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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
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.
©2018 Fluidigm Corporation. All rights reserved. Fluidigm, the Fluidigm logo, CyTOF, EQ, Helios and Maxpar are trademarks and/or registered trademarks of Fluidigm Corporation in the United States and/or other countries. All other trademarks are the property of their respective owners. 04/2018
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Olga KarpusField Applications Specialist, BeNeLux & [email protected]