TransFix/EDTA treated samples compared to fresh.
The results suggested that TransFix/EDTA can be used successfully
to stabilise CSF samples for screening purposes.
203/B82
Development of 8 Color Panel for Lymphoma
Diagnostics with Minimal Compensation
Requirements
Ivan Vorobjev1,2, Olga Khoudoleeva2,3
Biological faculty, Moscow State University, Moscow, Russia,
2
A.N. Belozersky Institute, Moscow State University, Moscow,
Russia, 3GeneTechnology, Moscow, Russia
1
Using new dyes BD Horizon V-500 and Brilliant Violet (BV)
we defined 8 color panel for the detection of small malignant
populations. The panel consists of 12 monoclonal antibodies (CD3,
CD19, CD45, CD5, CD23, CD38, CD20, CD22, CD43, CD10,
KAPPA, LAMBDA) and was developed to detect non-Hodgkin
lymphoma cells in small cell samples (hypoplastic bone marrow,
fine needle aspirates, or small lymph node biopsy specimens)
using 4 lasers (488, 640, 561 and 405 nm) from BD FACSAria
instrument. MAbs conjugates were selected to identify specific
antigen expression profiles characteristic for major nosologies (CLL/
SLL, mantle cell lymphoma, marginal zone lymphoma, follicular
lymphoma). This panel was tested on peripheral blood and bone
marrow and provides a significant improvement of diagnostic
potential. To minimize compensation problems we used 2 channels
for each laser, i.e. FITC and PerCP-Cy5.5 for blue laser, APC and
168
APC-Cy7 for red laser, PE and PE-Cy7 for green-yellow laser, and
BV and V500 for violet laser. Significant compensation using
standard filter setup was needed only for the following pairs of
channels: AmCyan/PacificBlue (~50%); FITC/AmCyan (~255); APC/
APC-Cy7 (~65%) and PerCP/PE (~25%). Using BV we obtained
staining index (SI) >15 for CD19 in the majority of samples. This
is significantly larger than obtained for this antigen using for the
same samples PE (~ 1.5 times) and APC (~2.5 times). Using PE
conjugates excited with the yellow-green laser allowed us to
obtain 1.5 time better resolution between positive and negative
population in compensated data set compared to the excitation
of same conjugates with the blue laser. Besides, use of green laser
makes compensation between PE and FITC channels negligible,
and significantly decreases compensation between PE and PerCP
channel and PerCP and PE-Cy-7 channel. The cell populations
were first plotted in a CD45-V500/SSC dotplot and different
subpopulations were gated subsequently. Signal obtained from
Horizon V500 required significant compensation with Pacific Blue
and FITC channels however after compensation it was sufficient to
discriminate CD45-bright and CD45-dim populations. We conclude
the use of new dyes and 4 lasers instead of three that are currently
standard will simplify data interpretation in troublesome cases and
benefit for diagnostics of lymphomas.
The authors thank BD Bioscience for providing CD45-V500, Dr.
N. Barteneva for helpful discussion and generous support, D.
Potashnikova for data handling assistance. This work was supported
in part by M.V. Lomonosov Moscow State University Program of
Development and by RFBR grants 11-01517a and 11-01749a to
I.A.V.
204/B83
Analysis of Skeletal Muscle: Correlated
Quantification of Mitochondrial Metabolic
Enzymatic Activities and Fiber Type-Specific
Biomarkers
Patrick McDonough1, David Reiner2, Tatiana Kostrominova3,
Richard Haas4
1
Biology, Vala Sciences Inc, San Diego, CA, United States,
2
WM&G Consulting, Imperial Beach, CA, United States,
3
School of Medicine - Northwest, University of Indiana, Gary,
IN, United States, 4Departments of Neurosciences & Pediatrics,
University of California San Diego, La Jolla, CA, United States
Background: Mitochondrial disease is the most common
neurometabolic disease of children, and is characterized by
impaired energy production resulting from genetically based
oxidative phosphorylation dysfunction.Defects in electron transport
chain enzyme complexes account for the majority of mitochondrial
disorders and most clinical diagnostics are based on assay of
catalytic activity mitochondrial metabolic enzymes in skeletal
muscle biopsies, which feature a high content of mitochondria.
Electron transport assays (most commonly for cytochrome C
oxidase (COX), succinate dehydrogenase (SDH), and NADHdehydrogenase) are performed on frozen tissue sections, yielding
colorimetric (bright-field) readouts of enzyme activity. In most
cases, researchers, physicians, and pathologists visually inspect
the biopsies and record a qualitative assessment of the enzymatic
activity, but the metabolic activities are not corrected for the fiber
types that are present. This is a problem as human muscle is
typically a mixture of Type I (oxidative) and Type II (glycolytic) fiber
types, which varies between patients.
Methods: We have developed methods to simultaneously label
skeletal muscle for metabolic enzymatic activities and fiber-specific
biomarkers, using a combination of bright-field and fluorescence
microscopy, and the methods to quantify the results on a fiber-byfiber basis, utilizing high-content analysis techniques.
Results: Preliminary results obtained from human skeletal muscle
biopsies indicate, for example, that NADH-dehydrogenase activity,
ISAC 2013 Program and Abstracts
205/B84
NK cells express activating and inhibitory receptors and the
resulting effect depends of the equilibrium of these engaged
receptors.
Background: Rheumatoid arthritis(RA) is a debilitating autoimmune
disease whose etiology remains unknown, but studies have
consistently implicated a plethora of inflammatory mechanisms
culminating in chronic symmetric and erosive synovitis, and
include a role for oxidative stress. The disease is consistently
associated with an increase in various pro-inflammatory factors that
includes cytokines (IL-1β, IL-6, tumor necrosis factor alpha TNF-α),
prostaglandins, reactive oxygen species (ROS) and nitric oxide (NO)
at sites of inflammation. As these reactive species contribute directly
towards the destructive, proliferative synovitis evident in RA, this
study aimed to correlate the degree of oxidative stress along with
downstream effects of oxidative damage in synovial infiltrated cells
and its correlation with disease activity score, so that measurement
of oxidative stress or its damage could help in monitoring the
disease progression of patients with RA.
In this work, we have developed 3 combinations to phenotype
NK cells receptors in order to access to inhibitory and activating
molecules on different types of patient samples. Healthy donors
were used as reference and pathological samples were also
analyzed (AML patients at different stages of the disease).
Conclusion: Taken together, raised levels of ROS and markers of
oxidative damage are a consistent feature of patients with RA. As
levels of ROS andmarkers of oxidative damage correlated positively
with the DAS 28, it suggests that monitoring of oxidative stress
could serve as a marker of disease severity in Rheumatoid arthritis.
Poster Session
Abstracts
Results: In SF of patients with RA, ROS and hydroxyl radical
correlated positively with oxidative damage markers. Theselevels
of ROS and hydroxyl radical also correlated positively with
Disease Activity Score 28 (DAS 28)and oxidative damage markers.
Furthermore, oxidative damage markers showed apositive
correlation with DAS 28.
Oral Session
Abstracts
Natural Cytotoxicity receptors (NCRs), very important in NK cell
activation, associate with different activating coreceptor and
transduce signaling that promote NK cell activation and lysis of
cancer cells. Evaluation of their expression should be of interest in
NK cell based or related therapy and it was shown that several NK
based therapy were successful in Acute Myeloid Leukemia (AML).
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The receptors NKG2D (CD314), the natural cytotoxicity receptors
(NCRs) NKp30(CD337), NKp44(CD336) and NKp46(CD335), the
activating form of KIR (Killer cell immunoglobulin like receptor)
known as KIR-S and CD16 provide activating signals enhancing
toxicity and production of cytokines.
Methods: The redox status of neutrophils sourced from
synovial fluid (SF) wasmeasured by Flow Cytometry in terms
of total reactive oxygen species (ROS) and hydroxyl radical.
Among the molecular damage markerproteincarbonylation
was detected by spectrophotometry and western blotting, lipid
peroxidationandadvanced oxidation of protein products (AOPP)
by spectrophotometry, nitrotyrosylation by western blotting and
S-nitrosothiolsby flurimetry.
Poster
Session
CD96, expressed on NK cells after activation, promotes cell
adhesion between NK cells and their target cells. CD96 is a cell
surface marker present on many leukemic stem cells in acute
myeloid leukemia
Sunanda Kundu1, Suhana Datta1, Parasar Ghosh2, Alakendu
Ghosh2, Subrata Chattopadhyay3, Mitali Chatterjee1
1
Pharmacology, Institute of Post Graduate Medical Education
& Research, Kolkata, India, 2Rheumatology, Institute of Post
Graduate Medical Education & Research, Kolkata, India, 3BioOrganic Division, Bhabha Atomic Research Centre, India.,
Mumbai, India
Wednesday,
22 May
The most studied inhibitory receptors are a family of
immunoglobulin (Ig)-like receptor with two (KIR2DL1 or
KIR2DL2/3) or three (KIR3DL1) domains. Inhibitory receptors
prevent host cells killing: KIRs recognize HLA class I molecules
that prevent killing of normal cells and NKG2A (CD159a) is also an
important inhibitory receptor that heterodimerizes with CD94.
Correlation of Oxidant Status and Molecular
Damage with Disease Activity Score in Patients with
Rheumatoid Arthritis
Tuesday,
21 May
The human lymphocyte subset of natural killer (NK) cells plays
a critical role in the innate immune response, particularly in the
control of tumor development and growth. The activation of NK
cells is the result of a balance between inhibitory and activating
signals.
206/B85
Monday,
20 May
Gaelle Bouvier 1, Florence Orlanducci 2, Cyril Fauriat 2,
Emmanuel Gautherot 1, Felix Montero Julian 1, Christine
Arnoulet2, Daniel Olive2
1
Global assay and applications development, Beckman
Coulter Life Sciences, Marseille cedex 9, France, 2Institut Paoli
Calmettes, Marseille, France
Data were analyzed with Kaluza Software version 1.2. These
results allowed obtaining an extended phenotype of the NK cells in
healthy donor and during AML disease. These combinations will be
a powerful tool to investigate the potential application (regulation
mechanisms of expression and activation of these receptors) related
to these diseases.
Sunday,
19 May
Extended NK Cells Phenotyping in Patients with
Acute Myeloid Leukemia
CD3-FITC, NKp30-PE, VIVID-ECD, CD56-PC5.5,(CD158a, h-PC7,
CD158b1/b2, j-PC7), CD159-APC, NKp46-APC-A700, CD45Krome Orange, CD57-Pacific Blue.
Saturday,
18 May
Disease Progression Monitoring
(B84 – B87)
CD3-FITC, NKp30-PE, VIVID-ECD, CD56-PC5.5,(CD158a, h-PC7,
CD158b1/b2, j-PC7), CD96-APC, NKp46-APC-A700, CD45-Krome
Orange, DNAM-Pacific Blue.
Special
Lectures
Conclusions: The approach will likely improve the ability to
diagnose mitochondrial disorders, and increase our understanding
of fiber-type specific protein expression and metabolism.
Congress
Overview
and the expression of TRMU (tRNA 5-methylaminomethyl-2thiouridylate methyltransferase), a protein whose mutation is
associated with mitochondrial disease, are highest in Type I fibers,
whereas “ragged fibers”, which occur in specimens from patients
with mitochondrial disease, can occur in either Type I or Type II
fibers.
The three following combinations were used for the study:
ISAC 2013 Program and Abstracts
Speaker/Author
Index
CD3-FITC, NKp30-PE, VIVID-ECD, CD56-PC5.5,(CD158a, h-PC7,
CD158b1/b2, j-PC7), NKG2D-APC, NKp46-APC-A700, CD45Krome Orange, DNAM-Pacific Blue.
169
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
207/B86
Significant Antigens for Detection of Minimal
Residual Disease in Patients with Mantle Cell
Lymphoma Using Flow Cytometry Approach
Jana Chovancova1,2, Michael Doubek2,3, Jiri Mayer2,3
Masaryk University Brno, Brno, Czech Republic, 2Central
European Institute of Technology, Brno, Czech Republic,
3
Faculty Hospital Brno, Brno, Czech Republic
1
Background: Mantle cell lymphoma (MCL) is a B-cell neoplasm
with an aggressive clinical course typically characterized by
CD5+19+10-23-20+43+ immunophenotype. The increasing
number of effective treatment options has brought needs for early
detection of complete remission and minimal residual disease
(MRD). In this study, we propose CD markers that could give an
opportunity in flow cytometric detection of MRD.
Methods: A group of 22 patients with new diagnosis of MCL
together with 16 healthy donors were included into the observation.
The samples of peripheral blood were analyzed employing eightcolor flow cytometry protocol. Cell surface were stained with
fluorescence labeled monoclonal antibodies (anti-CD5, 10, 19, 20,
21, 22, 23, 24, 27, 38, 43, 45, 79b, 196 and 200). Flow cytometric
acquisition was performed on a FACSCantoII flow cytometer
(Becton Dickinson, NJ, USA).
Results: There were found significantly lower expressions of
antigens CD21, CD23, CD196 and CD200 on MCL B lymphocytes
compared to healthy controls (p<0.01). Furthermore, negative
expression of CD24 was measured in 5 patients (23 %), negative
expression of CD79b (both 5% cutoff) in 2 patients (9 %), positive
expression of CD43 was found in 3 patients (14 %) and positive
expression of CD10 (both 95% cutoff) in 3 patients (14 %).
Conclusions: Variations of typical immunophenotype of MCL have
been observed, which complicates both diagnostics and MRD
detection in a way as it has been employed in chronic lymphocytic
leukemia, where a standardized protocol was developed. The
solution of MCL MRD observation might be to design an individual
flow cytometry panel for each patient. Suggested antigens could
help to determine suitable MRD flow cytometry panels.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
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Poster
Session
Acknowledgement: This work was supported by grant
MUNI/A/0723/2012.
208/B87
Detection of Elevated Monocyte Proinflammatory
Cytokine Responses by Flow Cytometry in Previously
Cryopreserved Samples from HIV+ Individuals at Risk
for Cardiovascular Disease
Emilie Jalbert1,2, Nisha Parikh3, Todd Seto3, Dominic Chow1,4,
Lishomwa Ndhlovu1,2, Cecilia Shikuma1,4, Jason Barbour1,2
1
Hawaii Center for HIV/AIDS, Honolulu, HI, United States,
2
Dept. of Tropical Medicine, University of Hawaii, Honolulu,
HI, United States, 3The Queen's Medical Center, Honolulu,
HI, United States, 4Dept. of Medicine, University of Hawaii,
Honolulu, HI, United States
Despite virologic suppression by HIV antiretroviral therapy, residual
inflammation associated with chronic HIV infection increases the
risk of developing cardiovascular disease. Monocytes have been
shown to be major players in the development of atherosclerosis
due to their proinflammatory responses to oxidized Low Density
Lipoproteins.
Monocyte functional assays using primary cells commonly feature
the use of freshly isolated samples. However, large cohort studies
require samples to be drawn and processed at different times and at
different national and international sites, making cryopreservation
and cell storage for future assaying the only viable option for
sample collection. Moreover, monocyte function is typically
170
assessed by quantifying secreted levels of cytokines by ELISA,
which does not provide information about single-cell functionality.
Our study sought to assess the functional properties of previously
cryopreserved monocytes from peripheral blood of HIV-infected
individuals by flow cytometry.
The cohort consisted of 33 HIV(+) subjects on HAART and 14
HIV(-) risk- and age- matched subjects. Our flow cytometry-based
functional assay measured monocyte production of IL-1β, IL-8 and
IL-6 in the absence of stimulation and in response to LPS or oxLDL.
Without stimulation, HIV(+) subjects had a greater frequency of
cells producing IL-1β and IL-8. In the presence of either oxLDL
or LPS, both groups increased the frequency of responding cells
compared to no stimulation, but HIV(+) subjects maintained a
higher frequency of IL-1β(+) and IL-8(+) cells compared to HIV(-).
There was no IL-6 production in either group in the absence of
stimulation, but upon stimulation with either oxLDL or LPS, there
was a higher frequency of IL-6 producing cells in the HIV(+) group.
The higher level of inflammatory cytokine production in HIV(+)
adults compared to HIV(-), both at rest and in the presence of
stimulation, may in part account for increased risk of cardiovascular
disease seen in the HIV(+) population.
DNA Damage and Repair (B88)
209/B88
Quantitative Imaging Analysis of Replication Vis-a-Vis Dna Damage- Sites in Cells Exposed to
Dna Targeting Anticancer Drugs and Oxidative Stress
Jurek Dobrucki1, Krzysztof Berniak2, Paulina Rybak2, Tytus
Bernas2,3, Agnieszka Waligórska2, Miroslaw Zarebski2, Ewa
Biela2, Hong Zhao4, Zbigniew Darzynkiewicz4
1
Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Krakow, Poland, 2 Faculty of
Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Kraków, Poland, 3Nencki Institute of Experimental
Biology, Polish Academy of Sciences, Warszawa, Poland, 4New
York Medical College, Valhalla, Ny, United States
Background: A recently described method [1, 2] of quantitative
analysis of spatial (3D) relationship between discrete nuclear
events detected by confocal microscopy was applied in analysis
of a dependence between sites of DNA damage signaling (γH2AX
foci) and DNA replication (EdU incorporation) in cells subjected
to treatment with topoisomerase inhibitors camptothecin (CPT),
etoposide (ETP), mitoxantrone (MTX), cis-platinum (CS) or hydrogen
peroxide (H2O2).
Methods: Newly synthesized DNA was fluorescently labeled
using a precursor EdU (5-ethynyl-2’-deoxyuridine) and a ‘click’
reaction. γH2AX foci were labeled by immunofluorescence.
Images of replication and histone H2AX phosphorylation sites were
recorded using confocal microscopy aided with deconvolution.
Nearest-neighbor and correlation analyses were performed using an
algorithm written specifically for this task and described in [1, 2].
Results: CPT induces γH2AX foci, likely reporting formation of
double-strand DNA breaks (DSBs), almost exclusively at sites of
DNA replication. ETP induces γH2AX in S-phase, with a moderate
tendency toward replication sites. MTX induces γH2AX foci with
no detectable preference for replication foci. Histone H2AX
phosphorylation induced by CS is detected 2 and 4 h after exposure
to the drug and largely coincides with replication foci. Oxidative
stress leads to induction of γH2AX in replicating as well as nonreplicating cells, and only a weak tendency toward damage at sites
of DNA replication.
Conclusions: High degree of colocalization of EdU and γH2AX sites
in cells treated with CPT is coherent with the known mechanism
of induction of DSBs by DNA topoisomerase I (topo1) inhibitors
at sites of collision of moving replication forks with topo1-DNA
ISAC 2013 Program and Abstracts
To help provide guidance in setting core strategy, iLab Solutions has
synthesized its experience in working with 45 institutions and 400+
core facilities to provide frameworks for considering each of the
questions. In addition, this analysis relies on the results of iLab's
2012 “Core Benchmarking Study,” which received input from 200+
core managers across the world.
The following are the key questions explored.
• Do we have the right set of cores?
• Should we move to a centralized model?
• Should we buy service contracts?
• How should we track core productivity?
• Should we accept external customers?
• Is the used subsidy level acceptable?
• Do we need a core management system?
The iLab Core Benchmarking Study shows continued significant
growth in the role that core facilities play in the research operation.
Cores must achieve continual improvement on several dimensions,
including the need to serve more customers, generate more
revenue, adopt more advanced technologies, and manage more
Speaker/Author
Index
ISAC 2013 Program and Abstracts
When developing a strategy for core facilities, all research
organizations face a common set of critical questions. There is no
universal “right” answer to these questions; instead, each institution
must consider its particular objectives and constraints and set its
strategy accordingly.
Poster Session
Abstracts
Background: The importance of evaluating usage and efficiency of
sorting services has always been recognised, but the significance of
implementing standardised algorithms that could be applied across
Tad Fallows, Heather Lorenz
iLab Solutions, Boston, MA, United States
Oral Session
Abstracts
Anna Petrunkina1,2
Department of Medicine, University of Cambridge,
Cambridge, United Kingdom, 2Unit for Reproductive Medicine
of Clinics, Clinic for Horses, University of Veterinary Medicine
Hannover, Foundation, Hannover, Germany
1
Identifying Challenges, Opportunities, and Strategies
for Core Operations
Commercial
Tutorials &
Exhibits
Algorithm for Calculating the Utilization and
Efficiency of Sorting Service
212/B91
Poster
Session
211/B90
Wednesday,
22 May
Children’s Research Institute at UT Southwestern (CRI) is an
innovative collaboration between Children’s Medical Center and
UT Southwestern Medical Center that combines the leading clinical
resources of Children’s with the outstanding research resources of
UT Southwestern. CRI’s mission is to perform “game changing”
research at the interface of regenerative medicine, cancer, and
metabolism. To support this research CRI established a flow
cytometry shared resource laboratory (SRL). The facility occupies
a 350 sq ft room, which has been transformed to accommodate
several state-of-the-art instruments. As the field of cytometry
continues to evolve at a rapid pace, CRI flow facility users will
receive continuing education to stay abreast of new flow cytometry
tools and techniques. The core also plans to closely collaborate
with principal investigators, through proactive initiatives such as
acquiring the latest instrumentation, discovering new resources
and ensuring the highest quality technical and scientific expertise.
The Flow Cytometry SRL is open to qualified self-operators 24
hours a day, 365 days per year for all scientists at CRI and UT
Southwestern. It complements the instruments available through the
UT Southwestern flow cytometry core facility. The Flow Cytometry
SRL now supports more than 40 scientists after only 6 months of
operation and its flow cytometers are used over 200 hours per
month.
Conclusions: General application of this formalism would provide
a unified method for analysis and potential comparison of sorting
facilities, as illustrated by a practical example for comparison of
utilization and efficiency of capacity realization for two facilities.
Obviously, this algorithm could be applied to evaluation of any
technical resources providing experimental services that needto be
quantified.
Tuesday,
21 May
Nicolas Loof
CRI Flow Cytometry Facility, Children's Research Institute at
UT Southwestern, Dallas, TX, United States
Results: From these, two standardised metrics based on practical
staff sorting capacity are defined: utilization U =Srecord ÷
(Coperator per day OD) · 100% and efficiency coefficient E = OD÷ (M·N);
where Srecord is the record of sorting hrs, M is the number of
contractual working days and N is the number of staff. In addition,
a simple flow chart-based algorithm has been developed to help
facility managers to initiate a more complex analysis and to make
clear and justifiable decisions with respect to applications for new
capital equipment or for changing staffing levels.
Monday,
20 May
Establishment of Flow Cytometry Shared Resource at
Children’s Research Institute
Sunday,
19 May
210/B89
Methods: In the vast majority of cases, the capacity of the sorting
service is defined as the equipment capacity for the existing sorting
resource, or, as the maximal capacity of staff working full working
hours. It is important to evaluate resources in the context of the
real working environment, by taking into account other duties
demanded by job descriptions, infrastructure and operational
strategy. A formalism has been developed that includes multivariate
factors in order to monitor the practical rather than the theoretical
sorting capacity of a resource.It is based on the calculation of
capacity per operator per day (Coperator per day, a numerical indicator
defined by the operational strategy and/orthe job profile) and the
cumulative number of practical operating days over a period (OD)
for all members of staff.
Saturday,
18 May
Facility Management (B89 – B92)
resources and facilities has not yet received sufficient attention.
Indeed, almost every facility providing sorting services will have
their own metrics for evaluating output and capacity. Given the
complex contributions that core facilities provide for research
institutions, it is not surprising how heterogeneous is the variety of
performance metrics and approaches.
Special
Lectures
Literature:
[1] K. Berniak, P. Rybak, T. Bernaś, M. Zare˛ bski, E. Biela, H. Zhao,
Z. Darzynkiewicz, J.W. Dobrucki. Relationship between DNA
Damage Response, initiated by camptothecin or oxidative
stress, and DNA replication, analyzed by quantitative image
analysis (submitted to Cytometry A).
[2] T. Bernaś, K. Berniak, P. Rybak, M. Zare˛ bski, H. Zhao, Z.
Darzynkiewicz, J.W. Dobrucki. Analysis of spatial correlations
between patterns of DNA Damage Response and DNA
replication in nuclei of cells subjected to replication stress or
oxidative damage (submitted to Cytometry A).
[3] D.A. Gilbert. Replication origin plasticity, Taylor-made:
inhibition vs recruitment of origins under conditions of
replication stress. Chromosoma, 2007;116:341-347.
Congress
Overview
“cleavable complexes” stabilized by CPT. The moderate or poor
correlation of replication sites with γH2AX foci as seen in the case
of ETP or MXT (also observed with these drugs in non-replicating
G1 and G2 cells), indicates on the mechanism of DNA damage
unrelated to replication. The increased number of replication foci
observed in cells treated with CPT suggests that stalling replicating
forks may trigger activation of new DNA replication origins. This
agrees with a postulated plasticity of replication origins [3].
171
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
The appropriate core strategy is dependent upon an institution's
objectives and constraints. Although, there is no “right” answer,
all levels within the institution must analyze the common set of
important questions to develop an appropriate cores program
strategy.
213/B92
The Purdue Cytometry Email Discussion List
J. Paul Robinson1, Bartek Rajwa2
1
BMS/BME, Purdue University Cytometry Laboratories, West
Lafayette, IN, United States, 2Purdue University
Purdue University Cytometry Laboratories developed a scientific
discussion list for flow cytometry in 1989 and the archive has
been maintained since 1990. This list is available to all members
of the community free of charge and is intended as a high quality
scientific discussion about all aspects of cytometry.
There are currently over 4000 active members on the list. The list is
a monitored list, which means that all messages are reviewed prior
to posting. The reason for this is that we do not accept advertising
by members of the list and our goal is to ensure that all messages
are valid scientific messages. Members should never see spam or
private messages as these are removed on a daily basis prior to
posting.
The list has now been operating continuously for over 23 years.
There is a large archive of questions and answers and this can be
searched. The current month listings as well as general information
about the list is available at: http://www.cyto.purdue.edu/hmarchiv/
index.htm . The same page will link users to the last 2 decades of
the archive which utilizes a Google based search engine to search
for selected search terms.
We encourage all members of the cytometry community to
participate in this electronic forum.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Wednesday,
22 May
To SUBSCRIBE: send an email to subscribe@flowcyt.cyto.purdue.
edu. You will be required to submit a survey and if accepted, you
will be sent posting instructions. Only members of the list may post
messages.
Poster
Session
Tuesday,
21 May
complex businesses. Additionally, Federal and state governments
are demanding that institutions deliver greater research results with
fewer resources and lower grant support. Ultimately, collaboration
between cores and institution administration is necessary to
appropriately address potential revenue, improve customer service,
and maintain the quality of research.
The list address is cytometry@lists.purdue.edu
Flow Cytometry Instrumentation
(B93 – B108)
214/B93
Detection of the Fluorescence Lifetime of Green
Fluorescent Protein Expressed in Yeast Cells with a
Time-Resolved Flow Cytometry
Frederick Crawford1, Bryan Sands2, Patrick Jenkins1, Ruofan
Cao1, Willaim Peria2, Roger Brent2, Jessica Houston1
1
Chemical Engineering, New Mexico State University, Las
Cruces, NM, United States, 2Fred Hutchinson Cancer Research
Center, Seattle, WA, United States
Background: The fluorescence lifetime is the average time a
fluorophore spends in an excited state before returning to ground
state. It is a useful fluorescence property because it can provide
additional quantitative information related to the environment of
the fluorescent species. In this contribution, we test the ability to
detect the fluorescence lifetime of green fluorescent protein with
a flow cytometer, when the protein is expressed in saccharomyces
172
cerevisiae. If detectable, we hope to then express fluorescent
proteins in yeast for signal transduction studies and measure the
fluorescence lifetime at a high throughput to quantify proteinprotein interactions.
