1 mobilizing mass cytometry - · pdf file02.10.2014 · mobilizing mass cytometry...
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Mobilizing
MASS
CYTOMETRY Where Do We Go From Here?
1
John Daley
Director of Longwood Medical Area CyTOF Core
New England Cytometry Users Group Meeting XX
October 2 2014
Starr Center
Mass General Hospital
Harvard Medical School
Boston, MA
Does Mass Cytometry have “Legs”?
(is it going to go anywhere?)
Will it become the preferred method of cytometric analysis or
will it be a niche oriented technology?
Will the instrumentation develop to become more efficient
and user friendly?
Will Highly Dimensional analysis be adopted as an accepted
unique and powerful tool in the Cytometry Community?
Will “Spin Off” Technologies evolve from Mass Cytometry?
2
Answer: A Definite Maybe! 3
Instrument Evolution
Assay Development
Community of Users
The Three Main Ingredients
A Work in Progress
Scottish Dawn
4
• Increase sample processing efficiency
from 40-50% to 90-99%
• Increase injection rate speed from 500 to
5000 cells/second
• Improve real time basic and advanced
acquisition and analysis software
CyTOF 1
Prototype
CyTOF 2
Auto sampler
Instrument Evolution
5 Assay Development
• Automate Staining Process via robotics
and microfluidic chip technology
• Improve Signal to Noise(S/N) of metal
reagents
• Create reagent panels and selection
tools
• Incorporate internal and external control
standards for every sample
• Improve gating strategies to be more
accurate and objective BAR CODE
• Offer Software modules for specific
applications
Panel Designer™
New
Polymers
6
Now if you make it faster, cheaper, and easier …
then the answer is…
Creating a
CyTOF Community LOCAL USER GROUP Meetings
CYTOForum :On Line Discussion Group
Company Sponsored
User Group meetings
And Most Importantly # 3
YES!
If you… Make it Faster
Flow rate from 500 to 5000/sec
Automate labeling and bar coding with machine robotics
Have real time analysis platform with experiment specific templates
Make it Cheaper Reduce instrument cost 650K to 65K
Sell 20-30 multiplex panels at same cost as single test
Reduce or remove service contracts/ depot swap system
Make it Simpler Automate all steps: staining/tuning/acquistion/analysis
Automate sample prep via robotics
Automate tuning of standards and beads
Automate sample processing with bead standard
Automate analysis via gating and single cell identification
7
500 Years
Brief Overview of Mass Cytometry
Uses rare earth metals(lanthanides) instead of fluorophores for Antibody Tags
Method of detection is based on Time of Flight(TOF) of ions in a chamber whose mass to charge (m/Q) ratio is resolvable to 1 mass unit
Cytometer is a modified ICP (Inductively Coupled Plasma)-TOF Mass Spectrometer made by Fluidigm : “CyTOF”(tm) “The IonMaker”
Signal overlap is minimal, but other factors need to be monitored
Isotope purity, M+16 Oxidation, N+1,TOF +1effect
Tagged single cell is heated to 7,500 Degrees Kelvin- Vaporized,Atomized,Ionized: Turns into an Ion Cloud
8
How Does it Work? Label sample
Inject sample
Acquire Data
Convert data to FCS Format
Analyze with third party software
9
Similar to Flow Cytometry labeling. Last step fix and
resuspend in water with DNA metal tag (No Scatter)
0.5ml loop or 96 deep well auto sampler
Cytobank
The First Experiment: May 1, 2013
Fluidigm/DVS Human PBL Training Kit using Cytobank SPADE
CD3-CD4-CD8-CD14-CD16-CD20-CD45
CD4+
CD8+
LO
CD4+
Fluidigm/DVS Human PBL Training Kit using Cytobank
SPADE Analysis ( Note Two Parameter Scale Adjustment)
CD3-CD4-CD8-CD14-CD16-CD20-CD45
Reproducibility: CyTOF to CyTOF
Sister Instruments
17 markers
Harvard: Nicole Carlson
Ragon: Katja Kleinsteuber
Dualing CyTOF(S) in Boston Instrument to Instrument : Operator to Operator : Tech to Tech
Comparison
13
Tuesday, May 20. 3:30 - 5 PM, Floridian Ballroom A
3:30 - 4:00
"Integrating mass Cytometry into an active flow
cytometry core: the Dana-Farber experience"
John Daley, Senior Director of Flow Cytometry, Dana-
Farber
4:00 - 4:30
"Navigating the computational challenges of high
dimensional mass cytometry data."
