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Cytof for All CyTOF for All The Top 8 Reasons to Use Mass Cytometry for Your Project Michelle Poulin, PhD Manager, MC/IMC field applications NA Chief aims of this presentation • Provide information on the ever-increasing use of CyTOF around the world. • Provide the reasons why CyTOF has become so popular Helios™, a CyTOF® system for high-parameter cytometry. • Clear up some misconceptions about the technology. 2 Summary Proven High-parameter capability Performance Array of reagents Simplicity Data quality Easy panels Community 3 Number 1: Mass cytometry is a proven and well-adopted technology Mass cytometry publication ramp Peer-reviewed* publications as of March 2020 900 66 800 Series2New 700 313 Series1Previous 600 500 167 810 400 300 147 497 200 72 330 44 183 100 67 67 111 0 2007– 2015 2016 2017 2018 2019 2020 2014 *Does not include commentaries or reviews Diverse study areas and panel sizes Areas of study Panel size distribution Infectious Transplantation/ disease stem cells Other diseases 41–50, 51–60, 1% 10% 4% 1–20, 8% 14% 15% Autoimmunity/ Method allergy development 12% 16% Data analysis ImmunoImmuno-- 12% 21–30, oncologyoncology 21% 14% 31–40, Immunology 49% Oncology 11% 13% Based on data from 186 peer-reviewed papers published in 2018 and 2019 6 Growing adoption of CyTOF in trials Clinical trials using CyTOF technology As of January 2020: 80 • 67 ongoing clinical trials 1 • 8 completed trials 70 18 60 Number Trial Phase 50 of Trials 17 Phase 1 13 40 74 Phase 2 26 Number 11 Phase 1/2 8 30 56 7 Phase 3 1 20 4 39 2 28 Phase 4 4 6 10 21 4 15 17 9 Other 5 5 23 0 0 (observational) Total 75 Cumulative totalTotal New Trialstrials Source: clinicaltrials.gov, January 2020 For Research Use Only. Not for use in diagnostic procedures. 7 Clinical trial trends Trials by research area Immuno-oncology 16% Infection and vaccine 7% Oncology 49% 7% Surgery 11% Autoimmunity 11% Other Source: clinicaltrials.gov January 2020 8 Number 2: Performance proven to equal or exceed that of fluorescence cytometry Validation of CyTOF single-tube cytometry RESEARCH ARTICLE ‘Validation of CyTOF against flow cytometry for immunological studies and monitoring of human cancer clinical trials’ Gadalla, R., Noamani, B., MacLeod, B. et al. Frontiers in Oncology 9 (2019): 415 Key findings • Side-by-side evaluation of human PBMC and tumor samples • Single-tube, 40-plus-parameter CyTOF panel Helios • 8 flow cytometry panels using same antibody clones • Cell population frequencies statistically equivalent for all populations • Equivalent staining quality and signal intensity as compared to background for >35 markers • CyTOF identified phenotypic, functional and activation/exhaustion proteins in major immune populations. LSRFortessa™ • Single tube X-20 • Limited sample size from tumor tissue 10 Mass cytometry excels at tumor proteomics REVIEW ARTICLE ‘Beyond the message: advantages of snapshot proteomics with single-cell mass cytometry in solid tumors’ Mistry, A.M., Greenplate, A.R., Ihrie, R.A., Irish, J.M. FEBS Journal 286 (2019): 1,523–1,539 Key information • MC has great utility for quantitative single-cell analysis of solid tumors. • Citation-driven comparison of MC to fluorescence cytometry including: o Multiplexing capability o Cost and throughput o Sensitivity and background noise • Advances in tumor biology enabled by MC: o ID of rare functional immune cell subsets in the tumor microenvironment o Characterization of cellular diversity within tumors o Single-cell signaling network mapping • Use to date in precision oncology 11 Key publications for comparison of CyTOF to fluorescence flow cytometry CyTOF is as sensitive as flow. Gadalla, R. et al. “Validation of CyTOF against flow cytometry for immunological studies and monitoring of human cancer clinical trials.” Frontiers in Oncology 9 (2019): 415. Advantages of CyTOF over scRNA-seq and flow cytometry for examining the tumor microenvironment Mistry, A. et al. “Beyond the message: advantages of snapshot proteomics with single-cell mass cytometry in solid tumours.” FEBS Journal (2018): 1,523–1,539. Advantages of CyTOF over multiparametric flow in a blood cancer analysis Pophali, P.A. et al. “Practical limitations of monocyte subset repartitioning by multiparametric flow cytometry in chronic myelomonocytic leukemia.” Blood Cancer Journal 9 (2019): 65. 52-parameter CyTOF panel Simoni, Y. et al. “Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates.” Nature 557 (2018): 575–579. Methods book for mass cytometry McGuire, H.M. and Ashhurst, T.M. Mass Cytometry, Methods in Molecular Biology. Springer Nature, Humana Press (2019) Spreading error issues in fluorescence cytometry Mazza, E.M.C., et al. “Background fluorescence and spreading error are major contributors of variability in high-dimensional flow cytometry data visualization by t-Distributed Stochastic Neighboring Embedding.” Cytometry A 93 (2018): 785. How spectral cytometry works Futamura, K. et al. “Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.” Cytometry A 87 (2015): 830. 