Methods: Yeast cells expressing green fluorescent protein (GFP)
and a fusion of green fluorescent protein with a “dark” (i.e. low
fluorescence) citrine-colored fluorescent protein (GFP-darkCitrine,
courtesy of the Brent laboratory at the Fred Hutchinson Cancer
Center) were analyzed with a laboratory-built, time-resolved flow
cytometer. The cytometry system was a modified FACSVantage™
SE (Beckton Dickinson) cell sorter. A custom-built data acquisition
system permitted real-time fluorescence lifetime analysis based on a
frequency-domain approach.
Results: After several populations were cultured and measured
with the time-resolved cytometery, the average lifetime of the
green fluorescent proteins expressed in the cells was found to
be 11.2 ns for the green fluorescent protein and 10.1 ns for the
green fluorescent protein when fused to the “dark” citrine-colored
fluorescent protein.
Conclusions: Future work is necessary to validate the fluorescence
lifetime values. If accurate lifetime values can be obtained from
within small yeast cells, then further work can be accomplished
where combinations of Forster Resonance Energy Transfer (FRET)
pairs are expressed in the yeast to study cell signaling and proteinprotein interactions.
215/B94
Polarizing Light-Scattering Profile – Advanced
Characterization of Non-Spherical Particles with the
Scanning Flow Cytometry
Dmitry Strokotov 1,2 , Irina Polshchitcina 1 , Alexander
Moskalensky1,2, Vyacheslav Nekrasov1,2, Andrei Chernyshev1,2,
Valeri Maltsev1,2
1
Laboratory of Cytometry and Biokinetics, Institute of Chemical
Kinetics and Combustion, SB RAS, Novosibirsk, Russia,
2
Novosibirsk State University, Novosibirsk, Russia
Background: Polarization measurements have been widely used
for analysis of particles. Usually only the intensity of light scattered
by a particle or an ensemble of particles is detected. An analysis of
individual microscopic particles assumes a solution of identification
and characterization problems. These problems can be solved from
measurement of light-scattering intensity just for particles described
by a simple optical model. To solve identification and especially
characterization problems for particles described by a complex
optical model it is necessary to measure additional information.
The polarizing measurement of scattering supports the solution with
extra independent information required. In particular polarization
of scattered light is sensitive to deviations of a particle shape from
spherical symmetry.
Methods: A scanning flow cytometer was improved to provide
large amount of data necessary for solution of such characterization
problems. The current device measures angle-resolved intensity of
light scattered by individual particles (light-scattering profiles, LSPs)
in regular and polarized states. It was fabricated by CytoNova Ltd.,
Novosibirsk, Russian Federation. (http://cyto.kinetics.nsc.ru).
Results: First, we measured regular LSPs of individual spherical
beads to verify proper alignment of the laser beam and the flow.
The solution of the inverse light-scattering problem was applied
for these LSPs to retrieve bead sizes and refractive indices. The
bead sizes were determined with uncertainty of about 10 nm –
an exeptionally high precision for optical methods. Second, we
developed a method to characterize polymer bead dimers, as an
example of non-spherical particles, based on regular and polarized
LSPs. Characteristics of a dimer, such as sizes and refractive indices
of constituent monomers, were successfully retrieved from the
solution of the inverse light-scattering problem. Orientation of each
ISAC 2013 Program and Abstracts
Utilizing Plasmon Surface Resonance for Flow
Cytometry
217/B96
Acoustically Enhanced Flow Cytometry for Remote
Plankton Monitoring
With the release and use of the Becton Dickenson FACS Diva
Software, the use of Area as the default parameter came into play.
As such, the use of area as a calculated parameter, methods needed
to be employed to ensure doublet discrimination and proper display
on standard FSC/SSC. Improper setting of forward area scaling can
alter the display of other two parameter histograms. This combined
with improper area gating strategy can lead to doublet inclusion
which in sorting rare events can compromise sort purity. In extreme
cases where area scaling with the individual lasers is ignored,
differences can exist between Area and Height where compensation
will likely not be optimal, particularly if one parameter – usually
height is saturated. In addition, area scaling can impact population
grouping. As FSC and individual laser area scaling is a function
of event size, the most common error is to accept the setting
determined by CS&T, which are 3.2 micron particles and proceed
with the sample(s) without regard to the sample’s actual size.
Events smaller or more likely larger than the CS&T beads, will
Speaker/Author
Index
ISAC 2013 Program and Abstracts
David Haviland1, Amy Hazen2
1
Methodist Hospital Research Inst, Houston, TX, United
States, 2Director, Flow Core, Univ. of Texas, HSC, Houston,
TX, United States
Poster Session
Abstracts
Background: In the ocean, the structure of the microbial community
determines the population and health of the higher trophic levels.
An understanding of the factors that regulate community structure
requires detailed and sustained observations of the plankton. A
submersible flow cytometer has been deployed to characterize
The Importance of Area Scaling with FACS DIVA
Software
Oral Session
Abstracts
Daniel M. Kalb1, Robert J. Olson2, Heidi M. Sosik2, Menake
E. Piyeasena3, Steven W. Graves4
1
Chemical Engineering, University of New Mexico,
Albuquerque, NM, United States, 2Woods Hole Oceanographic
Institution, Woods Hole, MA, United States, 3University of
New Mexico, Albuquerque, NM, United States, 4Center for
Biomedical Engineering, Department of Chemical and Nuclear
Engineering, University of New Mexico, Albuquerque, NM,
United States
218/B97
Commercial
Tutorials &
Exhibits
Using this we have answered the question to which extent the
observed effects can be utilized to create more detection channels
with less or no compensation problems.
Poster
Session
We have investigated the feasibility to make use of the effect of
surface plasmon resonance: Nano-Gold particles of a certain size
and shape are selectively absorbing light of certain wavelength. We
have mesured to which degree Nano-Gold particles (functionalized
with monoclonal antibodies) bound to PBMC subsets are changing
their absorbance and light scattering properties at different
wavelength. Based on these findings a method has been developed
to detect these changes measuring differential absorption and
scatter signals at different wavelength.
Conclusion: We have demonstrated the ability of acoustic focusing
to increase the volumetric delivery rate of the submersible
flow cytometer. Our environmental focusing and optimized
preconcentration systems will increase the sample throughput of
the system to 10 mL/min and enable the system to increase its
sampling rate 40 fold over its existing limit. This will enable the
system to sample much large portions of the microbial community
in the ocean and improve the information content for critical
environmental studies.
Wednesday,
22 May
To overcome this, new technologies (e.g. CyTOF) have been
developed to provide more detection parameters. However, so
far none of the new technologies has been developed to a level
that matches flow cytometry with regard to speed, sensitivity and
affordability.
Tuesday,
21 May
In the daily routine, currently up to 10 parameters are
simultaneously investigated. Even though more have been
successfully demonstrated, the difficulties arising from complex
compensation settings and issues with sensitivity have prevented
the simultaneous routine usage of more detection parameters.
Results: A proof of concept system has demonstrated sample
throughout of 10 µm beads at 10ml/min volumetric delivery rates
while maintaining excellent two dimensional focusing that restricts
the beads to a flow core (~15 µm across). We have examined
the focusing efficiency in each dimension as a function of input
power and flow rate for both beads and plankton samples. We
have demonstrated improved focusing for plankton samples and
can deliver such samples at several ml/min to a flow cell for
image flow cell. We will present our work to improve the focusing
performance of the system in relation to PZT orientation, cylinder
material, length and power. We will also present our most recent
efforts in environmental feedback control, focusing optimization,
automation, and integration into the submersible flow cytometer.
Monday,
20 May
In the past two decades, flow cytometry has become the swiss
army knife for many researchers in hematology or immunology.
Due to the increasing complexity of questions to be answered by
researchers, the demand for more available detection parameters is
a constantly increasing.
Sunday,
19 May
Martin Buescher, Anne Esslinger, Juergen Krieg, Christian
Peth, Markus Nagel
Biophysics, Miltenyi Biotec, Bergisch Gladbach, Germany
Methods: We have fabricated a cylindrical acoustic focusing
system to pre-concentrate the cells into the existing cytometer’s
flow chamber. We have developed a model to account for acoustic
frequency changes due to temperature and salinity variations found
in coastal waters. To minimize power requirements for our system
we are using simple environmental sensors and our model to drive
our focusing system at optimal frequencies. We have tested this
system by focusing beads and plankton at varying temperatures and
salinities in laboratory experiments. The two dimensional focusing
performance of our acoustic focusing capillary has been quantified
by video analysis of the flow cell.
Saturday,
18 May
216/B95
the phytoplankton and microzooplanktonic organisms of coastal
water. To effectively monitor the dilute phytoplankton population
it is desirable to maximize the volumetric sample throughput of
the existing system, currently limited to 0.25 mL/min. Simply
increasing the sample delivery rate widens the core and optical
focus is lost. Raising the sheath rate to tighten the focus increases
the linear velocity and blurs the images. Here we use acoustic
focusing to pre-concentrate the cells prior to entering the imaging
flow cell, allowing an increased volumetric sample rate without
increasing the linear velocity.
Special
Lectures
Conclusions: Measurement of the polarized LSP opens the way
for optical characterization of particles with complex shape and
internal structure. For example, this approach looks promising for
detailed characterization of red blood cells (RBCs). Implementation
of this approach into a hematological analyzer should lead to
substantial decrease of systematic errors in RBC indices. The
polarized LSP can also be used for assessing the homogeneity of
cell nucleus and for analysis of blood platelets microaggregates.
Congress
Overview
dimer in a flow relatively to the direction of the incident laser beam
were also determined. Both ordinary and polarized measured LSPs
are in good agreement with T-matrix simulations, which leads to
average uncertaintly of 50 nm for determined bead sizes in a dimer.
173
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
make the area scaling settings less than optimal. Proper FSC and
laser area scaling must be determined empirically for each sample.
Examples of the effects of sample size on area scaling will be
presented in addition to gating and templates for determining area
scaling.
219/B98
Nano-View Version II : An Improved Novel Approach
to Microparticle Cell Sorting
Vasilis Toxavidis1, John Tigges2, Kaitlin Groglio3
1
BIDMC, BOSTON, MA, United States, 2Beth Israel Deaconess
Medical Center, 3Flow Cytometry, BIDMC, BOSTON, MA,
United States
There is great interest in both medical and scientific communities
in submicron cell-derived particles, termed microparticles
or microvesicles. Although competing techniques have been
developed, flow cytometry remains the dominant approach. The
hurdle in analysis has always been the ability to accurately measure
the size characteristics of small particles, especially when only
considering scatter properties. Due to advances in microscopy and
the ability to identify the existence of <1um cellular particles, flow
cytometry instrumentation has been developed to have the ability to
identify populations from 400nm to 1um. However, the accuracy
of these measurements and the validity of the results are frequently
questioned. Therefore, we propose a hardware upgrade that will
not only improve accuracy, but will allow for validation of the
procedure by recovery of the microparticles.
In this study, we present the results of our independent testing of
the new Propel Labs’ Nano-View forward scatter detector (FSC)
integrated onto a Beckman Coulter MoFlo XDP* cell sorter. The
Nano-View design has improved the optical and electrical systems
over the standard FSC diode or PMT for the purpose of extending
the detection range down to < 200nm particles. The new optical
system design utilizes a custom aspheric imaging lens that has
been optimized to collect the scattered light from the core stream
and image it onto a pinhole. The collection angles in the FSC
direction extend up to 18 degrees, which is double the maximum
collection angle of a standard MoFlo FSC detector. The pinhole
serves to align the system and remove the stray laser light that has
not been generated by the particle of interest and greatly reduces
the background light that is received at the detector. The Nano-View
design has further improved the detection system by replacing the
photodiode with a much higher sensitivity PMT detector in the FSC
path.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
220/B99
8 Way Fluorescence-Activated Cell Sorting on the BD
Influx™
Suat Dervish, Frank Kao, Steven Allen, Adrian Smith
Advanced Cytometry Facility, Centenary Institute, Sydney,
Australia
Background: As the complexity and resolution of cellular
populations increases with complementary advancements in
reagent, antibody & instrument selection a need for increasing
simultaneous fluorescence-activated cell sorting (FACS) capability
is required. We describe the utilisation of the flexible and modular
architecture of the BD Influx™, in conjunction with 3D printing, to
permit the simultaneous sorting of 8 populations.
Methods: A 10 laser BD Influx™ (355nm, 405nm, 445nm, 488nm,
514nm, 532nm, 561nm, 594nm, 635nm, 786nm) with a 6 way
sort module installed (5kV deflection plates, increased vertical
displacement collection chamber), small particle option and
polarization sensitive detectors located at the Advanced Cytometry
Facility at the Centenary Institute, Sydney, Australia was used. The
sort devices .xml file called on by BD FACS™ Sortware was edited
to allow a custom 8 way sorting program to be selected. 3D printing
174
with acrylonitrile butadiene styrene (ABS) was used for the rapid
prototyping and optimisation of an 8-tube-capable sorting block. 8
way sorting parameters were optimised for both 70um and 100um
nozzles.
Results & conclusions: Instrument setup was critical in obtaining
high purity sorts. Aspects that required optimisation included
collection tube height, angle and spread, nozzle diameter
considerations, the influence of static on collection tubes, sidestream alignment in respect to deflection plates and charge
deflection. Careful attention to these parameters is also critical for
successful 6-way sorting.
Our results show that 8 populations can be successfully sorted
simultaneously and with high purity on the BD Influx™,
highlighting the power of combining a flexible sorter platform with
the utility of rapid prototyping tools such as 3D printing.
221/B100
A High Throughput Flow Cytometric Assay for Rapid
Quantitation and Detection of Mouse IgG
Robert Danielzadeh1, Melinda Krusemeier2
1
Charisela Technologies, Inc., Menlo Park, CA, United States,
2
UCSF, Monoclonal Antibody Core, San Francisco, CA, United
States
Background: Mouse IgG detection and quantitation are considered
important screening techniques used in pharmaceutical and
biotechnology industries. Charisela Technologies, Inc. has
developed a mouse IgG reagent kit for use in flow cytometry.
The quantitative determination of mouse IgG in hybridoma
supernatants, ascites, mouse sera, etc.; where the intensity of the
signal is independent of the IgG subclass and light-chain type.
Advances in hybridoma techniques increases the need for a Mouse
IgG assay for today’s research market needs, and
Method: Charisela has developed one of the fastest bead based
mouse IgG assay which allows for a rapid, reliable, accurate and
cost effective reagent for quantitative screening of secreted or
manufactured mouse IgG in solution (i.e. serum or cell culture
media) without only a one (5min) wash step and no dilution
of samples requirement and total preparation time of about 45
minutes.
Results: Competitive Bead based assay allows for a low
background, high intensity assay which allows for a wide dynamic
range of 1ug/mL -1mg/mL mouse IgG quantitation. This poster will
further depict plots which show no debris or doublets associated
with the assay along with the calibration curve results and samples.
Quantitation of IgG is a rapid 45 minutes and using the Charisela
Analysis software allows for a rapid analysis of FCS files. Charisela’s
assays are instrument agnostic and work on any flow cytometer.
Conclusion: The Quantifier Mouse IgG assay is geared specifically
for institutions that require quality control or screening of mouse
IgG, secreted via harvesting of hybridoma cells for quantitating
mouse IgG. Charisela’s Quantifier Mouse IgG Assay would clearly
be a logical alternative to current assay reagents in the market today
by providing multiple advantages over current methodologies;
such as high fluorescence, low background, non-fluorescent or
magnetic particles and rapid accurate results. This poster will depict
the principle, methods and performance of Charisela’s Quantifier
Mouse IgG Assay and advantages thereof.
ISAC 2013 Program and Abstracts
Nahla M. El Sharkawy1, Wafaa M. Radwan2, Enas S. Eissa2,
Samia H. Kandil2, Azza M. Kamel1
1
National Cancer Institute, Cairo University, Cairo, Egypt,
2
Faculty of Medicine, Menofeya University, Menofeya, Egypt
ISAC 2013 Program and Abstracts
Fluorescent activated cell sorting is a common method to isolate/
purify particles, cells and other populations of interest. For such
purposes, many different cell sorters are commercially available.
Therefore, questions arise concerning which cell sorter is best for
each application.
In general, the setup of a cell sort or a flow cytometry experiment
should be optimized for the experimental conditions. Many articles
are published regarding the setup of multicolor experiments like
antibody titer/concentration, buffer composition and appropriate
compensation. In most cases a successful staining is influenced by
the titration of the antibody and an optimal flow cytometer setup
with the appropriate controls.
However, very critical and often unrecognized parameters are
not discussed in detail. Some examples are temperature control,
protection from light, reagent stability, duration of the experiment,
cell reliability, and fluidic parameters (e.g. pressure, speed, sample
Speaker/Author
Index
Background: Image-based flow cytometers utilize high-quality CCD
camera systems in place of traditional PMTs. As detectors, CCD
cameras excel in the far-red range, where their quantum efficiency
(QE) is at least 10-fold greater than traditional PMTs. Increased farred QE may allow dyes to be titered at higher dilution factors (DF),
while still maintaining sufficient signal to distinguish positive from
negative cells, thereby greatly reducing the cost associated with
each stain.
Hanna Ulrich, Immanuel Andrae, Lynette Henkel, Dirk Busch,
Matthias Schiemann
Institute for Medical Microbiology, Immunology and Hygiene,
TU München, Munich, Germany
Poster Session
Abstracts
Brian McFarlin1, Karen Clise-Dwyer2, Kathryn E Ruisaard3,
Kimberlyn J Acklin3, Adam Venable1
1
Applied Physiology Laboratory, University of North Texas,
Denton, TX, United States, 2Univ of Texas, 3South Campus
Flow Cytometry & Cell Sorting Core Facility, University of
Texas, Houston, TX, United States
Sorted or Agonized?
Oral Session
Abstracts
Enhanced Far-Red Fluorescence Sensitivity in CCD
Camera-Based Cytometers Increases Threshold
Antibody Titration
225/B104
Commercial
Tutorials &
Exhibits
223/B102
224/B103
Withdrawn.
Poster
Session
Keywords: BORIS; Breast cancer; Neutrophils
Conclusions: When using abundantly expressed CD markers, a
camera-based image cytometer is capable of greater sensitivity
than lower priced benchtop instruments and has sensitivity
comparable to high-end instruments, at least in the far-red region
of the spectrum. Increased dilution of antibodies reduces the
cost of doing science and may eventually allow a camera-based
image cytometer to pay for itself. More research is needed to test
fluorescence sensitivity differences in cell-surface receptors with
lower cell-surface expression, and in the green to far-red channels
excited at 488.
Wednesday,
22 May
Conclusion: Increased BORIS expression in peripheral blood
neutrophils is associated with both benign and malignant breast
lesions; apparently, increased proliferation of breast tissue is the
determining factor. This might exclude BORIS as a tumor marker but
it will not jeopardize its value as a potential therapeutic target.
Tuesday,
21 May
Results: High level of BORIS was detected in all the malignant and
benign cases with mean positive percent of 64.4±16.6 & 67±12.3
and MFIR of 7.2±4.1 & 7±3.5 respectively. However in the control
group, the mean positive percent was 13.4±11.5 and MFIR 1.8±0.7.
Comparison between the three groups (malignant, benign and
control) showed that there is statistically significant difference in
the BORIS +ve% and MFIR between the malignant and control
group and between the benign and control group (p: 0.0001) but
no significant difference exists between the malignant and benign
group (p:0.934). There was no correlation of BORIS expression with
ER/PR status HER-2/neu expression or tumor stage.
Results: Consistent with our hypothesis the camera-based image
cytometer was able to separate positive from negative populations
at much greater antibody dilutions than any of the PMT-based
flow cytometers. Of the PMT-based instruments, the BD Aria IIu
demonstrated that greatest ability to resolve between positive and
negative at the highest dilution, while the 8HT outperformed the
Fortessa at a DF of up to 75.
Monday,
20 May
Patients and methods: The study was conducted on 85 female;
52 with breast cancer, 13 with benign breast lesions and 20 agematched apparently healthy females as normal controls. BORIS
expression was detected by Flow Cytometry in the peripheral blood
neutrophils of the patients and controls.
Sunday,
19 May
Aim: To confirm the previous results, verify if BORIS would
discriminate between benign and malignant breast lesions and to
verify if BORIS expression would correlate with disease stage, ER/
PR status or HER-2/neu expression.
Saturday,
18 May
Background: Early detection of breast cancer is the keyword for
improved survival or even cure. The identification of markers to
distinguish between normal cells, tumorigenic cells and different
stages of cancer is of critical importance for cancer diagnosis
and prognosis. BORIS is a paralog of the multifunctional CCCTCbinding factor (CTCF) gene. BORIS expression pattern is restricted
to testis and normally BORIS is not present in females. It has
been found to be aberrantly activated in various human cancers,
including female cancer such as uterine (endometrial) and breast
tumors.
Methods: The purpose of the present study was to compare the
detection capability of a camera-based image cytometer (MilliporeAmnis FlowSight) to other traditional PMT-based flow cytometers
(Millipore 8HT, BD Fortessa, BD Aria IIu, BD Influx). In addition
to detection technology, these instruments also differ in their red
laser power output, their flow cell type, psi/flow rate, optical layout,
and signal processing method. To complete these comparisons,
measurements were made in two laboratories and the PMTs of all
instruments were adjusted to the same peak fluorescence channel
on detectors for APC and APCeFluor780 using matched URF single
peak beads (Spherotech). Unless otherwise noted, all antibodies
and solutions were purchased from eBioscience. Whole blood (100
μL) was stained with titered doses of anti-CD66b-APC (DF=20,
50, 75, and 100) or anti-CD45-APCeFlour780 (DF=20, 50, 75,
and 100). An additional volume of cells was reserved to generate
“cells only” fractions as a control. After a 30-min incubation in the
dark on ice, RBCs were lysed using a commercially available fix/
lyse solution and washed cell pellets were resuspended in 150 μL
of a commercially prepared staining buffer. After blood cells were
prepped, calibrated mouse IgG antibody capture beads (Bangs
Lab) were labeled with the same dilutions of CD66b and CD45. A
minimum of 10,000 CD marker specific events was acquired for
each sample. All compensation and analysis was completed postacquisition using FCS Express.
Special
Lectures
Detection of Brother of the Regulator of Imprinted
Sites Expression (BORIS) in Peripheral Blood
Neutrophils by Flowcytometry in Benign and
Malignant Breast Lesions
Congress
Overview
222/B101
175
Congress
Overview
line performance). These parameters often have a dramatic impact
on the success of the sort experiment.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
Special
Lectures
For this purpose, we used a bench to bench comparison with
three commercially available cell sorters, and analyzed the purity,
recovery, yield and viability of different sorted cell populations.
226/B105
Concentration Measurements Using Acoustic
Cytometry
Jolene Bradford, Bradley Dubbels, April Anderson, Yu-Zhong
Zhang
R&D Molecular Probes Labeling and Detection Technologies,
Life Technologies, Eugene, OR, United States
Flow Cytometry offers accurate relative number of cells and
particles from a variety of sample types. A technical limitation
of cytometers that utilize conventional hydrodynamic focusing
precludes an accurate cell concentration due to lack of precise
measurement of sample volume analyzed. This limitation has been
mitigated by the addition of precisely measured counting beads
combined with a calculation to determine concentration. However,
counting standards are limited by their cost, technical complexity,
requirement for additional calculations or software, and potential
errors due to variations in the procedure such as pipetting and
gating. Using a volumetric sample and sheath delivery system, the
Attune® Acoustic Focusing Cytometer provides highly accurate
cell concentration data without the addition of counting beads,
resulting in a streamlined workflow. In no-wash assays, the absolute
cell number can also be calculated. Each sample acquired gives the
concentration in µl, automatically calculated for any population in
a given experiment. The Attune® Acoustic Cytometer can measure
cell concentrations in a variety of samples, and three applications
are highlighted: CD34+ cell concentration measurements in human
whole blood, absolute human CD4+ cell count using a no-wash
whole blood lysis procedure, and determining the concentration
of fluorescent marine microbes. Comparison of the acoustic
cytometry method to that using a counting standard will also be
presented. The syringe pump system used in the Attune® Acoustic
Focusing Cytometer makes it possible to obtain direct volumetric
measurements of diverse cell concentrations without the use of
counting standards. For Research Use Only.
227/B106
Low Cost Precision Pulsed LED for Flow Cytometer
Calibration in Statistical Photoelectron Units
James Wood
Comprehensive Cancer Center, Wake Forest University,
Winston-Salem, NC, United States
Pulsed LEDs are an attractive light source for the characterization
of flow cytometers, and for the calibration of the relative intensity
scale in real physical units. By measuring the means and CVs of
LED light pulse intensities over a wide range of pulse amplitudes,
detection efficiency, the scale linearity, and the statistical
photoelectron units generated per pulse can be measured. Since
the statistical photoelectron units and detection efficiency are
calculated from the measured CV, it is important to minimize other
sources of variation like the intrinsic CV. In contrast to calibrated
fluorescent microspheres, the pulsed LEDs produce more precise
pulses of light with very low intrinsic CVs (0.2% vs. 2% for
microspheres) thereby extending their use into the upper end of the
scale. Thus a pulsed LED can be used over a greater dynamic range
than calibrated fluorescent microspheres.
Typically, laboratory function generators are used to drive the
pulsed external LEDs, and ND filters are used to vary the pulse
amplitude. The expense of quality function generators limits the
widespread use of pulsed LEDs to characterize and to calibrate flow
176
cytometers. In order for more laboratories to have access to the
benefits of pulsed LEDs to characterize and to calibrate their flow
cytometers, an LED pulse driver has been designed that is low cost,
precise and portable. Driving a white LED with 2 microsecond
pulses at an approximate 1kHz repetition rate, the light pulses from
the pulsed LED can be directed at the flow cytometer flow cell
across from the fluorescence pick-up lens. Unattenuated pulses are
very bright and produce a peak with a CV of less than 0.2%. Using
an ND filter to attenuate the pulses increases the CVs of the peaks
by the square root of 1/attenuation as predicted from the reduction
of photons reaching the photodetector.
To reduce the cost further, the possibility to eliminate the need for
ND filters is being explored. The LED light output is approximately
linear with the current driving the LED. Precise control of the
current flowing through the LED can control the light output of the
LED in a way similar to an ND filter. Since a precision voltage to
current modulator is used in the low cost pulse driver, changing the
peak drive voltage to the modulator can change the peak current
that passes through the LED and control the LED light output. To
maximize the dynamic range of the pulsed LED light output, the
linearity of the light output vs. current of different LEDs is being
measured and circuitry designed to correct for any observed nonlinearity.
In conclusion, an LED pulser is being designed that will be
low cost, precise, and portable with the capability to rapidly
characterize a flow cytometer and calibrate a histogram scale into
statistical photoelectron units.
228/B107
Improved Light Scatter Detection for Small Particle
Analysis and Counting
David Houck, Bob Cao, Dale Lindseth
BD Biosciences, San Jose, CA, United States
Background: Current light scatter detection on the BD FACSCanto™
and similar products works well for discriminating lymphocytes,
red cells, platelets and particles greater than 0.5 microns in size.
