Mass Cytometry (CyTOF):

Modern way to analyse >40 markers on hundred thousands of cells on a single cell level using the Helios and Hyperion™ Imaging System

Olga Karpus, PhD

Field Applications Specialist, BeNeLux & Nordics

olga.karpus@fluidigm.com

MassCytometry or CyTOF(Cytometry Time of Flight)

Origin of a term

Cytometry the term "cytometry" can apply to any

method used to extract quantitative information from

individual cells, often antibody-based method.

Inductively coupled plasma mass spectrometry (ICP-MS)

is an elemental analysis technology capable of

detecting most of the periodic table of elements. It is

used in a variety of industries including, but not limited

to, environmental monitoring, geochemical analysis,

metallurgy.

Time of flight (TOF) detection in mass spectrometry.

• Extremely low detection limits;

• A large linear range

• Possibilities to detect isotope composition of elements

• High sample throughput – speed

Using the atomic mass spectrum

Each vertical bar is a stable isotope that can be measured.

• 135 channels• >35 metal tags• Additional isotopes possible

• Rare

• Nonbiological

• Nonradioactive

Lanthanides:13 metals, 35 isotopes

127I

IdU – S phase

194Pt 198Pt

Cisplatin – Live/Dead

102Pd

106Pd105Pd

104Pd

108Pd

DNA intercalator

103Rh

Barcoding

195Pt

89Y

CD45

209Bi

CD16

110Pd

Using the atomic mass spectrumLanthanides – 35 isotopes for antibody labeling

Metal-Tagged Probes

Cell-ID™

• Barcoding

• Intercalator: Identifies single-cell events

• Cisplatin: Dead-cell indicator

• IdU: S-phase

Maxpar IgG Antibodies

• >600 preconjugates

• Human and mouse

• Phenotyping and functional applications

• Individual or part of panel kits

• 35 labeling kits

• Conjugation service available

Lanthanide labeling of antibodies

Maxpar™ IgG antibodies or self-conjugated antibodies with labeling kits fluidigm.com

Cell-ID Pd Barcoding

BarcodedSamples

1 tube

= Benefits:- simplified sample prep- savings of antibodies- robust data- improved data quality by:

better cell doublet discrimination

Event#1: from Sample 1

Event#2:from Sample 1 & 11

vs.

17

• PBMC Phenotyping Kit (17)

• Additional targets (9)

• Barcoding (6)

• S-phase (1)

• Normalization beads (6)

• Cytokine kit (11) • DNA (3)

• Dead cell (2)

• Environmental monitoring (4)

28374346474950Channels ChannelsRemaining 8154

Panel Cell-ID Other

Experiment Design by Fluidigm

CyTOF TechnologyThe new standard for high-parameter protein detection

CyTOF® technology overcomes the limitations of fluorescence-based detection modalities by separating signals based on differences in mass instead of wavelength.

Separate and distinct signals | Uniform staining | No background

Highly pure, rare metal isotope labels that minimize background

Bi (1) Pd (6)

Dy (4) Pr (1)

Er (4) Pt (4)

Eu (2) Rh (5)

Gd (5) Sm (4)

Ho (1) Tb (1)

I (1) Tm (1)

Ir (2) Y (1)

Lu (1) Yb (5)

Nd (7)

89Y 209Bi141Pr 150Nd 161Dy110Pd 191Ir

Metal (isotopes)

Antibody-mediated multiparameter protein detectionToday’s gold standard

Violet405 nm

Blue488 nm

Yellow-green561 nm

Red640 nm

Staining intensities and backgroundEmission: fluorescence spillover

Fluorochrome-conjugated antibodies are widely used but have limited utility for high-parameter studies. These limitations impart significant complexities in experimental design and interpretation.

Fluorescence ‘spillover’ | Variable staining intensities | Background signal

La

ser

UV 355 nm

Mass Cytometry Workflow

Staincells using protocols and buffers validated by Fluidigm.

3

Acquirehigh-parameter data for millions of cells with the Helios™ mass cytometer.

4

Analyzedata using proven analytical tools.

DesignMaxpar® panel and prepare mixture from individual antibodies.

