Mobilizing

MASS

CYTOMETRY Where Do We Go From Here?

1

John Daley

Director of Longwood Medical Area CyTOF Core

john_daley@dfci.harvard.edu

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

14

John Daley B.S.

Director of Longwood Medical Area CyTOF Core

Harvard Medical School

Boston, MA

USA

john_daley@dfci.harvard.edu

WHAT DO YOU DO WHEN YOU

BLOW UP CELLS ???

CANT SORT THEM

CANT SEE THEM

CANT SAVE THEM

15

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?

17

• 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