Demands for smaller particle analysis by microbiologists have been
growing and special products to analyze particles at the 0.2 micron
level have been produced. Increasing interest in detection and
counting of cellular microparticles(MP’s) has pushed the need for
small particle analysis and standardization as well. We evaluated
some simple changes to the current system to see how the forward
and side angle light scatter detectors could be improved for small
particle analysis.
Methods: A breadboard cytometer was put togethersimilar to
the BD FACSCanto™ analyzer to testmodifications made to
the lightdetection system including the optics and electronics.
Evaluation of changes to the system was based on the ability to
resolve standard particles ranging in sizes from 0.1 to 0.5 microns.
Results:
SSC: Clear separation of 0.1 micron particles from background
noise.
FSC: Separation of 0.3 micron particles from 0.2 micron particles
with a photo diode detector.
Conclusions: Some modifications were made to the light scatter
detector system that could be used on the BD FACSVerse™, BD
FACSAria™ and BD FACSCanto™ flow cytometers. Improvements
will aid small particle analysis in microbiology and the analysis and
counting of cellular microparticles.
For Research Use Only. Not for use in diagnostic or therapeutic
procedures.
ISAC 2013 Program and Abstracts
Optimal Baseline PMT Voltages for Multicolor
Staining: Optimizing CST Settings for Cellular Samples
A 75-year-old female developed pneumonia after a party. She was
recovered after treatment. Since then she felt weak and had marked
weight loss (1/3 of the body weight) in seven months. Physical
examination showed generalized lymphadenopathy. The lymph
nodes were moveable, nontender and ranged from 1 to 2 cm. There
was no significant hepatosplenomegaly.
The excisional biopsies of right axillary and right inguinal lymph
nodes were performed. H&E sections showed altered nodal
architecture with paracortical expansion by intermediate sized
lymphoid cells, focal vascular hyperplasia, medullary plasmacytosis
and no well formed germinal centers. Immunohistochemistry
revealed appropriate distribution of CD3+ T cells and CD20+ B
cells. T cells were composed of mixed CD4+ and CD8+ cells.
CD21 highlighted scattered extrafollicular dendritic cell meshwork.
CD10+ cells were mostly in follicular areas.
Sunday,
19 May
Flow cytometry studies detected three populations of CD3+ T cells,
two of which were CD2+/CD4+ cells with high and intermediate
CD3 expression. They are positive for CD2 and CD5 with partial
loss of CD7. Interestingly, all T cells were negative for CD10. There
was an increased CD4:CD8 ratio. No evidence of monotypic B
cells was seen.
Monday,
20 May
PCR studies detected clonal T-cell receptor (TCR) gene
rearrangement and no clonal immunoglobulin heavy chain gene
rearrangement. The diagnosis of AITL was made.
Tuesday,
21 May
The patient was treated but deteriorated and died three months later
after the diagnosis.
In summary, flow cytometry study is a powerful and sensitive tool
to detect abnormal T cell population. It helps to make a diagnosis
of AITL with combination of clinical information and PCR for TCR
gene rearrangement.
Wednesday,
22 May
Methods: To determine the optimum voltage settings for a multiparameter staining panel, lyophilized human PBMCs were stained
separately for twelve different surface markers representing an
immunophenotyping panel for human T cells from endometrial
tissue samples. These single stained samples were acquired at a
range of PMT voltages from 300mV to 850mV to determine the
voltage for each marker and stain that offered the best resolution
of positive populations over background in each detector. This
experiment was carried out using a four laser Fortessa and a three
laser LSRII, and extended to include stained antibody capture beads.
We report a case of AITL detected by flow cytometry study and
confirmed by clonal T-cell receptor gene rearrangement via PCR.
Saturday,
18 May
Background: Proper cytometer calibration is essential to ensure data
quality and reproducibility between experiments. Futher, choosing
PMT voltages that optimize the resolution of each marker over
background reduces ambiguity in the interpretation of multicolor
stained samples. For Becton Dickinson multi-laser cytometers,
bead-based quality control is simplified using the Cytometer Setup
and Tracking module in the DIVA cytometer control software,
allowing for automated laser delay timing and baseline detector
voltage setup. These baseline voltage determinations for each
fluorescence channel are determined by incrementally increasing
the applied voltage to each PMT while determining the fluorescence
precision and resolution of a set of fluorescent microspheres
representing three different labeling intensities. Optimum operating
voltages are suggested based on these calculations and a cutoff of
ten times the standard deviation of electronic noise, as determined
by the CST baseline assessment routine for each cytometer. While
these suggested voltages represent a starting point for PMT signal
amplification and data collection, they may not result in the best
possible resolution of fluorescence measurements made on cells
from various tissue sources. In particular, for fluorophores with red
emission characteristics, a higher PMT voltage often results in better
resolution of stained populations.
cytometry study of AITL usually shows a mixture of CD4+ and
CD8+ T-cells with an increase of CD4:CD8 ratio.
Special
Lectures
Bridget McLaughlin
Medical Pathology and Laboratory Medicine, University of
California, Davis, CA, United States
Congress
Overview
229/B108
Poster
Session
Results and Conclusions: In most cases, the voltages recommended
by the baseline instrument calibration using BD CST beads returned
an appropriate starting voltage, however, for long red emitting
fluorophores, a higher operating voltage was determined to give the
best results.
Commercial
Tutorials &
Exhibits
Hematological Disorders
(B109 – B114)
230/B109
Important Role of Flow Cytometry Study in Diagnosis
of Angioimmunoblastic T-Cell Lymphoma
ISAC 2013 Program and Abstracts
Angioimmunoblastic T-cell Lymphoma, Flow Cytometry (dot plot)
There are three subsets of CD3+ T cells. Two of them are CD4+ with
high and intermediate CD3 expression. They are positive for CD2
and CD5 with partial loss of CD7. All T cells are negative for CD10.
Speaker/Author
Index
The neoplastic T cells are postulated to comemostly from TFH
cellsthat aretypically positive for CD4 and CD10. The flow
Poster Session
Abstracts
Angioimmunoblastic T-cell lymphoma (AITL) is a mature T-cell
lymphoma comprising 15-20% of peripheral T-cell lymphomas.
The AITL usually involves either middle-aged or elderly with no
gender preference. It is a systemic disease with an aggressive course
and median survival of less than three years. Morphologically,
AITL is characterized by a polymorphous lymph node infiltrate by
neoplastic T cells in a background of mixed mature T cells and B
cells with a marked increase in follicular dendritic cells and high
endothelial venules. The early diagnosis of AITL is important, but it
is difficult due to the polymorphic infiltrate in lymph node.
Oral Session
Abstracts
Xiaohong Zhang
Geisinger Health System, Wilkes-Barre, PA, United States
177
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
ligation-dependant probe amplification (MLPA) using 42 probes
for the following regions: 1p32-31, 1p21, 1q21.3, 1q23.3, 5q31.3,
12p13.31, 13q14, 16q12, 16q23 and 17p13.
232/B111
Results: A total of 245 alterations were detected in 79 cases (98%).
Investigating the same aberrations, the two methods showed a
congruency of higher than 90%. Low proportion of cells harboring
the abnormality, focal copy number alterations (CNAs) and
unmatched probes were responsible for the discrepancies. MLPA
revealed 95 CNAs not detected by iFISH providing additional
information in 53 cases (65%). These abnormalities not detected
by commercial FISH probes were successfully validated using
specifically designed BAC clones. Scrutinizing the CNAs on
chromosome 1 using more than 20 probes, significant heterogeneity
in size and location, variable intra-chromosomal and intra-clonal
rates of loss or gain were found.
Novel Approach Using Imaging Cytometry to Monitor
Red Blood Cell Surface Area Loss and Splenic Retention
Marie Nguyen 1, Innocent Safeukui 2, Pierre A. Buffet 3,
Guillaume Deplaine2, Sylvie Perrot2, Valentine Brousse4,
Alioune Ndour3, Odile Mercereau-Puijalon2, Peter H. David2,
Genevieve Milon5, Narla Mohandas6
1
Plate-forme de Cytometrie, Institut Pasteur, Paris, France,
2
Immunologie Moleculaire des Parasites, CNRS, Institut Pasteur,
Paris, France, 3Department of Parasitology, INSERM/UPMC,
AP-HP Pitie-Salpetriere Hospital, Paris, France, 4Department
of Pediatrics, AP-HP, Necker Hospital, Paris, France,
5
Immunophysiologie et Parasitisme, Institut Pasteur, Paris,
France, 6New York Blood Center, New York, NY, United States
Splenic sequestration of RBCs with reduced surface area and
cellular deformability has long been recognized as contributing
to pathogenesis of several RBC disorders, including hereditary
spherocytosis. However, the quantitative relationship between the
extent of surface area loss and splenic entrapment remains to be
defined. To address this issue, in the present study, we perfused
ex vivo normal human spleens with RBCs displaying various
degrees of surface area loss and monitored the kinetics of their
splenic retention using imaging flow cytometry. Treatment with
increasing concentrations of lysophosphatidylcholine resulted
in a dose-dependent reduction of RBC surface area at constant
volume, increased osmotic fragility, and decreased deformability.
The degree of splenic retention of treated RBCs increased with
increasing surface area loss. RBCs with a > 18% average surface
area loss (> 27% reduced surface area-to-volume ratio) were rapidly
and completely entrapped in the spleen. Surface-deficient RBCs
appeared to undergo volume loss after repeated passages through
the spleen and escape from splenic retention. The results of the
present study for the first time define the critical extent of surface
area loss leading to splenic entrapment and identify an adaptive
volume regulation mechanism that allows spherocytic RBCs to
prolong their life span in circulation. These results have significant
implications for understanding the clinical heterogeneity of RBC
membrane disorders.
233/B112
Donat Alpar1, Bela Kajtar1, Danielle de Jong2, Suvi Savola3,
Pal Jakso1, Laszlo Kereskai1, Laszlo Pajor1, Karoly Szuhai2
1
University of Pecs, Pecs, Hungary, 2Leiden University, Leiden,
Netherlands, 3MRC-Holland, Amsterdam, Netherlands
Background: Multiple myeloma (MM) is a genetically
heterogeneous disease with diverse clinical outcome. The low
proliferation of plasma cells hampers the effective karyotyping,
thus interphase fluorescence in situ hybridization (iFISH) is the
most commonly used approach to detect recurrent cytogenetic
abnormalities in this entity.
Speaker/Author
Index
Poster Session
Abstracts
Commercial
Tutorials &
Exhibits
Combined Cell and DNA Based Analysis of Genetic
Abnormalities in Multiple Myeloma
Oral Session
Abstracts
Poster
Session
231/B110
Withdrawn.
Aim: We aimed to assess the performance of a cost-effective
combination of cell and DNA based techniques to reveal molecular
cytogenetic aberrations in MM.
Methods: Diagnostic bone marrow samples from 81 patients were
analyzed for the presence of hyperdiploidy, deletion/monosomy
of chromosome 13, deletion of the TP53 gene, disruption of the
immunoglobulin heavy-chain gene, t(4;14), t(11;14), t(14;16),
t(8;14), gain of 5q and abnormalities of chromosome 1 using
low-cost iFISH array. All samples were also screened by multiple
178
Conclusion: Our study proves for the first time the reliability and
power of this combination of iFISH and MLPA to detect multiple
genetic abnormalities in multiple myeloma. Since both balanced
and unbalanced aberrations have high importance in the prognostic
classification of MM, MLPA and iFISH should be applied as
complimentary techniques in diagnostic pathology.
234/B113
Plasma Cell Heterogeneity in Normal Bone Marrow
Jeroen van Velzen1, Monique Minnema2, Andries Bloem1
Laboratory of Translational Immunology, University Medical
Centre Utrecht, utrecht, Netherlands, 2Department of
Hematology, University Medical Centre Utrecht, utrecht,
Netherlands
1
Multicolor flow cytometry (MFC) is becoming increasingly
important for both the diagnosis and minimal residual disease
(MRD) assessment of patients with plasma cells (PC) dyscrasias.
Inclusion of novel agents in standard treatment protocols has
resulted in induction of more complete remissions associated with
extended survival. Recent studies imply that absence of clonal PC
as detected with MFC with a sensitivity of ≤1:104 to 105 associates
with increases progression-free survival. It is therefore imperative
to develop a reliable MFC procedure to detect normal and clonal
PC. Classical markers for identification of PC include CD138,
CD38 in combination with cytoplasmic light chain expression 1.
A recent study by Hosen et al however 2 showed that CD138- PC
with tumorigenic properties do occur in some multiple myeloma
patients. Furthermore, hallmark myeloma PC characteristics like
presence of CD56 and absence of CD19 are also found on normal
bone marrow (BM) derived PC 3 and circulating PC include both
CD138+ and CD138- cells 4. These data warrant a search for
alternative markers for the identification of PC.
Vs38c (DAKO) recognizes a rough endoplasmic reticulum-associated
protein also known as p63, is commonly used in pathology for
immunostaining and distinguishes PC from other hematopoietic
cells due to their high secretory activity 5. Here we investigated
whether Vs38c can be used as a marker for the identification of PC
by MFC. We demonstrate that the combination of Vs38c and CD38
identifies approximately 20% more (Mean 22%SD 8.6% n=10)
PC in normal BM as compared to standard MFC using CD138 and
CD38. Further analysis of Vs38c+CD138+ and Vs38c+CD138- PC
show that both populations of PC express comparable levels of
intra-cytoplasmic light chains, CD184, CD38 and are both negative
for CD20. Vs38c+CD138- PC have a significant lower expression
of CD45, CD19, CD27, HLA-DR and BCL-2 as compared to
their CD138+ counterparts. These results illustrate 1. that Vs38c
indentifies significantly more PC than CD138, 2. that Vs38c
introduces additional heterogeneity in the BM PC compartment 3.
suggesting that Vs38c+CD138- PC, based on CD45 and HLA-Dr,
are more mature as compared to their CD138+ counterparts.
ISAC 2013 Program and Abstracts
236/B115
ISAC 2013 Program and Abstracts
Manipulating 30-50 Parameters in Real Time: High
Content Analysis of Flow Cytometry Data
J. Paul Robinson
BMS/BME, Purdue University, West Lafayette, IN, United States
Flow cytometry as a high throughput and high content screening
technology is an emerging field. While manipulation of high
parameter image data is mature, the management of flow cytometry
data has remained somewhat static for many years. However, the
capacity to collect molecular and functional data by analyzing one
Speaker/Author
Index
Analysis of multidimensional flow cytometry data is usually
performed in two steps. In primary analysis, variables such as
the proportion of events having given properties are extracted
from individual Flow Cytometry Standard (FCS) listmode files.
Secondary analysis involves statistical comparison of the tabulated
variables between groups or treatments. Whether markers are
238/B117
Poster Session
Abstracts
Albert Donnenberg1, Vera S. Donnenberg2, Daniel Normolle3
Medicine, University of Pittsburgh School of Medicine,
Pittsburgh, PA, United States, 2Univ of Pittsburgh, 3Biostatistics,
University of Pittsburgh Graduate School of Public Health,
Pittsburgh, PA, United States
1
Oral Session
Abstracts
Application of 5 Multivariate Classification
Methods to Compare Conventionally Analyzed
Multidimensional Flow Cytometry Data
Understanding the relationship between chemical structure and
biological properties is critical to systems biology, and subcellular
localization is an important property of any molecule. The ability
to accurately predict this relationship would also greatly assist in
the development of targeted markers and therapeutics. Significant
strides have been made over the past 15 years in automatically
identifying subcellular localization from fluorescent imaging data
using machine learning algorithms. Though this approach has
been shown to be faster and more accurate than the traditional
visual annotation approach, it does not address the question
of how molecular structure relates to its localization. Here we
construct a model to link automated methods of image analysis
and subcellular localization features to chemical structure using
images of a combinatorial library of styryl probes previously
collected by Sheddon et al. (J. Chem. Inf. Comput. Sci. 2003). For
this study we used the same combinatorial library of 1344 styryl
probes consisting of eight pyridinium/quinolinium groups and 168
aldehyde groups acquired using high content screening methods
and their various chemical features. We then build a model to
predict subcellular localization features from the various chemical
features.
Commercial
Tutorials &
Exhibits
High Content Analysis
(B115 – B119)
Devin Sullivan1, Evan Cesanek2, Gus R. Rosania3, Robert F.
Murphy4
1
Carnegie Mellon Univ, Pittsburgh, PA, United States, 2Vassar
College, Poughkeepsie, NY, United States, 3Univ of Michigan,
4
Carnegie Mellon Univ
Poster
Session
Conclusions: A detector with more FSC channels improves the
platelet – RBC separation and the platelet count can be determined
with confidence using scatter information only.
Modeling Subcellular Distribution from Chemical
Structure
Wednesday,
22 May
Results: We compared the platelet count obtained with our 5
channel forward detector to a CD61 platelet count and found very
good agreement. Even in samples with low platelet count, small
RBC size or large number of RBC fragment, the platelet count with
the 5 channel detector is very close to the CD61 count.
237/B116
Tuesday,
21 May
Method: In order to get better separation between RBCs and
platelets we added more forward scattering detectors. Our
prototype detector had a total of 5 detectors, covering axial light
loss (ALL) and 4 different forward scattering angles. In particular
our FSC detector covering the smallest angles plotted vs the FSC
detector covering the largest FSC angles showed good results for
separating platelets from RBCs or RBC fragments.
Monday,
20 May
Background: For a Complete Blood Count in Hematology we want
to distinguish platelets form RBCs and RBC fragments. In normal
samples, this task is not too difficult: The platelets separate fairly
well form the RBCs in a FSC vs SSC plot. However in a low platelet
sample with small RBCs or RBC fragments the separation between
the two populations sometimes becomes difficult.
Sunday,
19 May
Martin Krockenberger
Abbott Hematology, Santa Clara, CA, United States
Saturday,
18 May
Separating Platelets and RBCs by FSC Amplitudes
identified by unsupervised classification or by traditional “manual”
methods employing Boolean gates and analytical regions, the
number of candidate variables detected may exceed the number of
specimens to be compared (p > n) and many of the variables may
be highly inter-correlated. Thus simple bivariate comparison, one
variable at a time, lacks statistical power to detect between-group
differences and is blind to correlations that may exist between
variables. Here we begin with a data set comparing 35 non-small
cell lung cancer tumors and adjacent lung tissue, generating 86
variables by conventional region/gate analysis of individual data
files. We compare the application of five multivariate classification
methods to discern subtle differences between the stem/progenitor
compartments in the two groups. 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 multiparameter cytometry
problems.
Special
Lectures
235/B114
Congress
Overview
Reference List:
1. Rawstron, A.C. et al. Report of the European Myeloma Network
on multiparametric flow cytometry in multiple myeloma and
related disorders. Haematologica 93, 431-438 (2008).
2. Hosen, N. et al. CD138-negative clonogenic cells are plasma
cells but not B cells in some multiple myeloma patients.
Leukemia 26, 2135-2141 (2012).
3. Peceliunas, V., Janiulioniene, A., Matuzeviciene, R., &
Griskevicius, L. Six color flow cytometry detects plasma cells
expressing aberrant immunophenotype in bone marrow of
healthy donors. Cytometry B Clin. Cytom.(2011).
4. Caraux, A. et al. Circulating human B and plasma cells. Ageassociated changes in counts and detailed characterization of
circulating normal C. Haematologica 95, 1016-1020 (2010).
5. Banham, A.H., Turley, H., Pulford, K., Gatter, K., & Mason, D.Y.
The plasma cell associated antigen detectable by antibody VS38
is the p63 rough endoplasmic reticulum protein. J. Clin. Pathol.
50, 485-489 (1997).
179
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Key to the success of this technology is the scalable development
ofa requisite analytical environment to quantify descriptors of
function at a single-cell level for accurate characterization of
heterogeneous populations. By using the complexity of the flow
cytometry systems approach it is possible to build functional
relationships between cell phenotypes, activation molecules,
phosphorylation states & drug responsiveness all in a single assay.
These complex relationships cannot be viewed using traditional
analytical tools and this presentation will outline how a completely
new approach has created interactive windows into complex
data sets. These new tools for processing, reduction, analysis,
and visualization of very high content cytometryfunction to
transformour understanding of cellular heterogeneity, by defining
the relationship between phenotype and genotype, and the nature
of complex cellular responses. The new technology developed can
in real time manipulate millions of cells into pathways of response
in a totally interactive process.
239/B118
A Novel High Content Microscopic Method for
Mitochondrial Morphometry
Anthony Leonard1,2, Craig Beeson1,3, Baerbel Rohrer1,3
1
Drug Discovery and Biomedical Sciences, Medical University
of South Carolina, Charleston, SC, United States, 2Medical
Scientist Training Program, Medical University of South
Carolina, Charleston, SC, United States, 3Ophthalmology,
Medical University of South Carolina, Charleston, SC, United
States
Background: The regulation of mitochondrial morphology (MM) has
been implicated as both an indicator and determinant of metabolic
dysfunction in neurodegeneration and toxicology. We report a
novel high throughput and high content microscopic method for
the analysis of MM.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
cell at a time using flow cytometry has established significance
in both clinical diagnostics and translational research and with
technologies such as CyTOF have reached levels of extreme
complexity.This presentation will focus on a rapidly developing
area of very high contentsingle-cell analysis. These new cytometry
techniques allow for collection of large numbers of descriptors
of cell-function providing data-rich, multifactorial datasets
that cancomprehensively characterizeall phenotypespresent
inheterogeneous cell populations.Mass spectroscopy-based CyTOF
allowsrapid collection of 30-50 molecular descriptors from each
cell, with promise to increase this number >100. Ouroverall goal
is to discover relationships between molecular compositions and
observable phenotypes in model cellular systems using an array of
newly developed tools and to develop highly interactive functional
analytical tools with which to understand these relationships.
Figure 1. Method Overview
180
Method: Following 24 h treatment with compounds of interest,
cells from the 661w photoreceptor cell line were stained with
MitoTracker Deep Red and microscopic images acquired on a GE
INCell Analyzer 2000 automated fluorescence microscope (Fig.
1). Following image preprocessing and segmentation in GE InCell
Investigator Developer Toolbox 1.9.1, we classifiedMM subtypes
into physiological (puncta, rods, networks) and pathological
(swollen) shapes based on numerical morphometric data (length,
area, form factor, etc.).Classification was achieved using machinebased learning trained on a dataset of 890 manually classified
mitochondria (R 2.14.2, ctree() function, party library). We achieved
quality control by classifying small and low signal to noise ratio
mitochondria as “Junk”.Failures of automated nuclear or cellular
area segmentation algorithms were excluded on a per-cell basis by
excluding cells and nuclei with outlying area measurements (<25
µm2 or >1000 µm2). Out of focus, contaminated, poorly stained,
or otherwise low-quality microscopic fields were excluded by a
variant of the Dixon’s Q test, the Rout algorithm (GraphPad Prism
6.01). Basal and uncoupled mitochondrial functionswere measured
via Seahorse XF96 respirometry.
Results: Known mitochondrial toxins - rotenone and antimycin
A (AA) – as well as the oxidant t-butyl hydroperoxide (TBHP) –
alldecrease interconnectedness of mitochondria as evidenced
by lowered average area of the networked population in a dosedependent manner. However, these agents differed in their effects
on other MM subpopulations (for instance, AA and TBHP both
increased swollen type, but rotenone produced a decrease).
These changes correlated with decreased mitochondrial oxygen
consumption capacity. We interpret these data as validation of
the method’s accuracy and utility as a predictor of mitochondrial
function. Low coefficients of variance were achieved in this
method (CV: 9.4± 4.2%; “gold standard” Seahorse XF96 for same
treatments CV: 23.6 ± 12.3%), as well as remarkable throughput:
8-16 biological replicates each of 56 treatments(2x105 cells) were
acquired in 2 h and analyzed in ~8 h on a single modern Intel Xeon
Core i7 workstation.
Conclusion: A high throughput method to quantify mitochondrial
healthmay prove useful to a wide audience, from mitochondrial
disease biologists to clinicians and from pharmacologists involved
in drug discovery to those selecting their “molecule of choice”
forclinical development. This method may be extended to evaluate
the health of other intracellular organelles, or to quantify the
susceptibility of mitochondrial morphological subpopulations to
autophagy.
240/B119
CyTOFAnalyzer: A High Content Analysis Pipeline
Developed via the PlateAnalyzer Environment
J. Paul Robinson1, Valery Patsekin2, Bartek Rajwa3
BMS/BME, Purdue University Cytometry Laboratories, West
Lafayette, IN, United States, 2Purdue University, 3Bindley
Bioscience Center, Purdue University, West Lafayette, IN,
United States
1
The process of integrating information about cellular responses
provided by very high content flow cytometry (VHC FC) with
biological models brings cytometry into the domain of systems
biology[1]. For example, recent work by Nolan has shown
that VHC cytometry is fully capable of characterizing complex
signaling pathways [2-4]. Therefore, VHC FC has become a tool
able to quantitatively describe an entire biological system (such
as a immune system) rather than simply any small component
(selected population of cells) as has traditionally been the focus of
previous-generation FC. Rapid analysis of multiple multivariate data
ensembles representing VHC FC experiments requires innovative
approaches to data handling and visualizationCyTOFAnalyzer
is an FC analysis toolkit specifically designed for VHC data
generated by the CyTOF instruments. CyTOFAnalyzer can read
CyTOF generated datasets, and seamlessly interpret the barcoding
ISAC 2013 Program and Abstracts
Congress
Overview
strategies for multiplexing. By using the unique Logic Map feature
(Figure 1 below) it is possible to outlay the assay analysis pipeline
in a visual programming environment of PlateAnalyzer Canvas.
CyTOFAnalyzer can process any number of FC parameters and
can create an unlimited number of Boolean gates or logical
relationships between variables.
Special
Lectures
545 nm, and from thulium at 450-490 nm, both excited by pulsed
infrared (IR) light. The lanthanide lifetime in each of these families
could be tuned to take one of several values. Thus by combining
three materials, one from each family, we produced populations
of microspheres characterized by three different and independent
values of lifetime that can be read separately in either red, green,
or blue. The multiplexing level in the entire microsphere set is
120 and equals to the product of the number of lifetimes (5, 6,
and 4) available in each of the three material families. The lifetime
populations of europium beads were demonstrated for simultaneous
multi-channel DNA assays, showing non-crosstalk between each
lifetime channels. Thus, this work unlocks the hidden potential of
suspension arrays as the only analytical technique able to cope with
the complexity challenges in life sciences and medicine.
Saturday,
18 May
Sunday,
19 May
Figure 1. The example of Plate Analyzer Logic Map – an
environment for visual programming and design of flow cytometry
data analysis pipelines.
High Throughput Instrumentation
(B120 – B122)
Mega-Multiplexing Suspension Arrays
Flow Cytometry is a well established technology for rapid, highly
sensitive, multiparameter analysis of a wide array of biological
information, receptor expression, DNA Cell Cycle, phosphorylation
state of proteins of interest etc. The recent development of
Fluorogen Activating Proteins represents an innovative approach
to utilizing the speed and sensitivity of flow cytometry to develop
signals that can be associated with a cellular geography. Similarly
to using GFP to identify the expression of a protein, a FAP can
be engineered to flag the protein of interest. Unlike GFP- which
fluoresces all the time when excited- the signal is dark until it is
bound by the fluoro-probe which does not fluoresce until bound
by the FAP. There are probes available that are cell permeant, cell
impermeant, and which fluoresce when in a low pH environment.
