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 [email protected] 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) Mass is an elemental analysis technology capable of Cytometry or detecting most of the periodic table of elements. It is CyTOF used in a variety of industries including, but not limited (Cytometry to, environmental monitoring, geochemical analysis, Time of metallurgy. Flight) Time of flight (TOF) detection in mass spectrometry. Origin of a term • Extremely low detection limits;
• A large linear range
• Possibilities to detect isotope composition of elements
• High sample throughput – speed Using the atomic mass spectrum
• 135 channels • >35 metal tags • Additional isotopes possible
• Rare Lanthanides: • Nonbiological 13 metals, • Nonradioactive 35 isotopes
Each vertical bar is a stable isotope that can be measured. Using the atomic mass spectrum
Lanthanides – 35 isotopes for antibody labeling
102Pd 104Pd 195Pt 105 106 Pd Pd 194Pt 198 209Bi 89 Pt Y 108Pd 110Pd 127I CD45 Barcoding IdU – S phase Cisplatin – Live/Dead CD16 103Rh DNA intercalator Metal-Tagged Probes
Maxpar IgG Antibodies Cell-ID™
• >600 preconjugates • Barcoding • Human and mouse • Intercalator: Identifies single-cell • Phenotyping and functional events applications • Cisplatin: Dead-cell indicator • Individual or part of panel kits • IdU: S-phase • 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
Barcoded Samples
1 tube
Benefits: = - simplified sample prep - savings of antibodies - robust data - improved data quality by:
better cell doublet discrimination
vs.
Event#1: Event#2: from Sample 1 from Sample 1 & 11 Experiment Design by Fluidigm
Channels Channels 502846495443473717 Remaining 81
Panel Cell-ID Other
• PBMC Phenotyping Kit (17) • Barcoding (6) • Normalization beads (6) • Cytokine kit (11) • DNA (3) • Environmental monitoring (4) • Additional targets (9) • S-phase (1) • Dead cell (2) CyTOF Technology The 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
Metal (isotopes) 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 110Pd 141Pr 150Nd 161Dy 191Ir 209Bi Highly pure, rare metal isotope labels that minimize background Antibody-mediated multiparameter protein detection Today’s gold standard
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
UV 355 nm
Violet 405 nm
Blue Laser 488 nm
Yellow-green 561 nm
Red 640 nm
Emission: fluorescence spillover Staining intensities and background Mass Cytometry Workflow
1 2 3 4 Design Stain Acquire Analyze Maxpar® panel and cells using protocols high-parameter data data using proven prepare mixture from and buffers validated for millions of cells analytical tools. individual antibodies. by Fluidigm. with the Helios™ mass cytometer. Panel Designer application Design
Input probes Generate panel that Save and share from Fluidigm or minimizes signal panels/catalogs personal catalog overlap into low with collaborators signal targets
Available at the Fluidigm web portal for mass cytometry www.dvssciences.com Signal and tolerance Design
Panel Designer uses signal values obtained from the technical data sheet. Stain: Buffers, Validated 2 Protocols and Beads Stain Buffers • Cell staining • Fix/perm for intracellular staining • Intercalation buffer
Validated protocols • Surface • Cytoplasmic • Nuclear • Phosphoproteins
Normalization beads Helios™, a 3rd generation mass 3 Acquire 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 CyTOF Technology Overview Acquire ICP Ionizes Cells Nebulizer
Masses separated by TOF
High-pass ion optic
FCS file Analysis
Integrate per cell 4 Analysis example: Cytobank Analyze
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 HIPC immunophenotyping panel A multitube, 8-color flow cytometry workflow
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 CD3 CD3 CD3 CD3 CD3 Same CD19 CD4 CD4 CD4 CD19 fluor CD20 CD8 CD25 CD8 CD20 CD38 CD45RO CD38 CD24 CD11c CD45RA CD127 CCR6 CD27 CD14 CCR7 CCR4 CXCR3 CD38 CD16 HLA-DR HLA-DR HLA-DR IgD CD56 Viability Viability Viability Viability CD123 HLA-DR Viability
Lymphocytes Treg T helper B cells Mono, DC, NK
23 unique markers 5 sample tubes 8 fluorescent tags
Maecker et al. Nature Reviews Immunology (2012) 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™ 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.