21

Panel Designer application

Input probes

from Fluidigm or

personal catalog

Generate panel that

minimizes signal

overlap into low

signal targets

Save and share

panels/catalogs

with collaborators

Available at the Fluidigm web portal for mass cytometry www.dvssciences.com

Design

Signal and tolerance

Panel Designer uses signal values obtained from the technical data sheet.

Design

Stain: Buffers, Validated Protocols and Beads

Buffers

• Cell staining

• Fix/perm for intracellular staining

• Intercalation buffer

Validated protocols

• Surface

• Cytoplasmic

• Nuclear

• Phosphoproteins

Normalization beads

Stain

2

Helios™, a 3rd generation mass cytometer

• Single-cell suspension input with average range 1,000 events/sec

• Inductively coupled plasma (ICP) time-of-flight (TOF) mass spectrometry discriminates metal-conjugated probes on a per-cell basis.

• 75–209 Da atomic analytical mass range (135 channels)

• .fcs (FCS 3.0) and .txt data output

3

Acquire

CyTOF Technology Overview

NebulizerICP Ionizes Cells

High-pass ion optic

Masses separated by TOF

Integrate per cell

FCS file Analysis

3

Acquire

Analysis example: Cytobank

Plot raw data

Histogram Biaxial plot 3D plot Radar

Reduce dimensionality

PCA GemStone™ SPADE viSNE Citrus

Summarize statistics

Box plot Heat map Network Sunburst Dose curve

4

Analyze

HIPC immunophenotyping panelA multitube, 8-color flow cytometry workflow

Tube 1

CD3

CD4

CD8

CD38

CD45RA

CCR7

HLA-DR

Viability

Tube 2

CD3

CD4

CD25

CD45RO

CD127

CCR4

HLA-DR

Viability

Tube 3

CD3

CD4

CD8

CD38

CCR6

CXCR3

HLA-DR

Viability

Tube 4

CD3

CD19

CD20

CD24

CD27

CD38

IgD

Viability

Tube 5CD3CD19CD20

CD11c

CD14

CD16

CD56

CD123

HLA-DR

Viability

Lymphocytes Treg T helper Mono, DC, NKB cells

Maecker et al. Nature Reviews Immunology (2012)

23 unique markers 5 sample tubes 8 fluorescent tags

Same fluor

Challenges with current flow cytometric immune profiling

• Limited cell population identification

• Multitube, labor-intensive assays

• Variability in site-to-site results

• Subjectivity of manual gating strategies

The Maxpar® Direct™ Immune Profiling Assay™

30 unique markers 1 sample tube 37 populations

Comprehensive

Profile 37 immune cell populations from PBMC or whole blood with an optimized 30-marker panel.

Efficient

Simple single-tube workflow with pushbutton reporting. Just add sample and go.

Reliable

Produce consistent results lot-to-lot, run-to-run and site-to-site.

Comprehensive 30-marker panel

Additional markers enable:• Identification of

o γδ T cells (TCRgd)o Neutrophils, eosinophils and basophils

(CD66b, CD294)o NK cells, early and late (CD57)o MAIT/NKT (CD161)

• Better definition of T cell subsets (CD28, CD161, CXCR5)

• Pan-leukocyte identification (CD45)

• Live/dead indicator (intercalator-103Rh)

CD3 CD28 CD161

CD4 CD38 CD294

CD8 CD45 CCR4

CD11c CD45RA CCR6

CD14 CD45RO CCR7

CD16 CD56 CXCR3

CD19 CD57 CXCR5

CD20 CD66b HLA-DR

CD25 CD123 IgDCD27 CD127 TCRgd

30 antibodies plus viability

indicator

30 unique markers 1 sample tube 37 populations

Markers

Metals

89Y103Rh

141Pr

142Nd

143Nd

144Nd

145Nd

146Nd

147Sm

148Nd

149Sm

150Nd

151Eu

152Sm

153Eu

154Sm155Gd

156Gd

158Gd159Tb

160Gd161Dy

162Dy

163Dy

164Dy

165Ho

166Er

167Er

168Er

169Tm

170Er

171Yb

172Yb

173Yb

174Yb175Lu

176Yb209Bi

CD45CD196/CCR6

CD123/IL-3R

CD19

CD4

CD8a

CD11c

CD16

CD45RO

CD45RA

CD161

CD194/CCR4

CD25

CD27

CD57CD183/ CXCR3

CD185/ CXCR5

CD28CD38

CD56

TCRγδ

CD294

CD197/CCR7

CD14

CD3

CD20

CD66b

HLA-DR

IgD

CD127

Maxpar Direct Immune Profiling

Assay

6+ open channels to customize

When markers are

added, Maxpar

Pathsetter software

can be used to build

a new automated

analysis model.