This allows one for example to monitor the expression of a receptor
on the cell surface (cell impermeant probe), and the intracellular
expression (cell permeant probe) at the same time.
The Primary Pharmacology Group Cytometry Facility- has the
technology to perform multiparameter flow and image cytometry
from a single tube- up to 96 and 384 well formats. The readers can
be integrated into the Hi Res automation suite for live cell sample
preparation and analysis. The FAP technology is particularly suited
for the analysis of GPCR expression, and cellular desensitization/
internalization/ trafficking as a result of ligand binding with
a potential impact on characterizing GPCR Ligand Bias. This
presentation will review our integration of flow and image
cytometry platforms with the Hi- Res Nano Cell automation, and
our preliminary experience with the Fluor Activating Proteins in a
GPCR based project.
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
David Gebhard1, Shawn Hallowell2, Eric Benvenuti2, Regis
Doyonnas2
1
Primary Pharmacology Group, Pfizer, Inc., Groton, CT,
United States, 2Primary Pharmacology, Pfizer, Inc., Groton,
CT, United States
Oral Session
Abstracts
Grand challenges of life sciences underpinned by biological
complexity require high-throughput analytical technologies,
capable of simultaneous identification and quantification of
thousands of biomolecular species, known as multiplexing.
Multiplexing diagnostics is important in the areas of genomics,
proteomics, metabolomics, cytomics, and across medicine.In
this work, we radically extended the multiplexing capability of
spectrally-coded suspension arrays by adding the yet untapped
temporal dimension. We experimentally demonstrate time-resolved
suspension arrays comprising beads carrying lifetime codes,
independent from those used in spectral multiplexing. When used
concurrently they bring the total available multiplexing level to
10,000 or more. To produce lifetime codes we used three specially
designed families of lanthanide materials each with spectrally
separate time-dependent emissions: from europium at 610-625
nm excited by pulsed ultraviolet (UV) light, from erbium at 525-
Development of FAP Technology: Fluorogen Activated
Protein in High Throughput Cytometry
Commercial
Tutorials &
Exhibits
Advanced Cytometry Laboratories, MQ BioFocus Research
Centre& MQ Photonics Research Centre, Macquarie University,
NSW 2109, Australia
2
Newport Instruments, 3345 Hopi Place, San Diego, California
92117-3516, USA
3
Purdue University Cytometry Laboratories, Bindley Bioscience
Center, Purdue University, West Lafayette, Indiana 47907, USA
email: jie.lu@mq.edu.au, dayong.jin@mq.edu.au
1
242/B121
Poster
Session
Jie Lu1, Yiqing Lu1, Jiangbo Zhao1, Ewa M. Goldys1, Robert C.
Leif2, James A. Piper1, J. Paul Robinson3, Dayong Jin1
1
Macquarie University, Sydney, Australia, 2 Newport
Instruments, San Diego, CA, United States, 3Purdue University,
West Lafayette, IN, United States
Figure 1. Gaussian populations of microspheres encoding Eu red
luminescence lifetimes.
Wednesday,
22 May
241/B120
Tuesday,
21 May
Any pathway on the logic map can be evaluated by selecting any
linked components as shown highlighted by the yellow lines.
Results of analyses can be displayed as dotplots, population
numbers or percentages, or if appropriate, dose response curves. All
data can be output to spreadsheets.
Monday,
20 May
The power of CyTOFAnalyzer is that we have developed a process
for visualizing the entire assay so that any particular pathway of
interest can be instantly identified and results extracted at any time.
This is demonstrated in the accompanying figure; on the left are
14 phosphorylation states linked to 14 cell populations which are
linked on the right to “drug boxes” linking 12 activation molecules
at 8 concentrations each.
181
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
243/B122
A Mix-and-Read Cell-Based Assay for Antibody
Screening against Epithelial Growth Factor Receptor
Diana Caracino1, Wayne P Bowen2, David Onley2, Paul Wylie2,
Tristan Cope2
1
TTP Labtech, Cambridge, MA, United States, 2TTP Labtech,
Melbourn, United Kingdom
Antibodies against a wide range of protein targets have been either
approved or are currently under development for therapeutics. The
use of flow cytometry to identify antibody-antigen binding has been
well characterised. While flow cytometry has been widely utilised,
it requires the use of suspension cells or adherent cells that are
removed from the well, so it is unable to analyse cells in situ. In
addition, as the cells are passed through a flow tube, low affinity
antibodies can dissociate from the antigen as the equilibrium of
antibody concentration to antigen is changed. Flow cytometry
is typically not a high throughput method for this kind of assay.
Though higher-throughput systems are now currently available,
they still have to remove cells from a plate which can lead to crosscontamination, and require wash steps which are not ideal for lowaffinity antibodies. We offer an alternative high-throughput method
for looking at antibody binding whereby the primary screen can be
run through the mirrorball® high sensitivity cytometer to rapidly
identify antibodies of interest.
Here we present a sensitive robust, mix-and-read method for
the screening of antibodies against cell surface proteins. The
mirrorball®high sensitivity cytometer is used to quantify the cellular
fluorescence in cultures in 384-well microplates. We describe its
use for the determination of human epithelial growth factor receptor
(EGFR) antibody binding in A549 cells which are known to express
high levels of EGFR. A549 cells were incubated with mouse antiEGFR antibody (Cat. No. GR01-100UG; Merck Chemicals Ltd) and
fluorescently-labelled anti-mouse IgG antibody. Without washing
away unbound antibodies, plates were scanned and fluorescence
of each cell quantified. Clear concentration-dependent antibody
binding was observed with low assay variability. Addition of
Vybrant™ DiO, a 488 nm-excitable cell stain, allowed detection of
cells irrespective of the amount of anti-EGFR antibody binding for
improved assay performance. With its simple operation, no-wash
format, and high sensitivity, this new method is well-suited for high
throughput antibody screening.
High Throughput Screening
(B123 – B128)
244/B123
Development of a Multiplex Oligonucleotide Ligation
(MOL)-PCR Assay for the Detection of Shiga Toxin
Producing E. coli in the Beef Chain
Travis Woods1, Steven W. Graves2, Alina Deshpande3
Center for BioMedical Engineering, University of New
Mexico, Albuquerque, NM, United States, 2University of New
Mexico, 3Los Alamos National Lab
1
Introduction: Shiga toxin-producing Escherichia coli (STEC)
have been identified by the USDA as a serious threat to the
nation’s health stemming from contaminations in the food supply,
specifically, the beef chain. By using Multiplex Oligonucleotide
Ligation-PCR (MOL-PCR) for detecting STEC DNA signatures in
the beef supply chain we will be able to offer a multiplex and
high throughput alternative to the current tedious monitoring
techniques that are labor intensive and time consuming. MOL-PCR
is a nucleic acid based assay patented at Los Alamos National
Laboratory (LANL) that uses flow cytometry and microsphere arrays
for detection. This research will be focused on DNA detection of 8
STEC serotypes (STEC-8): O26, O45, O103, O104, O111, O121,
182
O145, and O157:H7. The goal is to produce a multiplex panel of
MOL-PCR probes for identifying DNA signatures corresponding
to each of the STEC-8 serotypes and ultimately strain specific
identification.
Methods: MOL-PCR pairs, or MOLigo pairs will be designed
computationally starting with published STEC single nucleotide
polymorphisms (SNPs) and other unique signatures using web
based alignment/search tools as well as other software designed
specifically for MOLigo pair design: MOLigoDesigner. The first
round of design will center on a multiple hit matrix identifying
each STEC serotype, and then testing for signatures for toxin genes
(stx1, stx2), other virulence genes and serotyping signatures in
the O-antigen gene cluster. Along side this method we will run
computational analysis of sequence data using PROSIG software
developed at LANL to identify new potential signatures. MOLigo
pairs designed in these ways will be evaluated using the Luminex
system for highly multiplexed analysis. Initial assay panels will be
tested for specificity by running against isolated genomic DNA from
commercial sources as well as reference strains from collaborators.
Conclusion: First results of the evaluation of our multiplex MOLPCR assay will be presented here. While this project is just getting
started, the MOL-PCR assay is already able to advance the current
STEC-8 serotype identification method of size based gel analysis
currently being performed. As more unique signatures are found
and tested the power of this assay will greatly increase. In addition,
the ability of this assay to simultaneously detect diverse signatures
such as SNPs and unique sequences provides a broader set of
options for developing a robust, highly specific assay that can be
sustainable.
245/B124
Single-Cell Nanotoxicity of Nanobarcoded
Superparamagnetic Iron Oxide Nanoparticles
Quantified by High-Throughput Scanning Image
Cytometry
James Leary1, Trisha Eustaquio2
1
Basic Medical Sciences and Biomedical Engineering, Purdue
University, West Lafayette, IN, United States, 2National Center
for Toxicological Research at FDA, Jefferson, AR, United States
Background: Oligonucleotide-functionalized nanoparticles
(NPs) are promising agents for nanomedicine, but the potential
nanotoxicity that may arise from such conjugates has yet to be
evaluated in a systematic manner. There is a need to evaluate the
intrinsic nanotoxicity, if any, of nanomedicine vehicles used for
drug/gene delivery from the therapeutic responses of those drug/
gene molecules. Since nanomedicine functions on the single-cell
level, nanotoxicity should also be performed at single-cell level on
these attached cells using high-throughput image analysis methods.
We report high-throughput in-vitro single-cell nanotoxicity assays
based on scanning image cytometry with LEAP™ used to study a
specific type of oligonucleotide-functionalized NP, “nanobarcoded”
superparamagnetic iron oxide NPs (NB-SPIONs) we have been
evaluating for use in animals as a precursor to humans. These
oligonucleotide-functionalized NPs allow the encoding of specific
experimental information onto individual NPs and the recovery of
this information in cell tissues using in-situ PCR techniques.
Methods: The major advantage of scanning image cytometry is its
ability to collect multiple fluorescent measurements from individual
cells in small cell populations, which makes this method amenable
to testing novel NPs that are often synthesized on a small scale.
LEAPTM scanning image cytometry allows for rapid screening of
large areas in microwells by using a large field of regard f-theta lens
that minimizes the number of stage movements necessary to scan
every cell in a microwell. Our selected panel of single-cell assays
measured common modes of nanotoxicity—apoptosis, necrosis,
generation of reactive oxygen species (ROS), and cell number.
Using these assays, the nanotoxicity of two sizes of NB-SPIONs
ISAC 2013 Program and Abstracts
246/B125
Flow Cytometry as a Drug Screening Platform
ISAC 2013 Program and Abstracts
Anne Dickson, Jolene Bradford
R&D Molecular Probes Labeling and Detection Technologies,
Life Technologies, Eugene, OR, United States
Characterization of white blood cells (WBCs) and other cellular
entities in whole blood using flow cytometry has historically
required removal of red blood cells (RBCs). This has been necessary
due to the abundance of RBCs. Two main strategies for RBC
removal have been density gradients or osmotic lysis. However,
these methods require additional costs, and have longer prep time,
selective cell loss, and general sample loss due to washes.
Speaker/Author
Index
Background: While flow cytometry (FCM) has increased over
the years in content, it has remained relatively low throughput
regarding sample number; several methods have recently been
developed to tackle this. Fluorescent cell barcoding (FBC) is where
Differences in Whole Blood Light Scattering from
Multiple Laser Excitation Sources: A Rapid NoLyse, No-Wash Method Using the Attune® Acoustic
Focusing Cytometer with Red or Violet Laser Option
Poster Session
Abstracts
Julia Tasset 1, Philip Hexley 2, Chad Robinson 1, Andrew
Osterburg3, Connie Fu1, George Babcock1,2
1
Surgery, University of Cincinnati, Cincinnati, OH, United
States, 2Research, Shriners Hospitals for Children, Cincinnati,
OH, United States, 3Research, Wright Patterson Air Force Base,
Dayton, OH, United States
248/B127
Oral Session
Abstracts
Development of a Multiplexing, High-Throughput
Technique for Initial Cell Screening
Commercial
Tutorials &
Exhibits
247/B126
Poster
Session
We have successfully been using IntelliCyts HyperCyt, a
commercially available innovative sampling device, attached to an
Accuri C6, a small two laser five detector basic analyser to analyse
96 and 384 well plates in minutes. This has allowed us to perform
high capacity flow cytometry with throughputs synonymous
with other platforms in a typical pharmaceutical drug screening
laboratory. Flow cytometry is now routinely used for identifying hits
tested in complex cell based assays that are normally run in later
drug discovery phases, but this time at scale.
Discussion: Here we have combined FCB and HTP acquisition,
in conjunction with PlateAnalyzer software, we have overcome
the bottleneck of data analysis and developed a rapid, efficient,
low-cost screening method which is amenable to automation. In
practice, we have successfully done FCB using 3 reactive esters
of 6x4x5 intensities respectively, if this could be translated to
the HTP method it would increase our multiplexing potential 20
fold, improving on the HTP demonstrated by Sklar et al., (2012)
potentially >1.6 million samples perday. In practice we have
struggled to overcome scaling this down due in part to pipetting
error. As this technique is amenable to automation the process
could be standardized and pipetting error reduced, allowing us to
reach this HTP not currently available, with minimal modification
of this protocol.
Wednesday,
22 May
We will describe some of the specific areas where high throughput
flow cytometry has been used, such as its routine use in target
based discovery programs, for example identification of compounds
that inhibit T cell intracellular AKT phosphorylation and how using
this data in combination with isolated enzyme activity aids hit
selection and their progression. We’ll also describe its application
to profiling large compound sets in phenotypic focussed screening
campaigns, multi analyte detection and for screening hybridoma
secreted antibodies using cell based assays.
Results: In under 60 minutes we can acquire and analyse 2304
samples, successfully identify treatmentsbased on shift in Ki67
staining from the control. By moving 1 gate to a different set of
barcoded populations we can again visualize the next 384 samples.
This initial screening allows us to rapidly identify samples of
interest, return to the original culture and re-acquire more cells
from a given sample, and further analysed based on G0, 1/S/G2,
M populations by ModFit LT, showing PHA and Concanavalin A
treatments stimulated proliferation and SB20358, P38, Cytochalasin
D treatments inhibited proliferation.
Tuesday,
21 May
The increasing use of disease relevant human primary cells
is driving the requirement for more sensitive high throughput
technologies that derive maximum information from fewer and
fewer cells. It is widely recognised that heterogeneous primary cell
populations are more suited to high content single cell analysis
techniques such as flow cytometry. New technologies are emerging
and high throughput flow cytometry sampling is now a reality.
Monday,
20 May
As a platform flow cytometers have proven their value over the
years in clinical laboratories throughout the world. The power of
flow cytometry with its multiparameter detection capabilities and
powerful analytical capability is evident. Although the analogous
HCS imaging technology has already established itself as a true HTS
platform in screening laboratories flow cytometry has struggled.
By far the primary reason for this is its low individual sample
throughput (often several minutes or more).
Sunday,
19 May
Rob Jepras, Poonam Shah, Metul Patel, Emma Sutton, Steve
Ludbrook
Molecular Drug Discovery, Glaxosmithkline, Stevenage,
United Kingdom
Method: 1152 different Jurkat cell samples were cultured in
12, 96-well microtiter plates for 24 hours, split into 6 groups:
control, SB20358 inhibitor, P38 inhibitor, PHA, Concanavalin A,
Cytochalasin D, to alter cell proliferation.Cells were fluorescently
barcoded with one of 3 intensities of pacific blue fluorescent and
one of 2 intensities of Alexafluor-350amine-reactive esters then
combined, allowing for identification of 6 samplesin 1 well. The
barcoded samples were stained with anti-Ki67 and FX cell cycle
dye then transferred into a 384 well microtiter plate. Each sample
was replicated, giving a total of 2304 samples; 384 wells, 6 samples
per well. Samples were acquired on a BD LSRII HTS acquiring
all 2304 samples in under 40 minutes. Using the PlateAnalyzer
software developed at Purdue University (Robinson et al., 2013)
we visualize a 384-well heat map based on user-defined variables,
allowing rapid identificationof populations of interest, which were
reanalysed by ModFit LT.
Saturday,
18 May
Conclusions: This study demonstrates that several modes of
nanotoxicity can be systematically and rapidly evaluated from small
sample sizes at the single-cell level using high-throughput scanning
image cytometry.
multiple samples are stained with a unique fluorescence intensity,
combined prior to antibody staining and analysis, reducing cost,
time, and staining variability (Nolan et al., 2006). Also systems such
as BD High Throughput Sampler (HTS) and Hypercyte by Intellicyte
have enabledFCM to increase sample throughput. Another
limitation has been analysis of vast amounts of datagenerated from
high throughput (HTP)FCM:recent software developments have
addressed this issue.
Special
Lectures
Results: The results suggest that the conjugated NB confers a
biocompatible coating that protects against nanotoxicity at very
high SPION doses, but both NB- and COOH-SPIONs of either size
generally have low in-vitro nanotoxicity at lower, physiologically
relevant doses.
Congress
Overview
(10 nm and 30 nm core size) was compared to the parent NP,
carboxylated SPIONs (COOH-SPIONs).
Conventional flow cytometry has prevented analysis of whole
blood due to long acquisition times. Even then, difficulties remain
in identifying WBCs by scatter. It has been reported that WBCs
183
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
can be resolved from RBCs in whole blood by using violet light in
combination with fluorescence labeling of WBCs. The maximal
absorption of violet light by oxyhemoglobin allowed discrimination
of WBCs in a forward scatter vs side scatter plot, in contrast to
conventional (488 nm) excitation sources.
In this study, we demonstrated differences in WBC scatter patterns
between violet, blue, and red excitation of whole blood. To
understand these differences, we labeled WBC subpopulations and
found that their spectral properties were radically altered using blue
or red (but not violet) excitation.
In addition to violet excitation, the ability of the Attune® cytometer
to analyze samples at high volume rates makes it the ideal
instrument for whole blood analysis. We have also developed a
procedure to enable the use of the Attune® cytometer with Red
Laser. The ability to analyze whole blood will provide researchers
with a simplified, fast, and reliable sample preparation procedure.
For Research Use Only.
249/B128
Complex Cell Based Assays with a Novel Imaging
Cytometry System
Oksana Sirenko, Steve Boege, Jayne Hesley, Dave Henderson,
Avrum Cohen, Max Starodynov, Evan Cromwell, Paul Comita
Molecular Devices, Sunnyvale, CA, United States
Background: An important trend in drug discovery is the use of
complex cell-based models offering more biologically relevant
information and higher predictivity for drug efficacy and toxicity
screening and development of disease models. There is a need
to increase throughput and simplify running such assays, while
maintaining data quality. Here we report results from an imaging
cytometer integrated into a microplate reader platform that provides
high content and high throughput analysis for cell-based assays.
Methods: Assays were performed using novel imaging cytometer
system in multiwell plate formats. The analysis software provides
cell-specific and averaged analysis outputs: cell count, average
and integrated cell area, averaged and integrated intensities of
fluorescent signal.
Results: We present several examples of cell-based assays:
Inflammation assay measured expression of adhesion molecules
VCAM, ICAM, HLADR on primary endothelial cells (HUVEC).
Cells were stained with FITC-conjugated antibodies and levels
of expression were analyzed via total or averaged fluorescence
intensity. Cells were stimulated with inflammatory cytokines and
treated with the panel of known anti-inflammatory compounds
(kinase inhibitors). Effects of anti-inflammatory compounds were
measured and IC50s were determined. The assay window was ~20,
and Z’ values were ~0.7.
Cell toxicity assay utilized induced pluripotent stem cell derived
hepatocytes. Cells were treated with a number of liver toxic drugs
for 72h. Cell viability was assessed with Calcein AM or AF-488
phalloidin. Toxicity was measured by decrease in cell count or total
cell area. IC50s for aflatoxins, staurosporine, etoposide and other
hepatotoxic compounds were determined. The assay window was
~200 and Z’ values were >0.9.
Differentiation of hematopoietic progenitors. We have developed
an assay suitable for testing the effects of growth factors and anticancer drugs on hematopoiesis. Hematopoietic stem cells cultured
in the presence of different growth factors or treated with anticancer drugs were stained using lineage-specific FITC-conjugated
antibodies or Cell Tracker Green. Numbers of cells expressing
lineage-specific markers or total cell numbers were detected and
EC50s for different factors determined. The assay window was >200
and Z’ values were >0.7.
184
Conclusions: We have demonstrated utility of a new imaging
cytometer for phenotypic screening and developed several assay
models that will be useful for both the academic and biotech
environment. We demonstrate that this add-on system to a plate
reader is useful for a wide range of assays, providing researchers
with cellular analysis capability, simplified workflow, high speed
and throughput, and the content of output information comparable
with more advanced imaging and analysis methods.
Image Processing and Analysis
(B129 – B131)
250/B129
Detecton of Internalized Exosomes by Tumor Cells
Using Amnis ImageStreamX
Patricia Simms1, Carrie Franzen2,3, Veronica Volgina2, Gopal
Gupta3
1
FACS Core Facility, Research Services, Loyola University
Chicago, Maywood, IL, United States, 2Oncology Institute,
Loyola University Chicago, Maywood, IL, United States,
3
Department of Urology, Loyola University Chicago,
Maywood, IL, United States
Microparticles (exosomes, microvesicles, apoptotic bodies, etc.)
are part of the endogenous communication system of the cell.
Theirstimulatory and inhibitory signaling activities are mediated
by the DNA, RNA, miRNA and proteinswithin the membrane
vesicle and bysurface molecules. Microparticles have also
been designed as drug delivery systems. Thus, detection and
analysis of these microvesicles are useful for disease diagnosis
and treatment.However, these particles are difficult to analyze
because of their small size (0.05-3 um). In this study, we analyze
exosomes produced by a bladder tumor cell line using the Amnis
ImageStreamX.
The limits of detection for the Amnis ImageStreamX for both beads
area and fluorescence were determined with Nano Fluorescent
Particle Size Standard Kit. Fluorescent particles as small as 0.22nm
were detected by their fluorescence signal, but the brightfield signal
did not distinguish bead from background. WithQuantum FITC-5
MESF beads, a signal with 15033 MESF FITC was detectable above
background.
Exosomeswere isolated from conditioned culture supernate by
ultracentrifugationand were labeled with the membrane stain PKH26 and analyzed on the ImageStreamX with an internal standard
added to allow for concentration determination. Exosomes were
co-cultured with tumor cells and analyzed for internalization of
PKH26+ particles in the tumor cells lines. Using the spot count
wizard (IDEAS software), we were able to determine the number
of PKH26 positive spots.At 17 hours incubation, the fluorescence
from the internalized exosomes was too bright to allow spot
counting. We also measured the total PKH-26 fluorescence
intensity of the cell, as a measure of the total exosome uptake.A
time course demonstrated that the uptake of exosomes increased
with longer co-culture times. We analyzed the effect of overnight
storage (+4 and -20)on exosomeuptake. The fluorescence intensity
of the labeled exosomes did decrease over time, but the number
of exosomes taken up by the recipient cells, as detected by spot
counting did not change. Using the internalization wizard, we
determined that the exosomes were internalized. Treatment with
trypsin did not decrease the exosome staining, suggesting that the
exosomes were internalized.A titer of exosomes demonstrated a
cutoff concentration for detection of5x10 6/ml.
This work demonstrates the feasibility of using the ImageStreamX to
detect and quantitate exosome uptake by tumor cells.
ISAC 2013 Program and Abstracts
Cellometer Image Cytometry as a Complementary
Analysis Tool to Flow Cytometry for Visual
Verification of Gated Cell Populations
To increase understanding of the cause of implant failure in patients
experiencing ALTR we investigate immune system contribution
to local tissue damage. MoM THRs shed metal nanoparticles into
periprosthetic tissue. We hypothesise that implant wear debris alters
antigen presenting cell function of dendritic cells leading to T cell
activation with the resulting increase in osteoclastic activity giving
rise to destruction of bone and tissue with subsequent implant
failure.
Methods: Thisstudy is designed to investigate immune cells in blood
and synovial fluid of MoM THR Ultima TPS patients. We also study
other implant types such as MoP (Metal-on-Poly), MoC (Metal–onCeramic) or others.
Wednesday,
22 May
Symptomatic patients are recruited at Norfolk and Norwich
University Hospital (NNUH) Orthopeadics department during their
routine visit to hospital. Asymptomatic patients are invited for a
checkup and asked to donate samples. Study cohort consists of
2 main groups namely; Operative Group (Primary MoP, revision
MoP, revision MoM, revision MoM-UltimaTPS) and Non-Operative
Group (Normal MRI scan, abnormal scan and atypical scan). A
healthy control group also included as comparison.
Poster
Session
The frequency and the composition of human leukocytes in the
body can be used as an indicator of health or disease. These cells
can be monitored in patients by identifying their surface markers
by immunophenotyping using flow cytometry. The protocol that we
developed uses a combination of 18 different antigenic targets or
antibodies to phenotype lymphocytes and Dendritic cells (DC) in
fresh whole blood and synovial fluid. Samples are processed and
results are generated on the same day.
Commercial
Tutorials &
Exhibits
Three 9 colour flow cytometry panels were designed to study
frequencies as well as absolute number of immune cells in this
cohort of patients. Targets including T cells (CD4+ and CD8+),
antigen presentation cells such as B cells, monocytes and DC as
well as detection of key chemokine receptors (CX3CR1 and CCR2)
and adhesion molecules (CD11b and CD62L) were detected using
flow cytometry.
Oral Session
Abstracts
Conclusion: Flow cytometry provides rapid and sensitive means
of detection and quantitation of immune cells especially rare cell
populations like DC.
Poster Session
Abstracts
It has long been acknowledged that occupational stress (as well
as other sorts of stress) causes biological changes in an individual.
We wished to evaluate how flow cytometry affects professionals
in the field as determined by differences in physical appearance
over time. In May of 2002, the ISAC convention was held in San
Diego, California at the Town and Country Hotel. At that time,
historical data for later stress evaluation was collected in the
following manner from many ISAC participants. Subjects were
randomly selected and those willing to volunteer were asked to
pose on a surfboard set up in front of a large paper mache ocean
wave and photographic data was collected. This historical data
will be presented in poster format so that any returning participants
from 2002 may empirically determine if the field of flow cytometry
has indeed been kind or cruel to the above mentioned wave riding
individuals. In addition, the data will provide a comic interlude
between other more rigorously scientific presentations for all poster
viewers. A partial list of ISAC participants volunteering for this
project in no particular order were, Zbigniew Darzynkiewicz, Ger
van den Engh, Carl and Sigie Stewart, Abe Schwartz, Greg Stelzer,
Howard Shapiro, Vincent Shankey, Ernie Wong, David Headley,
Scott Cram, Robert Leif, John Martin, and Jake Jacobberger.
Background: Total Hip Replacement (THR) with Metal-on-Metal
(MoM) bearings have demonstrated high rates of failure and are
associated with development of pseudotumors which can present
with symptoms of pain, limp, or mass as the result of significant
soft tissue destruction arising from adverse local tissue reaction
(ALTR). The Ultima TPS 28mm head MoM THR (Depuy) has been
associated with a lifetime failure rate of 20%. The UK Medicine and
Healthcare Regulatory Agency (MHRA) alert recommends annual
clinical follow up for the lifetime of MoM implants as well as
blood metal ion levels (cobalt and chromium) and cross sectional
imaging.