30 unique markers 1 sample tube 37 populations Comprehensive 30-marker panel
CD3 CD28 CD161 Additional markers enable: CD4 CD38 CD294 • Identification of CD8 CD45 CCR4 o γδ T cells (TCRgd) CD11c CD45RA CCR6 o Neutrophils, eosinophils and basophils CD14 CD45RO CCR7 (CD66b, CD294) CD16 CD56 CXCR3 o NK cells, early and late (CD57) CD19 CD57 CXCR5 o MAIT/NKT (CD161) CD20 CD66b HLA-DR • Better definition of T cell subsets CD25 CD123 IgD (CD28, CD161, CXCR5) CD27 CD127 TCRgd • Pan-leukocyte identification (CD45) • Live/dead indicator 30 antibodies (intercalator-103Rh) plus viability indicator
30 unique markers 1 sample tube 37 populations Markers 6+ open channels
CD45 CD127 CD196/ to customize CCR6 IgD 89Y 209Bi CD123/ HLA-DR 103Rh 176Yb IL-3R 175Lu 141Pr CD66b 174Yb 142Nd CD19 173Yb 143Nd CD20 Metals CD4 172Yb 144Nd
When markers are 171Yb 145Nd CD3 CD8a added, Maxpar 170Er 146Nd CD11c Pathsetter software 169Tm Maxpar Direct 147Sm CD14 168Er Immune Profiling 148Nd CD16 can be used to build CD197/ 167Er Assay 149Sm CD45RO CCR7 a new automated 166Er 150Nd CD294 CD45RA analysis model. 165Ho 151Eu 164Dy 152Sm CD161 163Dy 153Eu CD194/ TCRγδ 162Dy 154Sm CCR4 161Dy 155Gd 160Gd 156Gd CD56 159Tb CD25 158Gd CD27 CD38 CD57 CD183/ CD28 CD185/ CXCR3 CXCR5 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 Technion-Israel Institute of Technology, Asya Rolls Lab • 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. Tissue Microenvironment Defining the Tissue Microenvironment
Phenotype Function
Cancer Stem Differentiated TIL: TIL: TIL: Fibroblast / Cell Cancer Cell B cells T cells Myeloid 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 Defining the Tissue Microenvironment
Phenotype Function
Cytokines / growth Cell Death/ Cell Cycle / Signaling / transcription factors Apoptosis 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 Hyperion Imaging System Highly 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 Capture Detection Preserve precious samples and reduce Hyperion™ CyTOF technology variability by eliminating dependence on Tissue Imager serial sections. Hyperion Imaging System Workflow
1 2 3 4 Design Stain Image Analyze panels using IHC-validated tissues (FFPE and frozen) biomarkers using precise using post-analytical imaging antibodies conjugated to or fixed cells with metal- laser-directed protein and secondary analysis metal tags. conjugated antibodies. capture and detection software tools. with CyTOF technology. IMC Verified Antibody Datasheet IMC 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 UV laser ablates tissue 1 μm2 at a time
UV laser at 200 Hz frequency
Plume
Helium gas fills the chamber
Motorized stage in sample ablation chamber Ablated sample is lifted by helium gas and introduced into Helios
Plume flow Argon gas flow to Helios
Motorized stage in sample ablation chamber Mass cytometry overview Masses separated ICP ionizes plumes High-pass ion optic by TOF
Downstream Data preprocessing Signal extraction of data analysis and image assembly measured markers Integrate per plume IHC vs IMC protocol Classical IHC Staining Workflow 1 2 3 4 5 6 7 Deparaffini Heat Primary Secondary Addition of Hematoxylin Visualization zation Induced antibody antibody of HRP stain and analysis Antigen stain HRP Substrate Retrieval conjugate
General FFPE IHC/IF Prep Antibody Staining
IMC Staining Workflow 1 2 3 4 5 Deparaffini Heat Primary Ablation Visualization zation Induced metal and analysis Antigen conjugated Retrieval antibody stain General FFPE IMC Prep Antibody Staining 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) Time required Nominal (in silico) As for routine IHC Variable (based on ROI) Variable (project-specific) 1 2 3 4 Design Stain Image Analyze panels using IHC-validated tissues (FFPE and frozen) biomarkers using precise using post-analytical imaging antibodies conjugated to or fixed cells with metal- laser-directed protein and secondary analysis metal tags. conjugated antibodies. capture and detection software tools. with CyTOF technology. Hyperion speed and throughput
Stained with 32 marker panel 1mm
140 samples with >37 markers acquired in approx. 1 week completely hands free Breast cancer tissue (FFPE) Quantitative single cell analysis IMLS Follow-on study: 44 markers + clinical data
190 mm
Box approximates area of typical ROI (~0.5 mm2 or ~1 TMA core; ~30 min acquisition time @200Hz) Breast cancer tissue (FFPE) Quantitative single cell analysis IMLS Follow-on study: 44 markers + clinical data
190 mm
Phospho-H3 2 Fibronectin Box approximates area of typical ROI (~0.5 mm or ~1 TMA core; ~30 min acquisition time @200Hz) Histone H3 Breast cancer tissue (FFPE) Quantitative single cell analysis IMLS Follow-on study: 44 markers + clinical data
190 mm
PanKeratin Cytokeratin 8/18 2 Cytokeratin 5 Box approximates area of typical ROI (~0.5 mm or ~1 TMA core; ~30 min acquisition time @200Hz) Breast cancer tissue (FFPE) Quantitative single cell analysis IMLS Follow-on study: 44 markers + clinical data
190 mm
CD68 2 CD45 Box approximates area of typical ROI (~0.5 mm or ~1 TMA core; ~30 min acquisition time @200Hz) Histone H3 Breast cancer tissue (FFPE) Quantitative single cell analysis IMLS Follow-on study: 44 markers + clinical data
190 mm
SMA E-Cadherin triMethyl-H3 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™
100 µm
1 2 3 View and Validate Image segment Higher order analysis Detect presence of protein(s) Segment specific cell Differential expressions of interest, produce high populations of interest. within a cell and between quality plots with chosen Quantify differential cell populations with combinations of markers and expression of the epitope statistical significance and confirm experimental within the cells and in spatial context. success. Export files in across cell population. various formats. Analysis Pipeline
Primary
MCD Viewer Preliminary View & Cell Segmentation & High Dimensionality File Conversion Characterization Analysis
Exported files compatible with many other commonly used image analysis platforms for downstream analysis Shapiro. et al. Nature Meth. (2017) Thank you.
Olga Karpus Field Applications Specialist, BeNeLux & Nordics [email protected]
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