Nima Aghaeepour, Stanford University
5:00 - 5:30
"Highly multiplexed imaging of tumor tissues with
subcellular resolution by mass cytometry"
Bernd Bodenmiller, SNSF Assistant Professor for
Quantitative Biology, University of Zurich
Pratip “27 Color” Chattopadhyay
Chair
Workshop
A
YEAR
OF MASS
CYTOMETRY LESSONS LEARNED IN CELLULAR IONIZATION
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John Daley B.S.
Director of Longwood Medical Area CyTOF Core
Harvard Medical School
Boston, MA
USA
WHAT DO YOU DO WHEN YOU
BLOW UP CELLS ???
CANT SORT THEM
CANT SEE THEM
CANT SAVE THEM
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DISRUPTIVE TECHNOLOGY AT IT’S FINEST!
Q: WHAT DO YOU DO WHEN
YOU BLOW UP CELLS ???
A: YOU THINK LONG AND HARD
ABOUT WHAT IS THE MOST
INTELLIGENT THING ONE
CAN DO TO GET THE MOST
USEFULL INFORMATION WHEN
DOING SO
16
Creative thought at it’s finest!
THE ULTIMATE QUESTION
WHY WOULD YOU WANT TO USE MASS CYTOMETRY
OVER FLOW CYTOMETRY?
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• No Compensation
• Can do a ton of Markers • IT’S NEW AND COOL
• I can get funding if no one else has it
• I can see the Forest and the Trees
• Flow is a hassle at this level of analysis
• If Stanford and Gary Nolan’s Doing it , It must be True
Vs.
THE ULTIMATE QUESTION WHY WOULD YOU WANT TO USE MASS CYTOMETRY
OVER FLOW CYTOMETRY?
“Christophe Benoist”
18
• No Compensation / minimal signal overlap
• Small sample size
• Less variation over multiple tubes (Bar Coding)
• In depth analysis of functionally distinct subpopulations
• Use as a primary screening tool for subsequent cell
sorting and downstream analysis
Selected Mass Cytometry Data
19
Where do you draw the line?
3 Examples
Data 1: 16 Markers: Human T Cell
Kit (Fresh vs. Frozen)
16 MARKER Human T Cell Phenotyping Kit Dr. Kazuyuki Murase M.D. Ph.D.
(Ritz Lab)
CD4+ T Regs (RO+ 127- 25+)
CD25
CD127
“Fresh vs. Frozen”
Cluster Gated 1%
CD45/CD3 GATED
Human Bone Marrow : 32 Markers SPADE Analysis
Jana Jakubikova /Anderson Lab/DFCI Bone Marrow Analysis from Multiple Myeloma Patients: 32 markers,1
viability, 2 DNA
Normal
Donor
CD3 CD4 CD8
CD56 CD19 CD 14
Human BM 5000 Node SPADE Analysis
Normal
Donor
HLA-DR CD11b CD45
T Reg analysis by Mass Cytometry
Kazuyuki Murase/Ritz Lab/DFCI
CD4 regulatory T cells in patients
receiving low dose IL-2 treatment
17 Cell Surface + 4 Intracellular T,B,NK
The “First Wave”
Community
CD4 T CELL EFFECTOR FUNCTIONS
Nick Teslovich/Raychaudhuri Lab/Brigham and Women’s Hospital
31 Markers: Lineage/ Activation, CCR’s,
Transcription Factors, Effector Molecules
LMA Repository and Fluidigm Panel Mix
Learned some techniques from Kazuyuki
and other early adopters
Shared Experiences with High Background
Ended up being Lot to Lot variation of
Fixative
Analysis using: FlowJo,Cytobank,viSNE
Lab Focus: autoimmune disease using
techniques in human genetics,
bioinformatics, and systems biology SPADE vISNE
Q: Is There a better way to gate out Doublets in Mass
Cytometry?