Tuesday,
21 May
C Kevin Becker, Margie Becker, Phoenix Rose Becker, Bianca
Becker
Phoenix Flow Systems, Inc., San Diego, CA, United States
Pinar Court1, Darren Ebreo2, Simon Donell2, Simon Carding1
Institute of Food Research, Norwich, United Kingdom,
2
Norfolk & Norwich University Hospital, Norwich, United
Kingdom
1
Monday,
20 May
Effects of Flow Cytometry on the Physical
Appearance of Flow Cytometry Professionals
Investigation of Immune System Involvement in
Prosthetic Implant Failure with Polychromatic Flow
Cytometry
Sunday,
19 May
252/B131
253/B132
Saturday,
18 May
Traditionally, many cell-based assays that analyze cell populations
and functionalities have been performed using flow cytometry.
However, flow cytometers remain relatively expensive, and require
highly trained operators for routine maintenance and data analysis.
Flow cytometer can process and generate a large number of events,
but the data gating strategies are often complex and are performed
without the visual confirmation of the cells processed, which
may lead to an incorrect gating strategy. Recently, a novel image
cytometry system (Cellometer) has been developed by Nexcelom
Bioscience LLC (Lawrence, MA) for automated cell concentration
and viability measurement using bright-field and fluorescent
imaging methods. The image cytometer is capable of capturing
bright-field and fluorescent images and generates fluorescence
intensity data of each analyzed cell. The system can perform gating
operations such as fluorescence intensity or cell size similar to
flow cytometry on the analyzed cell population. The ability to
visually observe the gated cell population allows the elimination
of data uncertainties generated using flow cytometry. Here we
report, using an enzymatic vitality and viability stain, Calcein AM
and propidium iodide, that image cytometry allows for a visual
confirmation that the population of cells gated using flow analysis
is indeed the population of interest. The image cytometry method
offers not only a direct method for performing fluorescence cellbased assays, but also may be utilized as a complementary tool to
flow cytometers for aiding the analysis of more complex samples.
Immune Monitoring (B132 – B141)
Special
Lectures
Leo Chan1, Dmitry Kuksin1, Christina Kuksin2, Jean Qiu1
1
Technology R&D, Nexcelom Bioscience LLC, Lawrence, MA,
United States, 2Veterinary & Animal Sciences, University of
Massachusetts Amherst, Amherst, MA, United States
Congress
Overview
251/B130
Speaker/Author
Index
ISAC 2013 Program and Abstracts
185
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
255/B134
Functional Defects in the Immune Cells Responses in
Erdheim-Chester Disease
Tumor Exome Analysis Reveals Neo-Antigen-Specific T
Cell Reactivity in an Ipilimumab-Responsive Melanoma
Wanxia Tsai 1, Kathryn Davis 1, Juvianee Estrada-Veras 2,
Runsheng Wang3, Bernadette Gochuico2, William Gahl2,
Massimo Gadina1
1
Translational Immunology Section, NIAMS, NIH, Bethesda,
MD, United States, 2NIH Undiagnosed Diseases Program,
NHGRI, NIH, Bethesda, MD, United States, 3Office of the
Clinical Director, NIAMS, NIH, Bethesda, MD, United States
Pia Kvistborg1, Nienke van Rooij1, Marit van Buuren1, Daisy
Philips1, Arno Velds2, Mireille Toebes1, Laura van Dijk1,
Sam Behjati3, Henk Hilkmann4, Dris el Atmioui4, Marja
Nieuwland2, Michael Stratton3, Ron M Kerkhoven2, Can
Kesmir5, John Haanen1, Ton Schumacher1
1
Department of immunology, Netherlands cancer institute,
Amsterdam, Netherlands, 2Genomics core facility, Netherlands
cancer institute, Amsterdam, Netherlands, 3Wellcome Trust
Genome Campus, Wellcome Trust Sander Institute, Combridge,
United Kingdom, 4Peptide synthesis facility, Netherlands
cancer institute, Amsterdam, Netherlands, 5Theoretical biology
and bioinformatics, Utrecht University, Utrecht, Netherlands
Introduction: Erdheim-Chester disease (ECD) is a rare, non-familial
multisystem disorder characterized by proliferation and infiltration
of non-Langerhans histocytes into multiple organs. Little is known
about the pathogenesis of the disease and the few published studies
are based on a very limited number of patients. Therefore, we
investigated if leukocytes of ECD patients exposed to innate and
adaptive immune stimuli exhibited a different secretive profile when
compared to normal individuals. Furthermore, we also assessed the
ability of patients’ monocyte-derived macrophages, and bronchial
alveolar macrophages to phagocyte fluorescent particles.
Methods: Cell differentiation and phagocytosis assay:
Monocyteswere magnetically-sorted from peripheral blood
mononuclear cells (PBMC), and then cultured in complete medium
supplements with macrophage colony stimulating factor (MCSF)
for 6 days. Expression of macrophages-specific differentiation
markers such as CD105 and CD33 were then assessed by flow
cytometry. The ability of monocyte derived macrophages as well
as of bronchial alveolar-derived macrophages was then assessed by
incubating the cells with fluorescent latex beads for two hours at
37C°. Fluorescent beads incorporation was then measured by flow
cytometry.
Cytokine release assays: Whole blood (1ml) was incubated with
lipopolysaccharide (LPS) (in the present or absence of ATP),
staphylococcal enterotoxin B (SEB), muramyl dipeptide (MDP), Poly
(I:C), and Pam3Cys for 3 hours and 24 hours. Supernatant were then
harvested and released cytokines measured using a luminex-based
multiplex assay.
Results: Macrophages derived from monocytes of ECD patients
as well as alveolar macrophages didn’t show any differences
in phagocytic activity compared to cells obtained from normal
individuals.In-depth analysis of 27 human cytokinesmeasured
on the supernatant of blood cells after stimulationswith different
ligands showedreduced cytokine secretion in ECD patients. Most
noticeably, the levels of IL-1β, IL-1ra, and IL-10 were significantly
lower after the LPS stimulation for ECD patients’ cells. The levels of
IL-9and IL-10 were also decreasedafterSEB andMDP stimulations,
respectively.
Conclusion: These preliminary results of the current ECD patients
suggested that histiocytes might not be the only cells involved in
the pathology of ECD. The lower secreting capability of adaptive
immune cells such as T cells suggested that their impaired
activation could also be at the basis of this disease. Ultimately, the
changes in cytokine section may be useful as biomarkers for the
diagnosis of ECD patients.
Poster Session
Abstracts
Oral Session
Abstracts
254/B133
The evidence for T cell mediated regression of human cancers
such as non-small cell lung carcinoma, renal cell carcinoma and
in particular melanoma following immunotherapy is strong. AntiCTLA4 (Ipilimumab) treatment has been approved for treatment
of metastatic melanomaand antibody-mediated blockade of
PD-1, a second inhibitory receptor on T cells, has shown highly
encouraging results in early clinical trials. While the clinical
activity of these treatments is apparent, it is still unknown which
T cell reactivities are involved in immunotherapy-induced cancer
regression. T cell reactivity against non-mutated tumor-associated
self-antigens has been analyzed in patients treated with Ipilimumab
or with autologous tumor infiltrating T cells, but the magnitude of
the T cell responses observed has been relatively modest. In part
on the basis of such data, recognition of patient-specific mutant
epitopes (neo-antigens) has been suggested to be a potentially
important component. A potential involvement of mutated epitopes
in T cell control of cancer would also fit well with the observation
that the mutation load in sun-exposed melanomas is particularly
high. Intriguingly, on the basis of animal model data it has recently
been suggested that (therapy-induced) analysis of T cell reactivity
against patient-specific neo-antigens may be feasible through
exploitation of cancer genome data. However, human data have
thus far been lacking.
To address this we have used MHC class I molecules occupied with
UV-sensitive ‘conditional’ peptide ligands allowing production of
very large collections of pMHC complexes together with a pMHC
‘combinatorial coding’ strategy that allows the parallel detection of
dozens of different T cell populations within a single sample.
From a stage IV melanoma patient who exhibited a clinical
response to Ipilimumab treatment we identified tumor specific
mutations via exome sequencing of matched tumor- and healthy
material. The exome showed a clear UV-induced mutational
profile and contained 1075non-synonymous mutations. Possible
MHC epitopes covering these mutations were predicted using a
set of filters; 1) predicted to bind the patient’s MHC; 2) predicted
to be cleaved by the proteasome; 3) genes of which the mutated
peptides arose had evidence of RNA expression. In this case the
analysis yielded a set of 1952 epitopes restricted to the HLA-A and
HLA-B alleles of this patient. To screen for T cell reactivity against
these epitopes we used the pMHC combinatorial coding approach
and tumor infiltrating lymphocytes. Our analysis revealed T cell
reactivity against 2 neo-antigens, including a dominant T cell
response against a mutant epitope of the ATR (Ataxia Telangiectasia
and Rad3 related) gene product. Analysis of PBMC samples
collected before and during Ipilimumab therapy showed that this
particular response increased strongly after treatment from 0.06% to
0.28% of CD8 T cells after being stable in magnitude for 10 months.
Speaker/Author
Index
These data provide the first demonstration of cancer exome-guided
analysis to dissect the effects of melanoma immunotherapy. With
this proof of concept we are now closer to patient specific immune
monitoring and getting a better understandingof which antigens are
important players in tumor regression.
186
ISAC 2013 Program and Abstracts
Altered Mitochondrial Functional Response to
Activation in T-Cells of the Neonate
Oral Session
Abstracts
Poster Session
Abstracts
Natural killer (NK) cells are a subset of lymphocytes that play a
pivotal role in the innate immune response to tumors and viral
infections. Upon recognition of non-self, activated NK cells
produce cytokines and kill the targeted cell. The functions of NK
cell subsets have not wellcharacterized in cynomolgus macaque.
Previously, we developed a IFN-gamma (IFNγ) ELISPOT assay that
enumerates functional NK cells in cynomolgus macaques. PBMCs
isolated from cynomolgus macaques were co-cultured with the
NK target K562 cell line, resulting in the production of IFNγ. IFNγ
secreting cells were observed at frequencies that generally ranged
from 100-200 IFNγ spots per 1000 NK cells and background
levels were negligible. Cell sorting assays confirmed the source
of IFNγ was NK cells and not other cells such as T cells. Wethen
performed intracellular cytokine measurements by flow cytometry
to determine whether specific subsets of NK cells were responsible
for the IFNg production. Both isolated PBMCs and whole blood
Commercial
Tutorials &
Exhibits
Carol Donovan, Scott Haskett, Caleb Homiski, Cris
Kamperschroer
Immunotoxicology Center of Emphasis, Pfizer Drug Safety
Research and Development, Groton, CT, United States
For the simultaneous analysis of multiple samples we established
an antigen-coated 96-strip-well culture systemto stimulate T cells.
This flexible ready-to-use format provides the opportunity to screen
either for a single antigen or in parallel for several specificities
by combining 8-well-strips possessing different viral antigens.To
reduce time and workload for the fluorescent staining procedure,
we developed a rapid and easy-to-handle protocol and reagents.
Compared to conventional staining protocols, all steps are executed
in the 96-strip-well plate. Without any washing step, cells are
fixed and stained with defined reagentsto identify virus-specific T
cells and to evaluate their cytokine pattern.Thereby, we drastically
diminished the overall processing time for up to 96 samples to
only 50 minutes. Moreover, we developed an automated flow
cytometric analysis process. This includes the possibility to measure
the stained samples in the plates hands-free using pre-defined
experiment settings and acquisition templates. We also applied an
automated gating strategy for data analysis. Areport summarizes
the results of the T cell response.Overall, hands-on time for multisample acquisition and analysis is minimized and the standardized
reagents/protocol and sample analysis process decrease inter- and
intra-assay variations.
Poster
Session
Assays to Measure Natural Killer Cell Function in
Cynomolgus Macaques
Functional characterizations of antigen-specific T cells are
performed to gain information on the immune status. Flow
cytometric analysis of T cells examined for intracellular cytokine
expression after a short-term in vitro antigen stimulus is wellestablished for research purposes. Despite the advantages of the
approach to qualify samples on a single cell level and by multiple
parameters, the broad use for immune monitoring purposes is
hampered. Furthermore screening of a lot of samples is timeconsuming, requires many manual handling steps, and especially
operator experience in flow cytometric analysis of stimulated
T cells. To overcome these hurdles, we worked out a complete
strategy to rapidly study in multiple samples cytokine and activation
marker profiles in T cells by a semi-automated process.
Wednesday,
22 May
257/B136
Manuela Herber, Michaela Niemöller, Katrin Lange, Silke
Weber-Lohmann, Susanne Höher-Peters, Mario Assenmacher,
Anne Richter
Research&Development, Miltenyi Biotec GmbH, BergischGladbach, Germany
Tuesday,
21 May
Conclusion: Our results may partly contribute to the knowledge
about neonatal T-cell activation. Our data suggest that reduced
Ca2+ signaling upon PHA induced T-cell activation and altered
mitochondrial membrane potential changes with elevated O2production contribute to impaired responsiveness of cord blood
T-cells.
Rapid and Semi-Automated Monitoring of AntigenSpecific T Cell Immunity
Monday,
20 May
Results: Cytoplasmic Ca2+-level during activation was lower in
cord blood CD4+ and CD8+ cells than in those from the adult.
Mitochondrial mass measured in unactivated CD4+ cells was lower
in neonates than in the adults. Similar, but not significant difference
was detected in case of CD8+ cells. Mitochondrial Ca2+-uptake was
comparable in T cells obtained from the neonates and the adults.
However, the same mitochondrial Ca2+-uptake was associated
with slower and increased mitochondrial depolarization in CD4+
cord blood T-cells compared to the CD4+ cells from the adult.
Superoxide generation was higher in cord blood CD4+ and CD8+
T-cells than in those from the adults.
258/B137
Sunday,
19 May
Methods: We used kinetic flow cytometry methods for the
determination of mitochondrial Ca2+ levels, mitochondrial
membrane potential and superoxide generation and to relate
those to that of cytoplasmic Ca2+ levels during short-term aspecific
activation of CD4+ and CD8+ T-cells. We compared cord blood
samples of 12 term neonates to 11 healthy adult volunteers.
Saturday,
18 May
Background: Previous studies support that in term of calcium
signaling elicited by phytohemagglutinin (PHA) in T-cells are
diminished in magnitude in cord blood samples. Now we
investigated other elements of intracellular machinery strongly
linked to lymphocyte calcium response upon activation.
were incubated with K562 cells overnight in the presence of
Brefeldin A (BFA), a golgi apparatus inhibitor. The cells were then
stained with Live/Dead Aqua dye, CD3, CD16, CD8 and CD159α
to separate live and dead cells as well as non-NK cells from the
different NK cell subpopulations. The cells were then fixed and
permeabilized with Cytofix/Cytoperm buffer and stained with
anti-IFNγ to separate those IFNγ secreting cells from non-cytokine
secreting cells. The cells were then analyzed using a Canto flow
cytometer. Of the observed NK cell populations, CD159α+/CD16cells secreted IFNγ upon activation while CD16+/ CD159α- cells
did not secrete cytokine. Upon activation, the population of
CD159α+/CD16+ NK cells shifts in immunophenotype towards a
CD159α+/CD16- NK cell subpopulation. The IFNg ELISPOT assay
combined with intracellular cytokine measurements examine NK
cell functional activity at the single cell level and can be used to
determine the functional capacities of different subsets of NK cells.
Special
Lectures
Gergo Meszaros1,2, Ambrus Kaposi2, Bela Gyarmati3, Barna
Vasarhelyi1,2
1
Department of Laboratory Medicine, Semmelweis University,
Budapest, Hungary, 2Research Laboratory of Pediatrics and
Nephrology, Hungarian Academy of Sciences, Budapest,
Hungary, 3Department of Obstetrics and Gynecology, Uzsoki
Street Hospital, Budapest, Hungary
Congress
Overview
256/B135
In summary, we have set up a fast and convenient procedure
to routinely monitor antigen-specific T cell responses by flow
cytometry.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
187
Congress
Overview
260/B139
Using Image-Based Flow Cytometry to
Simultaneously Measure Granulocyte Phagocytosis
and Oxidative Burst: A Beginner’s Guide
Following Reconstuitution of Cmv Immunity in
Allogeneic Hematopoietic Cell Transplantation
Patients
Special
Lectures
Randall Williams, Adam Venable, Brian McFarlin
Applied Physiology Laboratory, University of North Texas,
Denton, TX, United States
Saturday,
18 May
Introduction: Granulocytes play a key role in innate immunity
and the most common functional measures are phagocytosis
and oxidative burst. Due to experimental limitations, these
measurements are rarely made on individual cells simultaneously.
Advances in image-based flow cytometry have made it possible to
develop assays that can measure multiple cellular functions in a
single assay. The imaging component provides visual confirmation
to support traditional flow cytometry dot plots, making this the ideal
platform for beginners.
Kivin Jacobsen 1, Liselotte Brix 1, Dalin Pan 2, Charlotte
Halgreen1, Theresa Hahn2, Philip McCarthy2, Paul K Wallace2
1
Immudex, Copenhagen, Denmark, 2Department of Flow
and Image Cytometry Department of Pathology Laboratory
Medicine, Roswell Park Cancer Institute, Buffalo, NY, United
States
Methods: The purpose of the present study was to validate
an image-based flow cytometry method for determining both
oxidative burst and phagocytosis of bacterial bioparticles at the
same time. Prior the experiments, all reagents were titered to
determine the lowest dose that resulted in the greatest effect or
most robust staining. All blood samples were collected from
subjects who consented to a testing protocol approved by the UNT
IRB committee. Separate mixtures of heparinized blood (100 μL),
E.coli or S.Aureus bioparticles (Life Technologies; 100 μL), DHE
(Sigma-Aldrich; 2.5 μg/mL) were combined in 1.2 mL library tubes
and incubated for 5, 10, 20, and 40-min in a 37°C water bath. A
duplicate set of tubes was also cultured in the presence of PHA
as a positive/maximal control. An additional tube kept on ice and
used as a negative control. All subsequent processing steps were
completed on ice in the dark to minimize additional activation
of cells. After the water bath incubation, N-ethylmaleimide (10
mM) was added to halt phagocytosis, preventing the uptake of
additional microparticles. Suspensions were labeled with CD66bAPC (eBioscience; DF: 50) and CD45-APCeFluor780 (eBioscience;
DF: 50) for 30-min. RBCs were then lysed and WBCs fixed using
a commercial Fix/Lyse solution (eBioscience). Prior to acquisition,
7AAD (EMD Millipore; DF: 20) was added to stain nuclear DNA.
Prior to collection, the Millipore-Amnis FlowSight was calibrated
each day measurement were made using standard calibration
beads provided by the manufacture. A minimum of 10,000
granulocyte (CD66b+/CD45-) events were acquired using the
FlowSight equipped with blue (60 mW) and red (100 mW) lasers.
Bioparticles, DHE, and 7AAD were detected off the blue laser,
while CD66b and CD45 were detected off the red laser. Side Scatter
(SSC) was detected using a dedicated SSC laser (785 nm; 10 mW).
Samples were compensated and analyzed post-acquisition using
Amnis IDEAS software (v.5.0.385).
Results: Our modified method allowed for rapid and accurate
detection of both phagocytosis and oxidative burst in granulocytes
from whole blood samples. Using this method we were able to
detect changes in granulocyte function that were induced by PHA
stimulation.
Conclusions: While this method appears promising for the
measurement of granulocyte function, more research is needed to
test and validate it in clinical conditions that impair granulocyte
function.
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
259/B138
Cytomegalovirus (CMV) infects and establishes persistent lifelong
infections in 50-85% of adults. Reactivation of the virus is a
frequently occurring complication of immunosuppression following
transplantation and can significantly contribute to morbidity and
mortality in such patients.
Reconstitution of CMV-specific T cell immunity after allogeneic
hematopoietic cell transplantation (alloHCT) has previously been
quantified using CMV tetramers, and shown to be a valuable aid
in predicting patients at risk of developing CMV reactivation. We
have developed an assay for quantifying CMV-specific CD8+ T cells
using CMV-specific Dextramers. Dextramers are MHC multimer
reagents that are used in flow cytometry to detect antigen-specific
T cells in the blood. Dextramers have much higher resolution than
conventional MHC multimers like Tetramers, and thus provide a
more reliable means for identification of antigen-specific T cells.
We here show that the CMV Dextramer assay including 7 alleles
(HLA-A*01,-A*02, A*03, A*24, B*07, B*08 and B*35) has high
specificity and sensitivity and accurately enumerates CMV-specific
T cells in both healthy donors and alloHCT patients, with a lower
detection limit of 0.08 cells/ul. The assay is highly reproducible
with low intra and inter assay variation.
Using the CMV Dextramer assay we were able to quantify
reconstitution of CMV T cell immunity post transplantation at day
30, 100, and 365 in 89 patients. Furthermore, in some recipients
receiving transplants from HLA-mismatched donors we could
measure CMV-specific T cells restricted by donors HLA and not
recipients HLA, indicating that adoptive transfer of CMV-specific T
cells can occur with alloHCT.
This study demonstrates that CMV Dextramers are reliable reagents
that can be used to monitor reconstitution of CMV immunity postalloHCT, and shows that adoptive transfer of anti-CMV immunity
can be quantified.
261/B140
Mass Cytometry for Multiparametric
Immunophenotyping of Seasonal Flu Vaccine
Recipients
Michael Leipold1, Mark Davis2, Holden Maecker1
Human Immune Monitoring Center, Stanford University,
Stanford, CA, United States, 2Stanford University School of
Medicine, Stanford University, Stanford, CA, United States
1
Background: Fluorescence flow cytometry is the current standard
method for immunophenotyping. The advent of mass cytometry
(CyTOF) has allowed a large increase in the number of markers
that can be used simultaneously, thereby reducing the number
of samples required. This has been used to investigate the initial
immune surface phenotype of recipients receiving seasonal flu
vaccination.
Speaker/Author
Index
Methods: We created a cocktail of 23 surface antibodies to look at
all major lineages in a single sample of cryopreserved PBMCs. The
cocktail also allows the further separation of important sublineages,
such as Naive and Memory T cells.
188
ISAC 2013 Program and Abstracts
Multi Color Flow Analysis of Subsets of PBMC for TLR
Ligand Stimulation
Oral Session
Abstracts
For Research Use Only. Not for use in diagnostic procedures.
www.yslbio.com
* Luminex is a registered trademark of Luminex Corporation
Poster Session
Abstracts
Background: Umbilical cord blood (UCB) is a promising alternative
for the treatment of hematological malignancies. The lower immune
reactivity of UCB lymphocytes is a well-known phenomenon;
however, immune tolerance mechanisms are not fully elucidated.
Galectin-1 has strong immunosuppressive properties and plays
a key role in the regulation of immune reactivity. We aimed
to determine the properties of intracellular galectin-1 (Gal-1)producing cells within CD3, CD4, CD8, regulatory T (Treg), and
natural killer (NK) cells in UCB compared to adult peripheral blood
(APB).
Cytokines function in a complex regulatory network. Assaying
multiple cytokines simultaneously by multiplex immunoassays
provides a better understanding fora given physiological
orpathological condition. We have developed a bead-based
multiplex immunoassay system utilizing multiple bead populations
for cytokine profiling. With multiple sizes of beads and multiple
levels of fluorescence intensity in each bead size, the system can
measure up to 24 analytes simultaneously in a single reaction. The
bead populations in the reaction are determined by a conventional
flow cytometer equipped with a 488nm laser.Cell culture
supernatant samples from human peripheral blood mononuclear
cells treated and untreated with phorbol myristate acetate (PMA)
+ ionomycine, lipopolysaccharide (LPS), R848 andLPS+R848
were tested by the human cytokine 18-plex assay panel (Eotaxin/
CCL11, IFNγ, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-8/CXCL8, IL-10/
CSIF, IL-12p70, IL-17A/CTLA-8, IL-22/IL-TIF, IP-10/CXCL10, MIG/
CXCL9, MCP-1/JE/CCL2, MCP-3/MARC/CCL7, RANTES/CCL5, and
TNFα). The majority of the assays can detect less than 1-5 pg/mL
of human, mouse, rat or non-human primate cytokines with assay
quantitation ranges from as low as 2 pg/mL to up to 5000 pg/mL in
cell culture supernatant, serum, plasma, bodily fluid and tissue/cell
lysate samples. The assays correlate well with Luminex* multiplex
immunoassays and require only 15 uL of sample in each reaction.
This multiplex assay system is affordable, easy-to-use and sensitive.
The authors will discuss further technical details of the technology.
Commercial
Tutorials &
Exhibits
Gergely Toldi1, Szonja Kollár1, János Rigó Jr2, Tivadar Tulassay1,
Barna Vásárhelyi3
1
First Dept of Pediatrics, Semmelweis University, Budapest,
Hungary, 2First Dept of Obstetrics, Semmelweis University,
Budapest, Hungary, 3Dept of Laboratory Medicine, Semmelweis
University, Budapest, Hungary
Yong Song, Shun Luo
YSL Bioprocess Development Co., Pasadena, CA, United States
Poster
Session
The Prevalence of Intracellular Galectin-1-Expressing
Lymphocytes in Umbilical Cord Blood in Comparison
with Adult Peripheral Blood
Simultaneously Measuring 18 Human Cytokines on a
Conventional Flow Cytometer
Wednesday,
22 May
263/B142
264/B143
Tuesday,
21 May
Immunology (B142 – B152)
Conclusions: The intracellular expression of Gal-1 may be
downregulated in neonatal lymphocytes due to the already reduced
immune reactivity of UCB. In contrast with previous findings, our
results indicate that the administration of exogenous Gal-1 failed
to decrease the rate of proliferation in T lymphocytes isolated from
either APB or UCB. This suggests that Gal-1-expressing lymphocytes
are unlikely to play a major role in mitigating the immune reactivity
of UCB.
Monday,
20 May
Toll like receptors expressed on immune cells recognize pathogen
associated molecules to initiate and activate innate and adaptive
immune responses towards invading pathogens. Peripheral blood
lymphocytes produce a spectrum of cytokines, when stimulated
with ligands of Toll like receptors. The response and type of
cytokine produced by subsets (T cells, B cells, Monocytes, pDC &
others) of PBMC population is specific based on TLRs expressed
by the subsets. A flow based immune assay is designed to
simultaneously monitor the receptor expression and receptor-ligand
interactions, by analyzing cytokines. Assay designed might be
useful in monitoring modified ligands, and their response on one or
other subset and changes in the cytokines produced. Our protocol
includes stimulation of either PBMC or Whole blood with specific
ligand in the presence of BFA, surface staining with specific markers
to identify subsets and intracellular staining for cytokines. A panel
of multi color antibody conjugates optimized to identify of positive
cells for specific (TLR) ligand stimulation.
Sunday,
19 May
Sriram Chitta1, Hyun-Ku Lee1, Payton Quintel1, Gita Singh1,
Jonathan Rosernberg2, Sujay Singh2
1
R&D, IMGENEX Corporation, San Diego, CA, United States,
2
IMGENEX Corporation, San Diego, CA, United States
Results: The prevalence of intracellular Gal-1-expressing CD3,
CD4, CD8, Treg and NK lymphocytes was lower in UCB than
in APB. However, their capability to produce Gal-1 reaches the
level seen in adults. The prevalence of naive cells was higher,
whereas that of central and effector memory T cells was lower in
UCB compared with APB. Lower Gal-1-producing cell proportion
might be due to the naivety of neonatal lymphocytes, as indicated
by the positive correlation detected between the number of CD3
lymphocytes expressing intracellular Gal-1 and the prevalence of
memory T cells.