12 Number 3: The simplicity of CyTOF A simple but powerful system • A single-detector system with a direct path from cell ionization to signal detection • Only one daily tuning and system optimization required • No assay- or metal-specific adjustments required • No compensation or single-stained controls needed • No impact of autofluorescence on signal sensitivity 14 Initializing the Helios for daily use System 30 min warm-up Daily QC and system 30 min optimization Acquisition* 20 min for 1M events 500 events/sec *No assay-specific optimization required before each new panel acquisition 15 Tools for streamlined, standardized analysis Automated instrument calibration and signal detection optimization (tuning) Data normalization beads and software Barcoding • Only high-parameter cytometric platform that Pd code enables barcoding • Improved single-cell discrimination • Eliminates sample-to-sample staining variation • Reduces processing and acquisition time Sample • Conserves sample and reagents 16 Number 4: The easiest high-parameter panel building you will ever experience Fluorescence vs. mass cytometry All you really need to know The overlapping emission spectrums of fluorescence tags: • Reduces overall sensitivity in channels where it occurs • Requires multiple control tubes of sample • Makes it difficult to use over 20 fluorophores routinely or change your panel later • Requires more iteration when building high-parameter panels • Is impacted by autofluorescence even when the autofluorescence is subtracted (sensitivity reduction) Helios has 135 truly distinct channels for signal detection. Fluorescence compensation and spectral unmixing are affected by instrument configuration and settings as well as use of appropriate controls. 18 Does CyTOF really require no compensation? A real-world comparison 22-marker immunophenotyping panel Flow Cytometry Target CyTOF Label Label Lymphocytes CD45 89Y Qdot 655 CD19 142Nd Pacific Blue CD127 (IL-7Ra) 143Nd PE-Cyanine5 Monocytes B cells CD8+ IgD 146Nd SB600 Naive, memory, Classical, CD11c 147Sm FITC Naive, nonclassical transitional, effector memory: CD16 148Nd Pac Orange plasmablasts central and CD194 (CCR4) 149Sm PE effector CD123 (IL-3R) 151Eu SB436 TCRgd 152Sm PerCP-eFl 710 CD185 (CXCR5) 153Eu SB702 γδ CD4+ NK CD3 154Sm AF 405 CD45RA 155Gd AF 700 T cells cells CD27 158Gd APC Naive, effector T regulatory CD45RO 165Ho SB780 memory: central cells CD197 (CCR7) 167Er SB645 and effector CD8a 168Er PE-eFl 610 Dendritic CD25 (IL-2R) 169Tm APC-eFl 780 cells CD20 171Yb eFluor 506 HLA-DR 173Yb Qdot 705 Myeloid, CD4 174Yb Qdot 605 plasmacytoid CD14 175Lu Qdot 800 CD56 (NCAM) 176Yb PE-Cy7 19 22-marker panel for Cytek® Aurora Source: spectrum.cytekbio.com 20 22-marker panel for CyTOF CHANNEL 89 151 171 143 147 153 167 173 174 175 176 142 146 148 149 152 154 155 158 165 168 169 89Y 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 142Nd 0 1 0.02 0 0 0 0 0 0 0 0 0 0.05 0 0 0 0 0 0 0 0 0 143Nd 0 0.01 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 146Nd 0 0.01 0 1 0 0.01 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 147Sm 0 0 0 0 1 0.02 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 148Nd 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 149Sm 0 0 0 0 0 0.01 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 151Eu 0 0 0 0 0 0 0 1 0.01 0.02 0 0 0 0 0 0 0 0 0 0 0 0 152Sm 0 0 0 0 0 0 0 0 1 0 0.01 0 0 0 0 0 0 0 0 0 0 0 153Eu 0 0 0 0 0 0 0 0.01 0 1 0 0 0 0 0 0 0 0 0 0 0 0 154Sm 0 0 0 0 0 0 0.01 0 0.05 0.02 1 0.02 0 0 0 0 0 0 0 0 0 0 155Gd 0 0 0 0 0 0 0 0 0 0 0.01 1 0 0 0 0 0 0.03 0 0 0 0 158Gd 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0.02 0 0 165Ho 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 167Er 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0.07 0 0 0 0 0 0 168Er 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.07 1 0 0 0 0 0 0 169Tm 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.03 1 0 0 0 0 0 171Yb 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0.05 0.06 0 0 173Yb 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.01 1 0.10 0.01 0.01 174Yb 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.02 1 0.01 0.01 175Lu 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 176Yb 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Source: dvssciences.com/paneldesigner/resources 21 A real-world comparison ‘Comparing fluorescent flow and mass cytometry optimization and implementation workflows’ Wightman, T., Houk, J., Misra, R.
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