A: Bar Code each cell so that a doublet yields an illegal bar
code combination
25
Courtesy of Dr. Michelle Poulin: Fluidigm Corporation
“20” is the Magic Number
Palladium is the metal
3 Binary Combos in a 6 metal Choice
Radbruch ?/ Bodenmiller : Answer
‘Doublet-free’ Barcoding • 3-digit barcoding enables elimination of cross-sample
doublets
SA
MP
LE
Pd Isotope
Event#1:
from Sample 1
Event#2:
from Sample 1 & 7
Bar Coding in Cytometry 27
Nature Methods - 3, 361 - 368 (2006)
Published online: 20 April 2006; | doi:10.1038/nmeth872
Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling
Peter O Krutzik & Garry P Nolan
2006 Fluorescence
Bodenmiller B, Zunder ER, Finck R, Chen TJ, Savig ES, Bruggner RV, Simonds EF, Bendall SC, Sachs K, Krutzik
PO,
Nolan GP. Multiplexed mass cytometry profiling of cellular states perturbed by small
molecule regulators. Nature
Biotechnology 30, 858
2012 Metal Tags
9 Surface Markers, 14 Intracellular
12 Stimuli at 8 concentrations
Measured 18,816 conditions
in a single analysis
Combinatorial Cell Labeling by DOTA-Maleimide
Mass-Tag Cell Barcode (MCB) Reagents
7 Metals :128 Binary Combinations Rachel Finck/ Nolan Lab Stanford
Getting Organized: Year Two 29
Dedicated Online Scheduler CyTOF Project Request Forms
Sample Report SOP’s For Self Run Users
What's Next? : Year Two
Implement Staining Training Program: Like Ragon Comparison Pilot
Organize High Dimension Data Analysis Workshops
Develop Antibody repository resource QC/QA
Ramp Up In house Experiments
QC/QA Validation
Flow-Mass experiments
Bar Code Pilots
Continue learning ,Keep Thinking
Major Personal Impressions
Pattern Profiles similar to Flow Cytometry
Instrument not as scary as I thought
Good sample prep is key to quality data
Experimenter needs to be willing to put heart and soul into it
Right dedicated operator crucial in optimal instrument and overall distribution of scientific communication
Good people at DVS: Small smart dedicated
Fluidigm appears to be the right parent of the adoption
Improvement needed in Speed, efficiency, sensitivity
Data analysis and interpretation
Cost
31
The Next Questions How does one use Mass Cytometry with Flow Cytometry ?
How to Integrate proteomics and Genomics at the Single
Cell Level ?
What other things can this technology do?
32
LMA CyTOF Consortium Harvard Medical School
Caroline Shamu Jodi Moore Noel Peters Christophe Benoist Hongye Liu
Dana-Farber Cancer Institute
Jerome Ritz Kai Wucherpfennig
Brigham and Women’s Hospital
Jim Lederer Josh Keegan
Ragon Institute
Katja Kleinsteuber Mike Waring
Beth Isreal Deaconess Medical Center
Harvard Stem Cell Institute
Fluidigm Inc. Michelle Poulin Leslie Fung Jeannie Gaylor Pankaj Chaudhari Nick Confuorto
Cytobank Nikesh Kotecha TJ Chen Geoff Kraker Don Sullivan Trish Ward
Dana-Farber Flow Cytometry Nicole Carlson
Suzan Lazo-Kallanian Tim Powers Kristen Leone Kristen Cowens Steve Paula
CyTOF Users
Nick Teslovich Jana Jakubikova Kazuyuki Murase Masahiro Hirakawa Josh Keegan Jon Sitrin
ACKNOWLEDGMENTS
Stanford Pioneers