Saturday,
18 May
262/B141
Methods: We took peripheral blood samples from 22 healthy
adults and cord blood samples from 19 healthy, term neonates.
Intracellular Gal-1 expression was determined by flow cytometry
(BD FACSAria) using in-house biotinylated anti-human Gal-1
mouse monoclonal antibody in the above subsets. Furthermore,
we assessed the prevalence of naive and memory T cells that play
a role in the regulation of immune reactivity. We also performed
functional analyses to assess the effect of exogenous Gal-1 on the
rate of proliferation of T lymphocytes isolated from APB and UCB.
Special
Lectures
Conclusions: Mass cytometry data is able to increase the number of
parameters that can be simultaneously measured in a single sample
and recapitulate known literature results for populations of widely
varying size.
Congress
Overview
Results: Prior to vaccination, baseline age-related differences
between donors could be identified, such as an overall decrease
in Naive CD4+, Naive CD8+ cells, and B cells with increasing
donor age. Additionally, temporal differences in immune response
could be identified, such as a peak in plasmablasts at Day 7
post-vaccination. Correlations with other measures of vaccine
responsiveness are on-going.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
189
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
265/B144
Technical Advancements Allowing Improved
Selection of True, Viable, Regulatory T Cells
John Tigges1, Vasilis Toxavidis2, Kaitlin Groglio3
1
Beth Israel Deaconess Medical Center, Boston, MA, United
States, 2 Beth Israel Deaconess Medical Center, 3 Flow
Cytometry, BIDMC, Boston, MA, United States
Regulatory T cells (Tregs) are critical to the maintenance of immune
cell homeostasis as evidenced by the catastrophic consequences
of genetic or physical ablation of the Treg population. Specifically,
Treg cells maintain order in the immune system by enforcing a
dominant negative regulation on other immune cells. Broadly
classified into natural or adaptive (induced) Tregs; natural Tregs
are CD4+CD25+ T-cells which develop, and emigrate from the
thymus to perform their key role in immune homeostasis. Adaptive
Tregs are non-regulatory CD4+ T-cells which acquire CD25 (IL-2R
alpha) expression outside of the thymus, and are typically induced
by inflammation and disease processes, such as autoimmunity and
cancer.
Tregs are highly influential in transplant medicine and at the
forefront of graft versus host disease. The ability of a Treg to halt the
immune response, even if only temporarily, is of great importance
from autoimmune responses to full organ rejection. Tregs forestall
the often-fatal graft versus host complication of bone marrow and
stem cell transplants, in which immune cells in the transplant attack
the recipient's cells. Several groups are planning clinical trials to
determine whether Tregs curb the rejection of transplanted organs
such as kidneys. To turn down the immune system, Tregs often latch
onto a protein called TIM-3 on the surface of helper T cells—which
orchestrate immune counterattacks against a pathogen—killing the
helper T cell in the process. In order to ensure the success of such
trials, an enormous amount of research has been done to isolate
and process Tregs. The majority of this isolation is done through
cell sorting. However, methods differ amongst researchers and
recovery of sample is not always optimal.
In this study, we propose the use of Propel Labs Avalon cell sorter
to isolate Tregs. It is our proposal that the jet-in-air system at low
pressure and larger 100um nozzle size will allow for a better
isolation system for Tregs. The FoxP3/GFP T cells will be labeled
with CD4 and CD62L to isolate Tregs. Using this method, we can
isolate the CD4+, GFP+, CD62L+/hi for Tregs that carry “true”
regulatory activity and separate them from the CD4+, GFP+,
CD62L-/lo Tregs which have an activated phenotype and are usually
short lived. However, it is not truly understood if this short life is
due to activity or cell viability.
Propel Labs Avalon system with its ease of use, small footprint,
and gentleness on cells during sorting can be a breakthrough
in bringing T cells closer to a clinical setting. By increasing the
potential number of sorted Tregs and their viability, greater testing
can be done to monitor the halting of an immune response in
transplantation.
266/B145
Identification and Functional Characterization of
Four Subsets of Renal Mononuclear Phagocytes in
Healthy and Diseased Kidney
Xin Wang1,2, Qi Cao2,3, Yiping Wang2,3, David Harris2,3
Flow Cytometry Centre, Westmead Millennium Institute,
Westmead, Australia, 2The University of Sydney, Sydney,
Australia, 3Centre for Transplant and Renal Research,
Westmead Millennium Institute, Westmead, Australia
1
Background: Renal mononuclear phagocytes (rMP), conventionally
comprising of macrophages and dendritic cells (DCs), play a central
role in health and disease of the kidney including homeostatic,
antimicrobial and immune responses. Although many studies over
190
several decades have characterized rMPs, and the classification and
role of rMP, especially their subsets, remain unclear.
Methods: A group of multi-markers (CD45, MHC-II, CD11c,
F4/80, CD103, CD11b) with functional assay (phagocytosis and
antigen presentation) were used to identify four subsets of rMP in
normal kidney. Adriamycin nephrosis (AN) was induced by 10 mg/
kg adriamycin in BALB/c mice. The distribution and phenotypic
plasticity of four subsets of rMP was assessed in different stage of
AN mice by flow cytometry.
Results: Four major subsets of rMP were identified in normal
and AN kidney, including macrophage like subsets: rMP1
F4/80+CD11c- and rMP2 F4/80+CD11c-; DC like subsets: rMP3
CD11c+CD103+ and rMP4 CD11c+CD103- (Table 1). rMP1 and
rMP2 displayed the macrophage like properties, including highly
expressed macrophage markers: CD68, CD204 and CD206,
and higher capability of phagocytosis than rMP3 and rMP4. In
addition, rMP3 and rMP4 were much more effective than subsets
rMP1 and rMP2 in presenting antigen to T cells and inducing T
cells proliferation. Interestingly, rMP1 were present in cortex and
medulla of normal and AN kidney, while rMP2, 3, 4 were mainly
present in cortex. rMP1 and rMP2 cells displayed M1 macrophage
phenotypes, including highly expression of inflammatory cytokines:
IL-6, TNF-a and MCP-1 in AN kidney, but rMP2 also express high
level of anti-inflammatory cytokine: IL-10. In addition, rMP3 and
rMP4 only highly express IL-6 in AN mice.
Table 1. Classification of renal mononuclear phagocytes subsets
according their surface marker and localization
Subset name
Mφ-like
rMP1
rMP2
DC-like
rMP3
rMP4
Markers
MHC-II
CD11c
F4/80 CD103
CD11b
Localization
＋(high)
＋(high)
－
＋
＋
＋
－
－
＋
＋
Cortex and medulla
Cortex
＋(low)
＋(high)
＋
＋
－
－
＋
－
－
＋
Cortex
Cortex
Conclusion: Four subsets of rMP were identified in normal and
diseased kidney. They displayed distinguished properties, including
distribution, phenotypes and in vitro functions, which indicated
they were functional separated rMP subsets, and may have various
roles in different renal diseases.The in vivofunction of four subsets
of rMP will be further examined by depletion and reconstitution
studies.
267/B146
Quality Assurance for Bone Marrow Aspirate
Specimens from Non-Human Primates
Constance Porretta1, Robert Siggins II2, Stephania Cormier3,
Gregory Bagby2
1
Medicine, LSUHSC, New Orleans, LA, United States,
2
Physiology, LSUHSC, New Orleans, LA, United States,
3
Pharmacology, LSUHSC, New Orleans, LA, United States
Background: The Comprehensive Alcohol Research Center
Core Laboratoryperforms FACS analysis for a longitudinal study
examining the effects of daily alcohol administration (13-14g of
alcohol /kg BW/week; 30%w/v in water) on disease progression in
simian immunodeficiency virus (SIV)-infected rhesus macaques.
We utilize an8-color phenotypingpanel for identifying mature
cell populationsin bone marrow aspirates (BMA), and a 13-color
panel for characterizing T, B, and myeloidcells and subpopulations
of peripheral blood leukocytes (PBL). BMA requires considerable
technical skill;specimens can become contaminated with peripheral
blood during the procedure. Therefore, we sought to establish a
flow-cytometry based assay to confirm BMA quality (i.e. lack of PBL
contamination).
Methods: BMA sample qualitywas assessed by measuring three
parameters in PBL and BMA:(1) cell cycle analysis using propidium
ISAC 2013 Program and Abstracts
Results:
Mean ± SD
p value
% G0+G1
75.18 ± 8.01
99.26 ± 0.36
<0.05
MG ratio
1.88 ± 0.95
1.48 ± 0.96
NS
% lin-
22.60 ± 11.40
ND
NA
ND = Non-detectable
18-Colour Flow Cytometry: Immunophenotyping
Cellular Infiltration during Flavivirus Encephalitis in
the Mouse
Oral Session
Abstracts
The aim of our workwas to study the MHC I expression of the
NLRC5-silenced B lymphocytes and the immunological synapse
during conjugation of NLRC5 silenced and overexpressed B cells
with CTL.
Poster Session
Abstracts
We determined that NLRC5 is expressed in JY and Raji cells and
human primary B-lymphocytes and this expression can be induced
with IFNg. According to our flow cytometry results the expression
level of MHC I showed a 2-3 times increase after treating with IFNg,
IL-4 and CpG B for 48 hours on primary B lymphocytes.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Nod-like receptors (NLRs) are intracellular pattern-recognition
receptors with similar domain characteristics to the membrane
bound Toll-like receptors (TLRs). In humans, the NLR family is
currently composed of 22 members, however only a few of them
have been characterized. The importance of the NLR family
members in inflammation and immunity is highlighted by a number
of infectious diseases (such as influenza, malaria, hepatitis),
also of cancers and autoinflammatory diseases that are linked
with polymorphisms, mutations or promoter hypermethylation
of NLRs (such as melanoma, breast cancer or allergy).Major
histocompatibility complex (MHC) class I and class II are crucial
for the function of the human adaptive immune system. An NLR
protein, CIITA (MHC class II transactivator), is a master regulator
of MHC class II gene expression as well as of some of the genes
involved in MHC class II antigen presentation. It has recently
been discovered that another member of the NLR protein family,
NLRC5, transcriptionally activates MHC class I genes, and thus acts
as “CITA” (MHC class I transactivator), a counterpart to CIITA. In
addition to MHC class I genes, NLRC5 can induce the expression
of beta2M, TAP1 and LMP2, essential components of MHC class
I antigen presentation. These findings indicate that NLRC5 and
CIITA are transcriptional regulators that modulate the synchronized
expression of critical components in the MHC class I and MHC
class II pathways, respectively.
Commercial
Tutorials &
Exhibits
Aims and Outcomes: The aim of this investigation was to design,
evaluate, and implement a flow cytometry panel of 15- to
18-colours, to simultaneously immunophenotype the major subsets
of leukocytes. Whilst our own investigations are conducted in a
model of viral encephalitis, our secondary aim was to develop
a panel that could be used in any tissue, of any disease model,
enabling widespread use of this technology. Because the detection
of 18-fluorescent parameters is only possible on certain state-of-theart cytometers, we sought to include a 15-colour variety that can
be used on most cytometers fitted with an octagon photo-multiplier
(PMT) array accompanying the violet laser.
Adrienn Veres1, László Mátyus2, Szilvia Benko3, Attila Jenei2
Dept. of Biophysics and Cell Biology, University of Debrecen,
Medical and Health Science Centre, Debrecen, Hungary,
2
Department of Biophysics and Cell Biology, University of
Debrecen, Debrecen, Hungary, 3Department of Physiology,
University of Debrecen, Debrecen, Hungary
1
Poster
Session
Background: Viral encephalitis is a major cause of morbidity and
mortality worldwide. The pathogenesis of flaviviral encephalitis
is a complex process, where the immune response elicited by
the virus contributes substantially to pathological damage in
the brain. As this process is still incompletely understood, there
is a critical need for a detailed analysis and characterization of
cellular infiltration in the infected brain, to elucidate the critical
mediators of immunopathology and viral clearance. Investigations
such as this have previously been impeded by limited numbers
of fluorescent parameters in flow cytometry, restricting detailed
profiling of immune subsets. Recently the technology has been
developed to allow the detection of up to 18-fluorescent parameters
simultaneously, which has enabled detailed immunophenotyping of
the immune response.
The Regulatory Role of NLRC5 in MHC I Expression
in B Lymphocytes
Wednesday,
22 May
Thomas Ashhurst1, Caryn van Vreden1, Mahmoud Karimi
Azardaryany1, Kelly Lundsten2, Steven Allen3, Suat Dervish3,
Frank Kao3, Adrian Smith3,4, Nicholas King1,4
1
Pathology, University of Sydney, Sydney, Australia, 2BioLegend,
San Diego, CA, United States, 3Cytometry and Imaging Facility,
Centenary Institute, Sydney, Australia, 4Advanced Cytometry
Facility, Centenary Institute/Bosch Institute/University of
Sydney, Sydney, Australia
269/B148
Tuesday,
21 May
268/B147
Conclusion: We have developed an immunophenotyping panel that
allows simultaneous investigation of all major subsets of leukocytes
that are relevant to immune infiltration during viral encephalitis.
This will provide an excellent research tool for the future.
Monday,
20 May
Supported by AA020312 (RS), AA009803 (CP, RS, SAC, GB) and
ES015050 and ES013648 and AI090059 (SC).
Sunday,
19 May
Conclusions: We found the percentage of resting (G0+G1) cells to
be a strong indicator of BMA quality. Therefore, for our longitudinal
study, we have established a cutoff value for BMA of 90% G0+G1,
using criteria of 2 standard deviations (SD) from the mean, to ensure
data integrity. BMA specimens with >90% of cells in G0+G1are
considered compromised with peripheral blood, and are not
included in our data sets until they can be re-sampled.In addition,
we noted an inverse relationship between the percentages of linand G0+G1 in BMA, indicating the presence of more immature
cells in specimens with a higher percentage of actively cycling
cells. In conclusion, our data demonstrate that cell cycle analysis is
a rapid, easy, and reliable methodto verify BMA sample quality and
maintain BMA data integrity.
Results: The panel allowed for simultaneous identification of B
cells (CD19, B220), CD4 and CD8 T cells (CD3, CD4, CD8), NK
cells (NK1.1), NKT cells (NK1.1, CD3), neutrophils and other
granulocytes (Ly6G, Ly6C, CD11b, SSc), monocytes, microglia,
and macrophages (CD45, CD11b, CD11c, Ly6C, F4/80, MHCII),
dendritic cells subsets (CD11c, MHCII, CD11b, CD8, CD4,
CD103), and plasmacytoid dendritic cells (CD11c, MHCII, B220).
Here we report our findings for the optimization of filters, bandpass, and laser power settings, and the quantification of spreading
error. Additionally we report the incorporation of the new Brilliant
Violet reagents into our panel, allowing for excellent population
resolution.
Saturday,
18 May
PBL
Mean ± SD
Method: Brains, blood, lymph nodes, and spleens were isolated
from C57BL/6 mice on day-7, following intranasal inoculation
with West Nile virus (WNV). Tissue was processed and stained
with a mixture of 12-17 antibodies, and a fluorescent viability dye.
Cells were analyzed on a 5-laser Becton-Dickinson (BD) LSR-IITM,
5-laser BD LSR-II FortessaTM, or a 10-laser BD LSR-IITM special order
research product (SORP) and the data analyzed using FlowJo v9
and v10 software.
Special
Lectures
BMA
Congress
Overview
iodide (PI) staining to determinepercentage of resting cells (G0+G1); (2)
ratio of mononuclear cells (CD66-SS lo) to granulocytes (CD66+SS
hi) (MG ratio); and (3) percentage lineage negative (lin-)cells.
191
Congress
Overview
Our first results showa correlation between NLRC5 and MHC I
expression levels on B lymphocytes.
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
Special
Lectures
270/B149
Modulating in Vivo T-Cell Activation: 15 Color
Immunophenotyping, Cytokine Analysis, and Cellular
Redistribution
Kelly Lundsten, Miguel Tam, Jeanette Ampudia, Naomi
Urbina, John Ransom, Gene Lay
BioLegend, San Diego, CA, United States
Understanding the mechanisms for modulating t-cell activation
and inhibiting activated dendritic cell migration is important in
our attempt to control key aspects of T cell regulation, from how
to most effectively combat immune response dysregulation to
the suppression of transplant rejection. The efficacy of models
for T cell activation and suppression can vary in vivo vs. in vitro.
In this application, modulation of T cell-specific activation was
achieved in vivo in a Balb/c murine model through the injection
of 50ug anti-CD3 low endotoxin azide free (LEAF) antibody with
or without 100ug LEAF purified anti-PD-1H/VISTA co-injected.
Using a 15 color flow cytometric assay utilizing all 7 Brilliant
Violet fluorophores, the kinetics of activation were monitored
through multiple cell surface markers, early to late, and changes
in cellular distribution from spleen to draining lymph nodes
were compared. Dynamics of cytokine production were also
monitored using ELISA assays for IL-4, IL-6, IL-10, TNFa and IFNg.
To complement previous ELISA and mRNA expression profiling
on CD3 in vivo stimulation, we show the kinetics of activation
and cellular redistribution. We also demonstrate that anti-PD-1H
antibody administration successfully modulates CD3-induced T cell
activation.
271/B150
A Longitudinal Flow Cytometry–Based Study on
Phenotypic Changes of Cryopreserved Murine T Cells
Using the BD FACSVerse™ System
Yibing Wang1, Shang Cai2, Weiguo Feng2, Rinku Jana1
BD Biosciences, San Jose, CA, United States, 2Cancer Center
and Stem Cell Biology, Stanford University, School of Medicine,
Stanford, CA, United States
1
Cryopreservation is commonly used to preserve fresh lymphocytes.
However, it is not clear whether the phenotype-distinct populations
can be cryopreserved equally effectively, nor how cryopreservation
contributes to variation in samples analyzed by flow cytometry.
Elucidation of the question has been limited by the issue of
instrument-related variations over time. Laboratories have been
attempting to establish standardization methods to eliminate such
variations.
The BD FACSVerse™ system is a high-performance flow cytometer
designed to address the need for instrument standardization,
specifically when conducting longitudinal and cross-site studies.
The flow cytometer provides an easy-to-use setup system designed
to maintain daily, consistent instrument settings for different
applications, including fluorescence compensation. To take
advantage of this new technical capability, we conducted a study
to compare cryopreserved murine lymphocytes to freshly prepared
ones, looking at the variability between the data sets. Freshly
prepared murine cells were initially phenotyped and then frozen
down. Afterwards, the cryopreserved cells were thawed on various
days and examined using the same phenotyping method.
We found that cryopreservation does have an impact on
different populations of lymphocytes in a longitudinal study,
which demonstrated the utility of the BD FACSVerse system’s
built-in standardization in detecting phenotypic changes of cell
populations.
192
272/B151
Defining CD4+ T-Cell Subsets Using Probability State
Modeling
Margaret Inokuma 1, Joe Trotter 1, Elizabeth Hill 2, Ben
Hunsberger2, Mark Munson2, Don Herbert2, Chris Bray2, Smita
Ghanekar1, Vernon Maino1, C, Bruce Bagwell2
1
BD Biosciences, San Jose, CA, United States, 2Verity Software
House, Topsham, ME, United States
In CD4+ T cells, the development of long-term memory is key
to effective protection upon subsequent antigenic encounters.
Formation of memory is complicated by the variety of polarized T
helper (Th) subsets, which can evolve and display plasticity. Naïve
CD4+ T cells have the ability to differentiate to four major distinct
fates. These four populations are Th1, Th2, Th17, and iTregs, each
having unique functions. In this study, a model was generated using
Probability State Modeling to map effector/memory populations,
using CD28, CD45RA, and CCR7. In contrast to CD8+ T cells,
CD4+ T cells have differences in the types of memory subsets as
defined with these markers. Using the transcription markers Tbet
(Th1), GATA3 (Th2), FoxP3 (Treg), and RORγt (Th17), the four
major Th subsets were identified and the memory populations of
each were characterized using the averaged model based on data
from 20 healthy donors. Additionally, changes in memory subset
populations and CD4+ T-cell subsets were monitored over time in
healthy donors. Findings from these models can be used to better
understand the role of CD4+ T-cell subsets in adaptive immune
responses and can be used to identify changes in disease states.
273/B152
Immunophenotyping in Clinical Translational Studies
Using Brilliant Violet Dye Conjugates
David Roumanes1, Christina Baker2, Shelley Secor-Socha2,
Yilin Qi2, Ashley Dunham2, Tim Mosmann2, Sally Quataert2
1
Center for Vaccine Biology and Immunology, University
or Rochester Medical Center, Rochester, NY, United States,
2
Center for Vaccine Biology and Immunology, University of
Rochester Medical Center, Rochester, NY, United States
Characterization of the immunophenotype of cell populations is
an important tool when assessing disease and immune status in
clinical translational research studies. Large-scale flow cytometry
offers an efficient method for interrogating single cells and
identifying a multitude of cell populations within a specimen. The
ability to stain with up to 16 fluorescently labeled markers poses a
challenge when designing phenotyping panels due to the limited
number of fluorescent dyes available. Our efforts to optimize
panels to efficiently detect the populations of interest have led us to
investigate many of the new brilliant violet dyes available. Utilizing
commercially available brilliant violet dye antibody conjugates,
we developed new immunophenotyping panels targeting immune
cell subsets and markers of their functional or activation status
within populations such as DC, NK, B, and T cells. Our approach
took advantage of the brightness and improved signal to noise
ratio for brilliant violet dyes compared with other options for the
violet laser; therefore resulting in improved signal for markers
found at low density as well as identification of small subsets.
The panels were applied to a vaccine response study comparing
young and old subjects. Our results show that the brilliant violet
dyes allow improved and consistent detection of challenging cell
differentiation markers over previous reagents. Importantly, the
use of brilliant violet conjugated antibodies provide better data for
standardization and normalization of results necessary when using
immunophenotyping panels across several clinical translational
studies.
ISAC 2013 Program and Abstracts
274/B153
Zhenjun Diwu1, Qin Zhao2, Yibo Wu2, Jinfang Liao1
AAT Bioquest, Inc., Sunnyvale, CA, United States, 2AAT
Bioquest, Inc., Sunnyvale, CA, United States
1
Background: Flow cytometry combined with fluorescence staining
is a powerful tool to analyze heterogeneous cell populations.
Among all the existing fluorescent dyes CFSE is the preferred
cell proliferation indicator. However, there are a few challenges
associated with the use of CFSE for monitoring cell proliferation.
1). CFSE indiscriminately reacts with all amino groups, thus often
causes cell cytotoxicity; 2). The CFSE fluorescence intensity of
the 2nd generation cells is decreased more than 10 fold from the
1st generation. You would have to wait for another generation to
start the cell proliferation analysis. 3). You would have to remove
medium for cell analysis with a flow cytometer since CFSE reacts
with medium components. 4). CFSE is not compatible with the
GFP-transfected cells or for the applications where a FITC-labeled
antibody is used due to their severe spectral overlap.
Monday,
20 May
Methods: Hela and Jurkat cells were plated in 96-well black wall/
clear bottom costar plate at 37 oC, 5% CO2 incubator for overnight.
The cells were dye-loaded at 37 oC for 15 to 30 min by adding
CytoTell Green, CytoTell Blue, CytoTell Red or CFSE in 100 μL
of 1X HBSS with 20 mM HEPES into each well (0.5-2 μM), and
washed twice with buffer after the dye loading. The cells were
analyzed with 1X71 Olympus fluorescence microscope (Olympus),
FlexStation fluorescence microplate reader (Molecular Devices) or
BD FACSCalibur flow cytometer (BD Biosciences).
Tuesday,
21 May
Wednesday,
22 May
Results: We report the functional analysis of cell proliferation using
a new panel of multicolor CytoTell™ dyes. CytoTell dyes are well
excited at major laser lines like 405, 488 or 633nm with multicolor
emissions. CytoTell Green has distinct advantages over CFSE. 1).
CytoTell Green has minimal cytotoxicity since it does not react
with critical intracellular proteins. 2). CytoTell Green exhibits much
faster response since there is no fluorescence intensity gap between
1st and 2nd generation of cells. As cells divide, CytoTell Green is
distributed equally between daughter cells that can be measured
as successive halving of the fluorescence intensity of the dye. 3).
CytoTell Green does not react with medium components, thus there
is no need to remove medium. 4). Nine generations of cell division
were visualized with CytoTell Green. 5). CytoTell Green is stable in
medium for a few weeks while CFSE readily hydrolyzes in medium
within 1 hour. 6). CytoTell Green has a peak excitation of 520
nm and can be excited by the blue (488 nm) laser line, making it
compatible with FITC filter set.
Poster
Session
Commercial
Tutorials &
Exhibits
CytoTell Blue and CytoTell Red work similarly as CytoTell Green
does. CytoTell Blue can be well excited with the violet laser line
(405 nm). It has a peak emission of 450 nm and can be detected
with a 450/20 band pass filter. CytoTell Red can be well excited
with the He-Ne red laser line (633 nm). It has a peak emission
of 660 nm and can be detected with a 660/20 band pass filter.
CytoTell Blue and CytoTell Red are compatible with applications
that utilize GFP or FITC antibodies for multicolor cell analysis.
Oral Session
Abstracts
These results highlight the importance of inducing HIVspecific antibodies at mucosal portal of HIV-1 entry to prevent
dissemination after sexual transmission, the major mode of HIV-1
transmission worldwide. Immature DC that are present in mucosal
surfaces are among the cells initially targeted by HIV-1. Various
types of DC are infected by HIV-1 in vivo and in vitro. Although
circulating and immature DC poorly support HIV-1 replication,
they are capable of efficiently transferring infectious viral particles
to permissive CD4 T cells during cell-to-cell contact. Thus the
viral particles transmitted efficiently across a “virological synapse”
represent a predominant mode of transfer and viral replication.
Neutralizing antibodies are known to inhibit viral infection and
replication in human CD4 T lymphocytes as well as in other
permissive cell types, such as macrophages and monocytes-derived
DC. The significance of this study is the demonstration that HIV1-specific antibodies are also capable of inhibiting early HIV-1
transfer from DC to permissive CD4 T cells.
Multiplexing Analysis of Cell Proliferation and
Cellular Functions Using a New Multicolor Panel of
Fluorescent Cell Proliferation Dyes
Sunday,
19 May
The ability of HIV-1-specific antibodies to inhibit the transfer of
HIV-1 from immature dendritic cells (DC) to autologous CD4 T
lymphocytes was investigated using a flow cytometric method
for intracellular detection of HIV-1 p24 core antigen. The assay
allowed for the detection of HIV infection in human primary CD4
T lymphocytes in the presence of HIV-loaded immature DC, and
a concomitant phenotypic characterization of the infected cells
according to their specific cell surface antigens. We analyzed the
kinetics of fusion, replication, and the ability of HIV-1-specific
antibodies to inhibit early HIV-1 transfer from immature DC to
autologous CD4 T lymphocytes. We found that HIV replication
was stimulated in immature DC when co-cultured with CD4 T
lymphocytes under condition of single cycle of infection. This
increased HIV replication was associated to the decrease of
expression level of SAMHD1, an HIV-1 restriction factor in DC.