Resource: immunophenotyping the naive mouse brain

RESEARCH ARTICLE

‘High-dimensional, single-cell characterization of the brain’s immune compartment’

Korin, B., Ben-Shaanan, T.L., Schiller, M. et al.

Nature Neuroscience 20 (2017): 1,300–1,309

Key findings

• CyTOF® analysis enabled broad analysis of immune cell subsets in immunological milieu of the murine brain.

• Previously undescribed immune populations, including some with frequencies <1%, identified

• Complexity of brain immune populations demonstrated

• Comprehensive map of brain immunity provides identification of specific cell subsets relevant for further disease or therapeutic study.

Technion-Israel Institute of Technology,Asya Rolls Lab

Tissue Microenvironment

Defining the Tissue Microenvironment

Phenotype Function

Cancer Stem Cell

Differentiated Cancer Cell

TIL:B cells

TIL:T cells

TIL: Myeloid

Fibroblast / vasculature

CD44 CD24 CD19 CD4 CD14 CD31

CD133 PD-L1 CD20 CD8 CD16 CD90

CXCR4 EpCAM CD22 CD25 CD11c CD248

Integrin a6 EGFR CD23 FoxP3 CD49d FAP

ALDH1 PDGFRa CD45R PD-1 CD80

Bmi-1 Her2 IgD LAG-3 CD86

Phenotype Function

Signaling / transcriptionCytokines / growthfactors

Cell Death/ Apoptosis

Cell Cycle / Proliferation

pNF-kB IL-6

p38 IL-10

p4E-BP1 pS6 GM-CSF IL-17A Caspase-3 CyclinA

pAkt pSHP2 IFN-g IL-17F Caspase-7 CyclinB1

Nanog pSLP-76 TNF-a IL-21 Cleaved PARP Ki-67

pERK1/2 pSTAT1 IL-2 IL-22 Granzyme B pH3

pLck pSTAT3 IL-4 VEGF Perforin pRb

Sox2 IL-5

Defining the Tissue Microenvironment

Hyperion Imaging SystemHighly multiplexed immunohistochemistry from FFPE, frozen tissue sections or cell smears

Comprehensive Highly multiplexed IHC enables simultaneous detection >37 protein markers.

Simple Stain samples with all antibodies simultaneously using conventional IF protocols

Contextual Get subcellular resolution while preserving information about tissue architecture and cellular morphology.

Powerful Preserve precious samples and reduce variability by eliminating dependence on serial sections.

CyTOF technologyHyperion™

Tissue Imager

Capture Detection

Hyperion Imaging System Workflow

Staintissues (FFPE and frozen) or fixed cells with metal-conjugated antibodies.

3

Imagebiomarkers using precise laser-directed protein capture and detection with CyTOF technology.

4

Analyzeusing post-analytical imaging and secondary analysis software tools.

Designpanels using IHC-validatedantibodies conjugated to metal tags.

1 2

IMC Verified Antibody DatasheetIMC antibody datasets independently assessed for:

✓ FLDM criteria for verification (tissues tested, clone selection, co-staining panel)