Monoclonal neutralizing antibodies prevented early HIV-1 transfer
from immature DC to CD4 T lymphocytes, whereas monoclonal
non-neutralizing antibodies did not. Interestingly, neutralizing
antibodies as well as some non-neutralizing antibodies also
significantly decreased HIV-1 replication in DC, even when added
2 hours after addition of HIV-1. This decreased HIV replication
was correlated with DC maturation and further associated with the
capacity of HIV-1-specific antibodies to bind to Fcgamma receptors
on DC. We propose that triggering of DC maturation by antibody
binding to Fcgamma Receptors participates to HIV-1 inhibition in
these cells.
275/B154
Saturday,
18 May
Vincent Holl1, Bin Su2, Alexandre Lederle2, Maryse Peressin2,
Valérie Glutz3, Mélanie Lambotin2, Christiane Moog2
1
Lab Science Dept., Covance, Meyrin, Geneva, Switzerland,
2
Inserm U748, Strasbourg, France, 3Hematology, Covance,
Meyrin, Geneva, Switzerland
Live Cell Imaging/Tracking
(B154 – B156)
Special
Lectures
A Flow Cytometric Method for Intracellular
Detection of HIV-1 P24 Core Antigen Used to
Investigate thePrevention of HIV-1 Transfer from
Dendritic Cells to CD4 T Cells by Neutralizing
Antibodies
Congress
Overview
Infectious Diseases (B153)
Poster Session
Abstracts
ISAC 2013 Program and Abstracts
Speaker/Author
Index
Conclusions: Cytotell dyes have minimal cytotoxicity and are well
retained in cells. They exhibit much faster response, and there is
no fluorescence intensity gap between 1st and 2nd generation of
cells. The combination of CytoTell Blue, Green and Red dyes can be
readily used for the multicolor applications with either GFP cells or
FITC-labeled antibodies.
193
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
276/B155
Determining Rna Expression at the Single Cell Level
in Live Cells Using Flow Cytometry
Don Weldon1, Alex Ko2, Yuko Williams2, Laura White2, Luke
Armstrong2, Victor Koong2
1
R&D, EMD Millipore, Temecula, CA, United States, 2EMD
Millipore, Temecula, CA, United States
The ability to measure the level of an RNA target in a single cell
provides researchers the ability to understand how individual cells
may differ from the rest of the population. This is of increasing
importance when studying the effects of stimulations or treatments
on cells in culture as individual cells may respond differently to the
experiment. Current methods to analyze cellular RNA require lysis
and amplification of the entire population to understand the effect
of the treatment as in the case of RT-PCR. The data is then a mere
average of the expression within the population and small numbers
of responding cells could easily be missed.
Here we present a novel live cell RNA detection technology
which allows for single cell RNA detection without the use of
any transfection methods. It is comprised of a gold nano-particle
core conjugated to duplexed oligonucleotides on the surface. A
fluorophore is linked to one of the strands and is quenched by
the gold core until it is displaced by its target RNA in the cell and
leaves the proximity of the gold allowing it to fluoresce. These RNA
detection probes are non-toxic, do not alter gene expression, and
utilize the cells own endocytosis mechanism.
Flow Cytometry can be used to detect the fluorescence which
is relative to the amount of target RNA present in each cell. This
provides a more complete understanding of the RNA expression
for each cell in the experiment as opposed to the average trends
seen in RT-PCR data. Furthermore, since the cells are unharmed
and unchanged after interrogation with the probes researchers
now have the ability to sort cells based on the expression level of
their target of interest and use the sorted products for downstream
experimentation.
277/B156
Simultaneous Tracking of Cell Type, Viability,
Proliferation and Expression of Intracellular Proteins
In Co-cultures of Leukemia and Hs-5 Stroma Cells
Using Multiparameter Flow Cytometry
Katarzyna Piwocka, Paulina Podszywalow-Bartnicka, Marta
Brewinska-Olchowik, Lukasz Bugajski, Monika Kusio-Kobialka
Laboratory of Cytometry, Nencki Institute of Experimental
Biology, Warsaw, Poland
Introduction: It is known that leukemia cells communicate with
the stroma and vice versa by the mean of secreted soluble proteins.
Previously we have discovered a novel, pro-survival pathway in
chronic myeloid leukemia cells, which is based on activation of
the PERK-eIF2alpha phosphorylation pathway (1). This signaling
pathway leads to the rearrangement of the protein translation
process what strongly influences the composition of expressed and
secreted proteins. We aim to study different parameters of the cellcell interactions detected in vivo in the co-culture conditions.
Methods: To this end we developed protocols to distinguish
different types of co-cultured cells and simultaneously analyze their
survival and proliferation rate together with detection of the level
of intracellular proteins using multicolor flow cytometry. We used
CellProliferation tracking compounds and Fixable Viability Dye
reagent to monitor cell division and apoptosis in combination with
dyes labeling each cell type. For a long term tracking of epithelial
cells we used a CellTracker Blue CMAC dye, whereas leukemia
K562 cells stably expressed proteins tagged with the green GFP
protein. Intracellular proteins playing a role in the prosurvival
194
pathways were detected by the antibodies previously stained with
the fluorochrome using the Zenon Labeling technology.
Results: We were able to show that each type of cells, which
were cultured together, responded in a different way to the
imatinib treatment and modification of the secretome. Moreover,
cells stained with both, CellTracker and Fixable Viability Dye,
unlike 7-AAD and propidium iodide, can be washed, fixed,
permabilized, and stained for intracellular antigens without any
loss of staining intensity of the dead cells. We found that the Zenon
labeling technology can be used together with the tracking dyes to
specifically stain intracellular proteins.
Conclusions: This novel combination of cell tracking and the
intracellular protein staining method allows for in vivo studies
of cells properties as well as cell signaling upon the co-culture
conditions. Thus it can produce a valuable information about the
influence of cell-cell interactions for biology of cancer and stroma
cells. Importantly, this methodology can be further improved and
modified dependently on needs, as the number and type of tracking
dyes available for different flow cytometry applications constantly
increases.
1. Kusio-Kobialka et al. 2012, Cell Cycle Nov 1; 11(21):4069-78.
doi: 10.4161/cc.22387.
This work was supported by grants from National Science Centre
(2011/01/B/NZ3/02145 to K.P.) and Ministry of Science and Higher
Education (IP2011 043071 to P.P-B.). K. Piwocka is an ISAC Scholar
Fellow 2012-2016
Microbiology and Aquatic Sciences
(B157 – B159)
278/B157
Scanning Flow Cytometry for Static and Dynamic
Characterization of E. coli cells
Anastasiya Konokhova1,2, Maxim Yurkin1,2, Valeri Maltsev1,2
Laboratory of Cytometry and Biokinetics, Institute of
Chemical Kinetics and Combustion SB RAS, Novosibirsk,
Russia, 2Department of Physics, Novosibirsk State University,
Novosibirsk, Russia
1
Background: Light scattering by a particle is determined by its
overall morphology, including shape and internal distribution
of the refractive index. Therefore, light scattering is a powerful
physical method for identification and characterization of bacteria.
In addition to identifying or distinguishing microorganisms by its
morphology, light-scattering can also potentially provide real-time
monitoring of bacterial growth in order to study cell cycle kinetics
or analyze growth rate for antibiotic sensitivity testing. This abstract
describes a method for high-precision characterization of individual
E. coli cells from light scattering measured with Scanning flow
cytometer and its implementation for static and dynamical analysis
of E. coli cells.
Methods: We used Scanning Flow Cytometer (SFC) – a technique
capable of measuring angle-resolved intensity light scattering
patterns (LSPs) of individual particles in flow (Novosibirsk,
Russia, http://cyto.kinetics.nsc.ru/).Characterization of particles
morphology from measured LSPs requires the solution of the
inverse light-scattering (ILS) problem. This solution is based on
fitting an experimental LSP by theoretical ones, calculated from the
modeling E. coli cell as a cylinder capped with hemispheres of the
same radius. To accelerate the fit we used a precalculated database
of 300 000 LSPs in a wide range of model parameters (length,
diameter, refractive index and orientation angle of cell in the
flow) and performed the nearest-neighbor interpolation on it. This
allowed us to determine length and diameter of individual bacteria
including errors of these estimates.
ISAC 2013 Program and Abstracts
Conclusions: The presented method allows characterization of a
population of rod-shaped bacteria by their length and diameter
distributions. It can be used for determination of dynamic
characteristics of bacteria, monitoring morphological changes of
bacteria cells over time. In particular, it can be used to study cell
growth or cell cycle kinetics. Another advantage or this method is
sensitive identification of bacteria cells in flow without fluorescent
staining. It is important to note, that the SFC-based method is not
specific to E. coli and can be directly applied to any rod-shaped
bacteria. The only additional effort may be needed for extension of
the database to larger or smaller bacteria sizes.
280/B159
Drags of the upper Chesapeake by were made at three benthic
levels of the shore and outer region of Swan Point located
within the upper Chesapeake bay. Size spectra of plankton
was compiled for species ranging from 60nm to 300um that
included dinoflagellates , diatoms and larger particles. The diatom
skeletonemacostatum is closely associated with oyster harvest.
We compiled a 3 dimentionalmap of the waters off Swan Point
that included a distribution of species considered important to this
local ecosystem. Conditions vary with seasons: less bioproduction
in winter and more eutrfication from over enriched water in late
summer. This causes low oxygen and loss of habitat.
Wednesday,
22 May
Nano-particles present unique challenges when analyzing and
sorting by flow cytometers. A detection limit of 60nm was achieved
with a specialized flow cytometer manufactured by PartecInc. by
utilizing 90 degree light scatter.
The Partec Space provides very limited sorting capability so we
developed an electro-optical system within a test bench that
included a quartz flow cell, specialized beam shaping optics and
an efficient scatter detection module with low noise PMT and
preamps. This subsystem was transferred to a FACVantage cell
sorter for sorting of nanometer sized phytoplankton.
Poster
Session
Commercial
Tutorials &
Exhibits
An assortment plankton were characterized by combining image
and pulse cytometry. Alterations of the FACSVantage optics and
detection channel allowed measurement and sorting of plankton
within the nanometer range. Large plankton were sorted with a
macrosort 400nm nozzle and flow cell.
Oral Session
Abstracts
Methods: Plankton species; Chlorella, Phormidium inundatum,
Phormidium persicinum, Cryptomonas, Rhodosorus,
Synechococcus, Skeletonema, Fremyella, were acquired from
the UTEX: The Culture of Algae and grown in photobioreactors
and cultured in specialized salt and fresh water media. Plankton
species Prochlorococcus marinus and Emiliana huxlei were grown
in sterile 2 L containers supplied with 0.2 µm filtered air in salt
water with fertilizer. Instant Ocean, ½cup per gallon of deionized
water, was added to the culture with Microalgae Grow Mass
Pack with Silicate. Cells were harvested by gentle centrifugation,
roughly 300 x g for 5-10 minutes. The supernatant was decanted/
aspirated and the pellet resuspended in a sterile saline solution to
achieve 1x106 cells/mL. Plankton were then stained with SYTOX
Green(Invitrogen, S7020), at a maximum concentration of 5 µM
for 10 minutes after vortexing. Samples were filtered with a 70 µM
Partec filter and kept on ice before flow cytometric analysis.
Microscopic plankton form the foundation of the food web in
aquatic ecosystem such as the Chesapeake bay. Data describing
the abundance and distribution of these organisms indicates a
direct response to specific forms of pollution. Phytoplankton are
the primary producers and vary in size while zooplankton are the
primary consumers and are generally larger. The goal of this study
is to index size, shape, and morphology from images obtained with
stage microscopy and AmnisImageStream then correlate this data
with scatter and fluorescence profiles generated by 2 specialized
cell sorters.
Tuesday,
21 May
Background: Phytoplankton conversion of light on the upper
limits of the ocean consists of half of the photosynthesis on the
Earth. The population densities indicate the health of not only the
phytoplankton, but the entire aquatic ecosystem. The isolation
and sorting of aquatic samples using flow cytometry allows for
quick and effective population analysis. The forward scatter on the
MoFlo Astrios allows for differentiation of small and large particles
from 0.2 to 30 µm on FSC. This design provides researchers greater
flexibility to isolate and sort specific phytoplankton of different sizes
while utilizing the 7 laser, 42 parameter MoFlo Astrios.
Gordon Wiegand
Estuary Biophotonics, Baltimoe, MD, United States
Monday,
20 May
Carley Ross, Alan Dean, Robin Morris
Research and Development for Cell Sorters, Beckman Coulter
Life Sciences Division, Fort Collins, CO, United States
Development of a Plankton Cell Sorter Utilized with
the Amnis ImageStream
Sunday,
19 May
Moflo Astrios™ Forward Scatter: Cell Sorting of
Nano and Large Phytoplankton Simultaneously with
High Purity
Conclusions: The forward scatter and optical collection design
of the MoFlo Astrios provide flexibility to detect large and small
populations and sort them with high purity.
Saturday,
18 May
279/B158
Special
Lectures
Results: Plankton populations were distinguishable using
fluorescence and scatter patterns on both log and linear scales
simultaneously. With the Astrios optical flexibility, the plankton
fluorescence spectra were optimized for signal to noise. Isolation
of the P. marinus, Synechococcus and other plankton species using
cell sorting achieved 99% purity for all populations.
Congress
Overview
Results: The developed method was tested by two strains of E. coli,
showing 135 and 15 nm median precision in determination of
length and diameter of single cells, respectively, which is very good
for optical methods. Obtained length and diameter distributions
showed a good agreement with microscopic measurements of
the same samples. The method was also applied for monitoring
morphological changes of E. coli cells during the exponentialgrowth phase. According to obtained distributions the decrease in
cell volume was observed as bacteria cells approached stationary
phase of growth.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
Poster Session
Abstracts
Flow Cytometry: The MoFlo Astrios was configured with 7
lasers and setup with a 100 µm tip to accommodate the larger
phytoplankton (Cryptomonas). Cells were selected on their “live”
status by being highly fluorescent in the red channels (chlorophyll)
and low in the green channels (Sytox -). For small particle analysis,
1 µm beads were run simultaneously with the P. marinus and
Synechococcus and analyzed on FSC-Log parameters. Populations
were sorted based on fluorescence and size as a 6-way sort into 5
mL tubes. The plankton were sorted at 25K eps to collect at least
100,000 events per each population using sort mode Purify 1-2.
195
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
Microelectro-Mechanical Systems
(MEMS) and Microfluidics
(B160 – B163)
281/B160
Human Organ-on-a-Chip BioMEMS Devices for More
Rapid Testing of New Multimodal in Vivo Diagnostic
Imaging and Therapeutic Strategies
James Leary1, Pierre-Alexandre Vidi2, Christy Cooper2, Sophie
Lelievre2
1
Basic Medical Sciences and Biomedical Engineering, Purdue
University, West Lafayette, IN, United States, 2Basic Medical
Sciences, Purdue University, West Lafayette, IN, United States
Background: BioMEMS human “organs-on-a-chip” can be used
to create artificial model human organ systems for developing
new diagnostic and therapeutic strategies. They represent a
promising new strategy for rapid testing of new diagnostic invivo imaging and therapeutic approaches without the need for
involving risks to human subjects. This approach has received
strong initial endorsements from both the FDA and NIH as a way
of speeding up new clinical therapies and providing ways to
testing of drugs, not testable in animals, targeted to human-specific
biochemical pathways in cells. We are developing multicomponent,
superparamagnetic and fluorescent nanoparticles as X-ray and
MRI contrast agents for noninvasive multimodal imaging and for
antibody- or peptide-targeted drug delivery of these nanomedical
systems to tumor and precancerous cells inside these artificial organ
BioMEMS devices.
Methods: Superparamagnetic iron oxide (SPIO) nanoparticles (NPs)
was done using the
method of Yu et al4. Iron oxide hydroxide (FeO(OH)), oleic acid,
and 1-octadecene. The SPIO NPs were physically loaded into
hydrophobically modified glycol chitosan (HGC) NPs by
sonication. Cy5.5 was added to the multicomponent nanoparticle
complex to produce near infrared fluorescence (NIRF) to produce
far red fluorescent NPs for maximal transmission through tissue.
Human mammary epithelial cells (HMT-3522 S1) were cultured at
37°C in 5% CO2 in H14 medium consisting of DMEM/F12 (Sigma,
St Louis, MO). Laminin 111 (BD Biosciences, Discovery Labware)
is used at a final concentration of 133 μg/ml which has been shown
to induce basoapical polarity in S1 cells important to the exposure
of previously hidden surface receptors during early neoplastic
transformation. Confocal microscopy images were obtained to
confirm intracellular uptake of the SPIO-loaded GC NPs at 60×
magnification using an Olympus IX70 confocal microscope (Center
Valley, PA).
Results: Magnetic fields were used to move the nanoparticles
“upstream” to find their target cells in an organs-on-a-chip model
of human ductal breast cancer. Unbound nanoparticles were then
removed by reversing the magnetic field to give a greatly enhanced
image of tumor cells within these artificial organ structures.
Nanomedical systems targeted to tumor cells were imaged by
multicolor confocal microscopy inside the artificial organs to test
both nanoparticle targeting and therapeutic responses, including the
differential viability of normal and tumor cells during treatments,
were assessed by multicolor confocal microscopy.
Conclusions: Using branched PDMS microchannels and 3D tissue
engineering of normal and malignant human breast cancer cells
inside those BioMEMS channels, we can mimic the early stages of
human ductal breast cancer with the goal to improve the sensitivity
and resolution of mammography and MRI of very small tumors and
test new strategies for treatments.
196
282/B161
Chip Based Impedance Flow Cytometer with
Integrated Acoustophoretic Sample Preconditioning
Carl Grenvall 1, Christian Antfolk 1, Christer Zoffmann
Bisgaard2, Thomas Laurell1
1
Department of Measurement Technology and Industrial
Electrical Engineering, Lund University, Lund, Sweden, 2FOSS
Analytical A/S, Hillerød, Denmark
Background: Here we present, for the first time, a microchip
impedance flow cytometer (MIC) with integrated acoustophoretic
sample preconditioning and prefocusing. By acoustically aligning
dense particles (cells) in a precise way, while removing cell-sized
less dense particles (lipids) prior to MIC-measurements, it is possible
to perform cytometry on raw milk without solvents or additional
instrumentation. Our group has previously reported chip integrated
2-dimensional acoustic pre-alignment of cells/particles prior to
impedance readout [1], avoiding the need for more complex sheet
flow pre-alignment and/or 3-D configured electrode configurations
[2-5]. We have also reported chip based acoustophoretic removal
of cell-sized lipid globules. This paper now demonstrates the first
fully integrated system that offers sample preconditioning by lipid
removal, cell pre-alignment and impedance readout. This platform
aims at opening a path to easier and less time consuming analysis
of raw milk, blood, and other complex bio-suspensions [6-8].
Method A microfluidic glass chip was fabricated by wet
etching, Fig. 1. The chip is divided into three parts; 1) an initial
preconditioning zone, which removes lipid particles in an acoustic
zone that operates in a 3l/2 standing wave mode where lipids are
focused in the pressure antinodes and routed to the side outlets,
2) a 2-dimensional acoustic standing wave pre-focusing zone that
aligns sample particles (cells) in the channel centre and, 3) an
electrode zone that measures the particle size. Acoustic actuation
was performed using piezoceramic transducers operated at 4.92
and 1.97 MHz, Fig. 1d & e. As a model system polystyrene beads
(3, 5 and 7 µm) were added to consumer cream, diluted to 0.5%
lipids using 0.9% NaCl in MQ water, and run through the chip.
Control samples with only 7 µm beads were run, evaluating the
improved measurement accuracy obtained by the 2-D acoustic
prefocusing.
Results: The chip was able to remove lipid particles while retaining
the polystyrene particles, Figure 2 a-b. Most importantly, the MIC
raw data pulse amplitudes from the 7 µm measurements showed
a significantly reduced size variation with the pre-focusing zone
activated, Fig. 2c & d.
ISAC 2013 Program and Abstracts
Congress
Overview
Special
Lectures
NanoCellect’s microFACS real-time control loop as it has fast
processing and calculation capabilities without electronic jitter.
High-level instrument automation can easily be achieved using this
processor in high-level C or LabVIEW development environments.
NanoCellect’s microFACS demonstrated high sorting accuracy and
sample enrichment, and better cell viability after sorting due to
its gentle displacement of fluid. A sorting chip is a closed system
that can be disposable after one-time use, thus, reducing potential
sample cross-contamination and bio-hazardous issues.
Conclusions: The chip performs lipid removal with and
2-dimensional acoustic prealignment enabling subsequent
impedance based cytometry, which opens the path towards simple
And Fast Analysis Of Raw Milk Samples In The Dairy Industry.
Oral Session
Abstracts
Up to five different fluorescent emission wavelengths including
GFP/FITC, PE, tdTomato, PerCP were successfully distinguished
by the COST coding technology using a single laser (l=488nm)
and single Hamamatsu PMT.The NanoCellects detection
architectureprovides an integrated solution to lab on a chip platform
multicolor flow cytometer and dramatically reduces the size and
cost of the whole system. Moreover, this detection architecture can
make the color 197compensation analysis easier for users who are
not familiar with its concept.
Poster Session
Abstracts
In conclusion, the COST architecture will provide an integrated,
optofluidic solution to multicolor detection, thus enabling the
construction of the next generation optofluidic flow cytometers that
are much portable and less expensive than existing commercial
systems.
Speaker/Author
Index
ISAC 2013 Program and Abstracts
An improved optofluidic flow cytometer system that can
differentiate multiple fluorescent wavelengths using a single
detector is presented in this work. The fluorescence detection
configuration NanoCellect has developed employs ‘color-spacetime’ (COST) coding in order to register signals from fluorescently
labeled cells or beads as they pass through a customized color
filter array. The filter array consists of five slits, two transparent slits
and three narrow bandpass color filter slits. Fluorescence signals
are transformed into time domain with narrow bandwidth by the
color filter array. The first two transparent filter slits establish the
reference for the overall fluorescence intensity and give information
on the cutoff frequency of digital filter. The following three color
filters modulate fluorescence emission waveform in a unique
way and create differently coded ‘color-fingerprint’ for each
fluorescence color that is picked up by a single PMT. By using the
in-house digital signal-processing (DSP) algorithm, the registered
fluorescence information is convertedto a matrix of fluorescence
intensity ratios normalized to the strongest peak (e.g. the signal
intensity from either the 1st or 2nd transparent filter slits).
Commercial
Tutorials &
Exhibits
Recent innovations in cell sorting technologies have yielded
increases in performance and capabilities of the standard
fluorescent activated cell sorting (FACS). NanoCellect Biomedical
Inc. has developed a lab-on-a-chip cell sorter (microFACS) with
improved performance and capabilities while significantly reducing
cost and size by employing ‘space-time’ coding and an on-chip
integrated piezoelectric actuator. We present how the microfluidic
FACS system highly integrated with optics, electronics, and
acoustics performs at a high level in terms of throughput, purity,
and ease of use. The ‘space-time’ coding technology allows for
precisely calculating the travel velocity of each cell of interest
and guarantees to trigger the on-chip piezoelectric actuator on
time, thus, resulting in high sorting accuracy and sample purity.
A real-time control loop, that is crucial for the high-accuracy
sorting, is realized by employing an off-the-shelf embedded
processor based on National Instruments’ (NI) Reconfigurable
I/O platform. An FPGA was chosen for implementation into
Sung Hwan Cho1, Phillip Poonka1, Kendall Chuang1, Peter
Buerki1, Melesio Arambula1, Zach Olson2, John Hanks2, YuHwa Lo3, Jose Morachis1
1
NanoCellect Biomedical Inc., San Diego, CA, United
States, 2National Instruments Corp, Austin, TX, United States,
3
University of California San Diego, La Jolla, CA, United States
Poster
Session
Melesio Arámbula1, Sung Hwan Cho1, Daniel Johnson1, Razi
Alon1, Peter Buerki1, Phillip Poonka1, Kendall Chuang1, YuHwa Lo2, Zach Olson3, Jose Morachis1
1
NanoCellect Biomedical Inc., San Diego, CA, United States,
2
Electrical and Computer Engineering, University of California,
San Diego, La Jolla, CA, United States, 3National Instruments,
Lake Forest, CA, United States
Optofluidic Flow Cytometer Employing Color-SpaceTime (COST) Coding: On-Chip Multiple Fluorescence
Differentiation
Wednesday,
22 May
Microfluidic Fluorescence-Activated Cell Sorter
(micro-FACS) Employing ‘Space-Time’ Coding and
On-Chip Piezoelectric Actuator
284/B163
Tuesday,
21 May
283/B162
Monday,
20 May
References:
1. C. Grenvall et al., CYTO conference, Program number 458,
2012
2. S. Gawad, Ph. Renaud et al., Lab Chip, 1, 76-82 (2001)
3. D. Spencer and H. Morgan, Lab Chip, 11, 1234-1239 (2011)
4. R. Rodriguez-Trujillo, G. Gomila et al. Biosens Bioelectron, 24,
290-296 (2008)
5. K Cheung, Tarnók et al, Cytometry A, 77, 648-66, (2010)
6. C. Grenvall and T. Laurell et al., Anal Chem, 81, 6195-6200
(2009)
7. C. Grenvall et al., CYTO conference, Program number 459,
2012
8. C. Grenvall, JR Folkenberg et al. Cytometry A, 81, 1076-1083
(2012)
The lab-on-a-chip cell sorting system along with the ‘space-time’
coded algorithm and real-time on-chip sorting have allowed
NanoCellect to generate a powerful microFACS system that is
portable, and affordable which can ultimately be used by everyone
without having to pay hundreds of thousands of dollars, lose
valuable lab space, or have to share core facility.
Sunday,
19 May
Saturday,
18 May
An improved user interface including a disposable sorting
chip mount was built by using a combination of off-the-shelf
components and custom-built parts. Our custom chip interface
uses magnets to pre-align the laser with the microfluidic channel
and final alignment is performed using a pico-motor driven XYZ
stage controlled via the LabVIEW-based control software. The
optical filter swapper uses a self-aligned magnetic system for simple
and rapid exchange of custom ‘space-time’ coding optical filter sets.
197
Congress
Overview
Multidimensional Image Cytometry
(B164 – B165)
Speaker/Author
Index
Poster Session
Abstracts
Oral Session
Abstracts
Commercial
Tutorials &
Exhibits
Poster
Session
Wednesday,
22 May
Tuesday,
21 May
Monday,
20 May
Sunday,
19 May
Saturday,
18 May
Special
Lectures
285/B164
Rapid Method for Evaluating Micronuclei Formation
Using ImageStreamX
A. Nicole White1, Ashley Sullivan2, Sherry Thornton1, Stefan
Pfuhler2
1
Rheumatology, Cincinnati Children's Hospital Medical Center,
Cincinnati, OH, United States, 2Global Product Stewardship,
Proctor and Gamble, Mason, OH United States
The micronucleus assay is a commonly used genetic toxicity
measure employed by toxicologists and safety experts to measure
and evaluate a cell’s response to environmental or chemical
compounds. Micronuclei (MN) originate from chromosome
fragments or whole chromosomes that are unable to migrate to the
poles during the anaphase stage of cell division. They are small
membrane bound DNA fragments that are formed in the cytoplasm
during interphase following exposure to the genotoxic compound
of interest. Current evaluation methods include manual evaluation
under a microscope, laser scanning cytometry (LSC), and flow
cytometry (FC). The manual method involves lengthy evaluation
times and low statistical power. LSC methods reduce the evaluation
time and provide both quantitative and qualitative analysis, but
lack the robust population statistics that can be gained by using the
Amnis ImageStreamX. Lastly, FC methods improve the statistical
ability of the assay but lack qualitative methods of evaluation.