✓ Expected positive and negative staining patterns

✓ Expected co- and counter-staining patterns with other targets

Motorized stage in sample ablation chamber

UV laser at 200 Hz frequency

Plume

Helium gas fills the chamber

UV laser ablates tissue 1 μm2 at a time

Motorized stage in sample ablation chamber

Plume flowto Helios

Argon gas flow

Ablated sample is lifted by helium gas and introduced into Helios

Mass cytometry overviewICP ionizes plumes High-pass ion optic

Masses separated by TOF

Integrate per plumeSignal extraction ofmeasured markers

Downstream data analysis

Data preprocessingand image assembly

IHC vs IMC protocol

General FFPE IHC/IF Prep

1

Deparaffinization

6

Hematoxylin stain

2

Heat Induced Antigen Retrieval

Antibody Staining

3

Primary antibody stain

4

Secondary antibody HRP conjugate

5

Addition of of HRP Substrate

7

Visualization and analysis

Classical IHC StainingWorkflow

IMC StainingWorkflow

General FFPE IMC Prep

1

Deparaffinization

2

Heat Induced Antigen Retrieval

Antibody Staining

3

Primary metal conjugated antibody stain

4

Ablation

5

Visualization and analysis

Hyperion speed and throughput

Hyperion Imaging System imaging speed is related to laser frequency (200 Hz) and capture size (1 mm).

Hyperion Imaging System imaging speed is NOT related to the # of markers.

Example IMC throughput per day

• ~12 mm2 tissue

• ~45 tissue microarray cores (0.6 mm diameter)

• ~15 tissue microarray cores (1 mm diameter)

Staintissues (FFPE and frozen) or fixed cells with metal-conjugated antibodies.

3

Imagebiomarkers using precise laser-directed protein capture and detection with CyTOF technology.

4

Analyzeusing post-analytical imaging and secondary analysis software tools.

Designpanels using IHC-validatedantibodies conjugated to metal tags.

1 2

Nominal (in silico)

Time required

As for routine IHC Variable (project-specific)Variable (based on ROI)

Hyperion speed and throughput

140 samples with >37 markers acquired in approx. 1 week completely hands free

1mm

Stained with 32 marker panel

Quantitative single cell analysisBreast cancer tissue (FFPE)

Follow-on study: 44 markers + clinical dataIMLS

190 mm

Box approximates area of typical ROI (~0.5 mm2 or ~1 TMA core; ~30 min acquisition time @200Hz)

Phospho-H3

Fibronectin

Histone H3

Quantitative single cell analysisBreast cancer tissue (FFPE)

Follow-on study: 44 markers + clinical dataIMLS

190 mm

Box approximates area of typical ROI (~0.5 mm2 or ~1 TMA core; ~30 min acquisition time @200Hz)

PanKeratin

Cytokeratin 8/18

Cytokeratin 5

Quantitative single cell analysisBreast cancer tissue (FFPE)

Follow-on study: 44 markers + clinical dataIMLS

190 mm

Box approximates area of typical ROI (~0.5 mm2 or ~1 TMA core; ~30 min acquisition time @200Hz)

CD68

CD45

Histone H3

Quantitative single cell analysisBreast cancer tissue (FFPE)

Follow-on study: 44 markers + clinical dataIMLS

190 mm

Box approximates area of typical ROI (~0.5 mm2 or ~1 TMA core; ~30 min acquisition time @200Hz)

Quantitative single cell analysisBreast cancer tissue (FFPE)

Follow-on study: 44 markers + clinical dataIMLS

SMA

E-Cadherin

triMethyl-H3

190 mm

Box approximates area of typical ROI (~0.5 mm2 or ~1 TMA core; ~30 min acquisition time @200Hz)

Hyperion Imaging analysis pipeline A three step workflow using MCD Viewer and histoCAT™

Image segmentSegment specific cell populations of interest. Quantify differential expression of the epitope within the cells and across cell population.

2 3

Higher order analysis Differential expressions within a cell and between cell populations with statistical significance and in spatial context.

View and ValidateDetect presence of protein(s) of interest, produce high quality plots with chosen combinations of markers and confirm experimental success. Export files in various formats.

1

100 µm

Analysis Pipeline

MCD Viewer

Preliminary View & File Conversion

Cell Segmentation & Characterization

High Dimensionality Analysis

Exported files compatible with many other commonly used image analysis platforms for downstream

analysis

Primary

Shapiro. et al. Nature Meth. (2017)

Thank you.

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Olga KarpusField Applications Specialist, BeNeLux & Nordicsolga.karpus@fluidigm.com