Here, we present a proof of concept study to demonstrate the
potential utility of the ImageStreamX in the MN assay. The study
was conducted using the CHO-K1 cell line and the standard in
vitro cytokinesis block MN method. Briefly, cells were treated with
various concentrations of a standard positive control, Mitomycin
C, for four hours followed by treatment with cytochalasin B for 20
hours to disrupt cell division. MN were then identified using the
ImageStreamX in divided cells, as indicated by binucleation, to
ensure only intact cells were evaluated.Boolean logic was used to
create combined spot, intensity and threshold imaging masks in the
IDEAS software to identify and calculate MN populations within the
binucleated subset. This approach enabled us to develop a rapid
method for evaluating micronuclei formation using population
statistics and qualitative confirmation. Ultimately, the Amnis
ImageStreamX may provide novel means for assessing genetic
toxicity as it allows for improvements upon current methodsfor
high throughput evaluation of both quantitative and qualitative
information of a population of cells.
286/B165
High-Throughput Image Analysis Software Applied to
High-Content Neuronal Screens
David Logan, Anne Carpenter
Imaging Platform, Broad Institute of Harvard and MIT,
Cambridge, MA, United States
Background: The scale of microscopy images that can be collected
automatically can be overwhelming; testing tens or hundreds
of thousands of samples is now feasible via screening facilities.
However, high-throughput screens of all but a few neuronal
phenotypes are in their infancy. Automating the analysis of these
complex cells, often co-cultured with other cell types, has been a
challenge. Commercial software exists to automatically measure
some phenotypes in certain neuronal assays, for example, to
measure the extent of neurite outgrowth under ideal assay
conditions. However, this software is often inadequate because
of the lack of algorithm adaptability, incapacity to compensate
for illumination and focus variations, requirement for manual
intervention, and cost. We have adapted our image analysis and
machine learning tools, CellProfiler and CellProfiler Analyst, to
198
neuronal assays including: multiple neurite outgrowth assays,
an RNAi screen to quantify axonal initial segments in a Bipolar
Disorder model, and synaptic formation screens.
Methods: Using typically nuclear, dendritic, axonal, and/or
domain-specific markers, we identified and segmented neurons
and glia. Next, we corrected for illumination artifacts and preprocessed images to flexibly enhance the features of interest.
After segmentation, we quantified the staining intensity, area,
shape, overall length and often branching patterns and relative
proximity for dendrite- and/or axon-specific staining. We found
it unnecessary to separately stain for glia, and instead used a
supervised machine learning method to train a Gentle Boosting
classifier to categorize neurons versus glia.
Results: These methods yielded dozens of reliable measures in
neurites under various treatment conditions. Many of the screens
are in the followup or validation phase after identifying compounds
or genes indicating interesting phenotypes. Post-hoc analysis is
underway to identify optimal doses, outliers, and hits.
Conclusions: Phenotypic assays of neurons in vitro are at a
nascent, but encouraging stage. We produce free, open-source
software (www.cellprofiler.org) that enables biologists, including
neuroscientists, to accomplish a broad range of cellular image
analysis projects without programming for each application. We
plan to package these neuronal specific methods and algorithms
into CellProfiler.
New Probes and Assays
(B166 – B174)
287/B166
PerFix-nc (No Centrifuge Assay): The New
Intracellular No-Wash Assay with Extended
Capabilities
Fabrice Malergue, Laeticia Khemici, Felix A. Montero-Julian
Life Science, Beckman-Coulter Immunotech, Marseille, France
Background: PerFix-nc, the novel, commercially available
permeabilization method was presented last year. Based on the use
of a new detergent and new fixative procedure, PerFix-nc allows
for simultaneous intra- and extra-cellular staining. The single-step
staining combined with elimination of the wash step streamlines
the work flow, from 2-3 hours down to 45 minutes. This method
has been developed for the direct treatment of human whole blood
or bone marrow specimens. Here, we present how the method has
been modified and optimized to support the processing of other
sample types, such as PBMC, cell lines, mouse whole blood and
mouse cells.
Methods: Several antibodies targeting various specificities and
conjugated to different fluorochromes were tested on a variety of
sample types using Perfix-nc. For cell surface markers, Versalyse
was used as the method of reference. For intracellular markers,
Intraprep (Beckman Coulter) was used as the method of reference.
Samples were acquired on Navios cytometer and analyzed with
Kaluza software (Beckman Coulter).
Results: Multiple rounds of optimization were performed, ultimately
resulting in two procedures: (1) when the sample contains a normal
concentration of RBC (Red Blood Cells), such as whole blood or
total bone marrow, the normal procedure is recommended, without
any adaption required due to the origin of the sample (human or
mouse); (2) when little or no RBC are present (PBMC, cell lines,
splenocytes, thymocytes, etc.), the modified procedure must be
applied whatever the origin of the sample (human or mouse).
This modified procedure requires resuspension of the initial
sample in full serum (calf or bovine: FCS/FBS), followed by
reduction to half volume of the reagents that are required in the
ISAC 2013 Program and Abstracts
Note: For Research Use Only. Versalyse, Kaluza and Navios are
trademarks of Beckman Coulter, Inc.
Development of Novel Metal-Chelating Polymers for
Mass Cytometry
New Fluorophore for Violet Laser Excitation
Jolene Bradford, Wenjun Zhou, Bradley Dubbels, Bradley
Bone, April Anderson, Kyle Gee
R&D Molecular Probes Labeling and Detection Technologies,
Life Technologies, Eugene, OR, United States
Flow cytometry instruments offering solid state violet laser diodes
are becoming more prevalent due to their small size, low power
requirements, cost effectiveness, and reliability over long periods
of time. Concurrently, the use of violet-excited fluorochromes
in multiparametric flow cytometry has been essential in the
expansion of multicolor flow cytometry, enabling greater numbers
of markers to be detected in one sample. We have developed
a novel violet-excited dye, Pacific Green™ dye, which has an
excitation maximum of 411 nm and an emission maximum of 500
nm. The development of this dye addresses the need to transfer
well-resolved markers off the common Blue 488 nm and Red 635
nm excitation lines and onto the Violet 405 nm excitation line,
thus enabling the detection of other markers with the 488 nm and
635 nm lasers. Pacific Green™ dye can be used for three color
immunophenotyping using violet laser excitation with Pacific
Blue™ and Pacific Orange ™ dyes with minimal compensation and
without 488 nm excitation. Data is shown for human lymphocytes
stained with anti-CD3 complexed with Pacific Green™ Zenon®
labeling reagent, Goat anti-Mouse seconday detection, and
Streptavidin secondary detection. Six color immunophneotyping
data is shown using direct conjugates of Pacific Green™, Alexa
Fluor® 488, phycoerythrin (PE), PE-Cy7, Pacific Blue™ and Pacific
Orange™ conjugates. Pacific Green™ dye can be used with the
far red emitting QDot® 605, 655, and 705 conjugates to enable
higher multiplexing using violet laser excitation. Finally, Pacific
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
ISAC 2013 Program and Abstracts
290/B169
Poster
Session
We will conclude with a prospectus on expanding the variety of
metals for mass cytometry. Attention is given to account for the
different chemistries of particular elements that require either new
protocols to adapt them to our current tags, or entirely new ligands.
These ligands must fully sequester non-lanthanide metals, but this
in turn raises new challenges in attaching the ligands to the polymer
backbone, loading the metal ions into the ligands, choosing the
order in which to do so, and finding a ligand system that will
maintain water solubility of the polymer.
Wednesday,
22 May
The structural characteristics of tags are shown to be linked to
their performance in mass cytometry assays. Results so far infer
that antibody-polymer/dendrimer conjugate performance does
not simply depend on the number of tags attached to an antibody,
but also on the effect(s) that the polymer or dendrimer has on the
antibody-antigen interaction.
Tuesday,
21 May
It seems obvious that an effective polymer tag should present
numerous chelating ligands, each capable of tightly binding at least
one metal atom of interest, and be monodisperse in order to allow
quantification. One would expect sensitivity to linearly increase
with the number of ligands per tag; however, the optimal number
of ligands and tag structure is difficult to rationalize. Additionally,
the tag should have one orthogonal reactive group for covalent
attachment to an antibody. Our first case study comprises polymers
in which the initial polymer backbone was prepared with reversible
addition-fragmentation chain transfer (RAFT) polymerization. A
number of post-polymerization modifications were performed to
create polymers with numerous repeat amino groups and a thiol
or disulfide terminal group. The subjects of the second case study
are based on polyamidoamine (PAMAM) dendrimers. We have
prepared tags using 3rd and 4th generation dendrimers, which
consist of 32 and 64 amino groups per cystamine core. In both
case studies DTPA and/or DOTA was added to the amino groups
for metal chelation, and a maleimide linker was added for antibody
attachment.
We have developed two novel probes for determining the level and
distribution of free thiols, such as reduced glutathione (GSH), in a
cell population. GSH provides vast majority of the reducing power
available in the cell, thus its oxidation status largely determines
the thiol-disulfide status of the cell by interchange reactions. The
concentration of GSH has been found to decrease upon induction
of apoptosis due to extrusion of GSH, also when using nonoxidative apoptogenic agents. We have investigated the use of two
novel thiol reactive agents as well as a well established GSH probe,
monochlorobimane, to explore the changes in the level of free
thiols in response to apoptosis induction. Jurkat cells were induced
to undergo apoptosis using camptothecin, and apoptotic traits
such as phosphatidylserine externalization, caspase activity and
mitochondrial potential were investigated at different time points
after induction. Along with detection of the classical apoptotic
features we also used the three thiol probes to determine changes in
the thiol level. Upon addition to the cells the probes permeate the
cell membrane and react with intracellular thiols, causing the cell
to fluoresce. Quantification of the cell fluorescence after staining
(without washing) can then be used to determine the population’s
cellular thiol level at the single cell level. We found that all three
thiol probes could be used to detect apoptosis. . Remarkably,
the two novel probes have different properties as one detects
early apoptosis and the other late apoptosis. The third probe,
monochlorobimane, has much slower staining kinetics compared
to the two new probes, which reach steady state within one minute.
Consequently, monochlorobimane is difficult to use for generating
consistent data. Based on the this study, we suggest adding
examination of the level of free thiols to the list of phenotypes
which may be measured in order to detect apoptosis, as this is a
fast, reliable and easy way of assaying apoptosis.
Monday,
20 May
Metal-chelating polymers (tags) are essential for the provision
of biologically-significant sensitivity in mass cytometry. The
development of tags with high sensitivity and low nonspecific
adsorption is, however, still a young field. In this work, we attempt
to understand and predict structural properties of tags with the
goal to design and synthesize improved antibody-tag conjugates.
We will discuss the importance of tag water-solubility, as well
as its charge state and effect on nonspecific adsorption. We will
describe how different structural characteristics of tags conjugated
to antibodies influence the sensitivity and nonspecific background
of the immunoassay. Finally, we will discuss the implications and
challenges of expanding the variety of tags by using metals outside
of the lanthanide series.
Lars Johansen, Søren Kjærulff, Mette E Skindersø
Chemometec, Allerod, Denmark
Sunday,
19 May
Daniel Majonis1, Xudong Lou1, Olga I. Ornatsky2, Vladimir
Baranov1, Scott D. Tanner2
1
DVS Sciences Inc., Markham, ON, Canada, 2DVS Sciences
Inc., Richmond Hill, ON, Canada
A Novel Rapid Apoptosis Assay Based on Thiol Redox
Status
Saturday,
18 May
288/B167
289/B168
Special
Lectures
Conclusion: With only minor adaptions to the normal procedure,
the innovative PerFix-nc kit can now be used on all types of
samples. Moreover, reducing the reagent volume required, the user
can effectively double the number of tests provided in the kit.
Congress
Overview
normal procedure. There are no other changes, thus preserving the
simplicity of the method, the reduced work load (less than 15 min)
and the streamlined workflow (45 min).
199
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
292/B171
291/B170
Barbara Seredick, Jolene A. Bradford, Mike Olszowy
R&D, Life Technologies, Eugene, OR, United States
Brilliant UltraViolet™ Dyes: A New Set of High
Sensitivity Fluorescence Reporters for Multicolor
Flow Cytometry with a UV Laser
Brent Gaylord, Yongchao Liang, Frank Uckert, Barry Leonard,
Haiqing Li, Lan Tran, Glenn Bartholomew, Yu Chen, Fengjun
Luan, Stephanie Widmann, Alan Stall
BD Biosciences, San Diego, CA, United States
While UV lasers have been available for many years, their utility
has been manly restricted to DNA/RNA binding fluorochromes
(e.g. DAPI, Hoescht 33342) for studying cell cycle or stem cell side
populations. The low fluorescence intensity of standard immunofluorescent dyes, such as AlexaFluor™ 350 or AMCA, have made
them impractical for routine cytometric flow analyses. The obvious
void in dye technology coupled with the historically high cost of
lasers has limited the widespread adoption of UV lasers in flow
cytometers and cell sorting systems.
Here we report the development of novel class of highly fluorescent
polymeric reporters which are excitable by a UV (355nm) laser.
The introduction of these dyes has the potential to dramatically
extend the functionality of the UV laser adding up to 6 totally new
colors for multi-color cytometric flow studies.
Methods: The Brilliant Violet™ dyes were the first to be introduced
from a new class of highly fluorescent reporters. Brilliant Violet™
421 (BV421™) and the 6 Brilliant Violet Tandems™ are based
on a well-defined, synthetic, light harvesting polymer structure.
Chemical adaptation of the underlying polymer backbone was
done to “tune” the excitation profile from 405nm (Violet) to 355nm
(UV) and thus create the framework for an entirely new family
of reporters for the UV laser. Corresponding UV Tandems were
generated by synthetic incorporation of orthogonal linking sites into
the base polymer structure which allow for discrete dye and protein
bioconjugation.
Results: The first Brillliant UltraViolet™ (BUV) polymer has an
excitation maximum near 355nm with emission below 405nm.
Fluorescence can be measured using a 390/18 band-pass filter
which excludes light from the violet laser (405nm) and minimizes
spillover from BV421™. Cross laser excitation is also minimal
with typical compensation into the BV421™ channel around 6%.
Brilliant UltraViolet Tandem™ examples will also be presented
highlighting the ability to span the entire spectrum from UV to
the near IR with high efficiency. The BUV polymer reagents
have moderate to high stain index values, some of which are
approaching levels equivalent to PE reagents. Comparative flow
performance will be presented as will multicolor staining panels
which highlight their compatibility with conventional flow labels
and BV421™.
Conclusions: A new family of reporters has been developed and
validated for significantly expanded utility of the ultra-violet
laser in multicolor flow cytometry. The initial examples show
unprecedented staining performance for UV-excitable immunofluorescent reagents. As with BV421™ these polymers can serve as
the basis for developing a wide range of bright, ultraviolet excitable
tandem reagents similar to the Brilliant Violet Tandems™. The
superior staining performance and unique excitation / emission
profiles of these new dyes offer a broader pallet of colors for those
designing multi-parameter flow panels.
Speaker/Author
Index
Poster Session
Abstracts
Green™ dye can be paired with fixable blue-emitting or fixable
yellow-emitting viability stain with violet excitation to exclude dead
cells. For Research Use Only.
200
Monitor Caspase 3/7 Activity without Cell Fixation: A
Novel Apoptosis Reagent from Molecular Probes®
Introduction: Apoptosis, or programmed cell death, is
characterized by cell shrinkage, membrane “blebbing”, and
genomic fragmentation. Apoptosis is morphologically and
functionally distinct from other mechanisms of cell death such
as necrosis and autophagy and it plays an important role in
various biological processes including cell turnover, embryonic
development and negative selection of cells during immune
system development. Disregulation of apoptosis is implicated in
various human pathologies including neurodegenerative diseases,
autoimmune disorders and cancer. Activation of enzymes known
as caspases is an early event in the process of apoptosis and results
in the cleavage of protein substrates and subsequent disassembly
of the cell. The CellEvent® Caspase-3/7 Green Detection Reagent
is a novel fluorogenic substrate designed for the detection of
activated caspases 3 and 7 in apoptotic cells. This cell-permeant
reagent consists of a four amino acid peptide (DEVD) conjugated
to a nucleic acid binding dye. During apoptosis, caspase-3 and
caspase-7 proteins are activated and are able to cleave the caspase
3/7 recognition sequence encoded in the DEVD peptide. Cleavage
of the recognition sequence and binding of DNA by the reagent
labels apoptotic cells with a bright, fluorogenic signal, without the
need for fixation and permeabilization. When used together with
the SYTOX® AADvanced™ dead cell stain, apoptotic cells can
easily be discriminated from live and necrotic cells.
Methods: In this study the CellEvent® Caspase 3/7 Green Flow
Cytometry Assay Kit was used to monitor apoptosis in multiple cell
lines using a flow cytometer. Use of the reagent was compared to
other methods to detect apoptosis, including antibody staining and
detection of membrane asymmetry.
Results: The CellEvent® Caspase 3/7 Green Flow Cytometry Assay
Kit detected an increase in caspase 3/7 activity during apoptosis as
confirmed by staining using an anti-active caspase 3/7 antibody.
When treated with a small peptide inhibitor (Ac-DEVD-CHO),
staining with CellEvent® Caspase 3/7 Green Detection reagent was
reduced as was staining with the anti-active caspase 3/7 antibody.
Detection of apoptotic cells using the CellEvent® Caspase 3/7
Green detection reagent was similar to staining with an annexin V
antibody conjugate.
Conclusions: The CellEvent® Caspase 3/7 Green Flow Cytometry
Assay kit represents a dramatic improvement over existing reagents
for caspase detection as it is compatible with live cells and doesn’t
rely on fixation and permeabilization. The reagent stains cells in
as little as 30 minutes, permitting fast and easy multicolor staining
with additional reagents for multiplexing. For Research Use Only.
Not for use in diagnostic procedures.
293/B172
Reactive Oxygen Probes — A Broad Range of Colors
with Easier Labeling and Compatibility with Fixation:
Novel CellROX® Reagents from Molecular Probes®
Barbara Seredick, Bradley Bone, Mike Olszowy
R&D, Life Technologies, Eugene, OR, United States
Introduction: A natural consequence of aerobic respiration is
the generation of highly toxic radicals called reactive oxygen
species (ROS). Once produced, ROS and other free radicals can
damage proteins, lipids and DNA. ROS have been implicated
in various human pathologies including cancer, atherosclerosis,
neurodegenerative diseases and diabetes. To combat oxidative
stress, cells are equipped with antioxidant defense systems that
correct imbalances in the concentrations of pro- and antioxidants.
In other systems ROS generation plays an important protective role
ISAC 2013 Program and Abstracts
Poster
Session
The advantages of this label-free technology are especially
significant fordrug discovery, cancer research and primary/stem cell
research since the cells can be studied in a non-toxic environment
which more closely reflects their natural condition.
Commercial
Tutorials &
Exhibits
References: Maiden, A. and Rodenburg, J. (2009). An improved
ptychographical phase retrieval algorithm for diffractive imaging.
Ultramicroscopy 109: 1256-1262
Maiden, A., Rodenburg, J. and Humphry, M. (2010). A new method
of high resolution, quantitative phase scanning microscopy. Proc.
SPIE 7729IL.
Oral Session
Abstracts
Poster Session
Abstracts
Recent advancements in the technology of high throughput flow
cytometry (HTFCM) have enabled the acquisition of large datasets
as well as saving a significant amount of time, resources and
money. One of the more powerful and underutilized techniques
that facilitate the high throughput capability of flow cytometry is
fluorescent cell barcoding. This technique allows for fluorescent
labeling of at least four different assay plates which are then pooled
together and carried out as a single assay. As a result, this reduces
the cost and duration of the assay by greater than four times. We
have successfully applied this technique to increase the throughput
in 384 well assay plates. This has enabled significantly faster
screening by allowing an equivalent of a 1536 well plate to be
read in the same time as a 384 well plate. We have accomplished
this technique by using lipophilic dyes that can be added to 4
unique plates of treated cells and then combined using standard
robotic equipment. The dyes are cost effective, non-toxic and
commercially available in several different colors. Utilization of
lipophilic barcoding allows for an increased throughput of flow
cytometry screening.
We demonstrate that this technology is appropriate for label-free
imaging of adenocarcinomic human alveolar basal epithelial cells
(A549 cells) and particularly for reporting the cellular changes
associated with mitosis. There is over a two-fold increase in cell
intensity during mitosisallowing dividing cells to be confidently
distinguished visually from non-dividing cells and automatically
segmented from non-dividing cells using image analysis software.
This change in cell intensity combined with the change in cell
area also allows dividing cells to be sub-divided into two further
populations: those cells entering cytokinesis and those returning as
daughter cells. In addition to this, due to localised changes in RI
within the cell, ptychographic images highlight the mitotic spindle
and chromosome arrangementduring mitosisenabling the different
phases of celldivision to be distinguished. Additionally cell ruffles
and apoptotic cells are easily identified from the raw images.
Wednesday,
22 May
Orzala Sharif, James Gilligan, Paul Anderson, Christopher
Trussell, Edward Ainscow, John Joslin
AD/Online Screening, Novartis (GNF), San Diego, CA, United
States
Here we report a novel, non-destructive, high contrast microscopy
technique which does not depend on the addition of stains. In
this technique the image producing step is transferred from the
microscope lens to a high-speed ptychographical algorithm
(Maiden and Rodenburg, 2009; Maiden et al., 2010). The algorithm
utilizes both amplitude and phase data from the sample to report on
quantitative changes in the refractive index (RI) and thickness of the
specimen. This information can be provided at any selected focal
plane using post-acquisition focussing, eliminating the requirement
for automated autofocussing during large scale screening or timelapse imaging.
Tuesday,
21 May
Barcoding with Lipophilic Dyes Can Further Increase
the Throughput of Flow Cytometry Screening
Use of fluorescent dyes has been a vital tool for biological
research to obtain information that is otherwise invisible to the
light microscope. Dyes have been engineered to minimise any
perturbation to the natural system, but this can never be completely
discounted. Imagingthe cell cycle and in particular the M phase
of mitosis and cytokinesis currently relies on the addition of
exogenous fluorescent stains or transfection of the cells with
fluorescent proteins to provide the contrast required to view the
essentially transparent cells. However, addition of these stains
candisturb the natural kineticsof the cell division process and even
induce cell death making them far from ideal in live cell imaging
experiments.Therefore there is a specific requirement for stain-free
tools to analyze cells throughout the cell cyclein vitro.
Monday,
20 May
294/B173
Peter O’Toole, Karen Hogg, Joanne Marrison
Biology, University of York, Heslington, York, United Kingdom
Sunday,
19 May
Conclusions: The CellROX® ROS detection reagents are bright and
stable ROS sensors that offer significant advantages over existing
ROS sensors because they are compatible with labeling in different
media and can be used with fixatives. For Research Use Only. Not
for use in diagnostic procedures.
Ptychography — Label Free Imaging of Dividing Cells
Using Quantitative Phase Information
Saturday,
18 May
Results: Cells treated with tert-butyl hydroperoxide or menadione
and stained with the CellROX® ROS detection reagents
demonstrated increased fluorescence as compared to control cells.
Treatment of cells under oxidative stress with the antioxidant,
n-acetyl cysteine, demonstrated diminished staining with the
CellROX® ROS detection reagents addressing the specificity of
the reagents. Detection of ROS by the CellROX® reagents was
increased as compared to ROS detection by H2DCFDA, and in
the case of CellROX® Orange and CellROX® Deep Red, offer
additional choices for multiplex flow cytometry.
295/B174
Special
Lectures
Methods: In this study we investigated the use of novel fluorogenic
ROS probes, the CellROX® ROS detection reagents, to monitor
ROS using a flow cytometry platform. Use of the reagents was
compared in multiple cell lines and conditions and compared to
results using another fluorogenic ROS probe, H2DCFDA.
Congress
Overview
in the early response of the innate immune system to pathogens and
low concentrations of ROS may serve as secondary messengers in
cell signaling. Although fluorogenic probes such as Aminophenyl
fluorescein (APF) and hydroxyphenyl fluorescein (HPF), and
2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) have been
widely used to detect ROS using flow cytometry and imaging
platforms, all are excitable by the 488nm laser and emit in the
fluorescein channel, and require that labeling occur in a proteinfree buffer. In contrast, the novel CellROX® ROS detection reagents
from Molecular Probes® offer increased flexibility for multiplex
experiments, with fluorescence emission in the fluorescein, PE, or
APC channels for CellROX® Green, Orange, and Deep Red. In
addition, the CellROX® reagents offer increased ease of use with
the ability to label cells in complete growth media, have increased
photostability as compared to H2DCFDA, and are compatible with
fixation (CellROX® Green and Deep Red).
Speaker/Author
Index
ISAC 2013 Program and Abstracts
201
Congress
Overview
Special
Lectures
Saturday,
18 May
Sunday,
19 May
Monday,
20 May
Tuesday,
21 May
Wednesday,
22 May
Poster
Session
Commercial
Tutorials &
Exhibits
Oral Session
Abstracts
Poster Session
Abstracts
Speaker/Author
Index
New Software Development (B175)
flow cytometric fluorescence resonance energy transfer and by
fluorescence microscopy using number&brightness analysis.
296/B175
Results: The dipole potential was successfully increased by
6-ketocholestanol and decreased by phloretin in SKBR-3, JIMT-1
and CHO cell lines. An increased dipole potential resulted in a
significant increase in ErbB2-ErbB2 homoassociation both in starved
and EGF stimulated samples while decreasing the dipole potential
had the opposite effect. The evaluation of experimental data for
ErbB1-ErbB2 heteroassociation is underway.
High-Fidelity Data Transfer: The Secret to Pipelining
and Automated Cytometric Analysis
Michael Stadnisky, John Quinn, Jay Almarode, Adam Treister
Tree Star, Inc., Ashland, OR, United States
Background: The exponential increase in the throughput and
content of flow cytometry assays is applying evolutionary pressure
on current data management and analysis solutions. Our work
with clinical trial data has revealed that development of an analysis
pipeline designed from FCS data file creation to finished report
was essential to address the needs of HT/HC flow cytometry; nextgeneration analytical methods rely on the high-fidelity transfer of
cytometry data to a server for storage and processing.
Methods: Herein, we assess the ability of a new application
to automate data movement from a cytometer to repository, to
manage quality assurance (QA) on data files and their transfer,
and to bridge cytometrists to cloud-based data storage. Based
on concepts developed at Stanford and our partner sites, we
designed instrument- and server-agnostic software that monitors
data generated by a cytometer, employs checksums and an XML
manifest for data integrity and transfer QA, and uploads the data to
a repository in an Analytical Cytometry Standard (ACS) container.
Results: This application facilitates the examination and comparison
of metadata, facilitates data sharing via data archives like
FlowRepository and ImmPort, and provides a bridge to analysis
automation leveraging the advantages of cloud computing.
Furthermore, we show that “protocol” XML objects direct adaptive
analysis of cytometry data, executing and querying the results from
expert-established gating s