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Insights Powered by Mass Cytometry: Investigators Share Their Experience

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Insights Powered by Mass Cytometry: Investigators Share Their Experience 41 42 43 44 45 47 48 49 Nb Mo Tc Ru Rh Ag Cd In 77 78 79 Insights Powered Ir Pt Au by Mass Cytometry 57 58 59 Investigators share La Ce Pr their experience 58 59 60 61 62 63 64 65 Ce Pr Nd Pm Sm Eu Gd Tb 64 65 66 67 68 69 70 71 Gd Tb Dy Ho Er Tm Yb Lu Improving care: It’s a mission that drives us all. Translating new discoveries into better clinical outcomes takes dedication and a desire to use new approaches to ask the most difficult questions. Mass cytometry enables you to deeply profile precious translational and clinical research samples across a range of cell surface and intracellular markers to uncover new insights in cellular phenotypes, function and signaling status. The scientific progress that has been made using mass cytometry is impressive, with hundreds of peer-reviewed publications across a range of disease research areas. Here are just a few examples of how mass cytometry is empowering researchers to ask new questions to advance our understanding of health and disease. We believe this is just the beginning, and we look forward to seeing your story added to this growing list soon. Chris Linthwaite President and CEO, Fluidigm CONTENTS Mass Cytometry for Discovery and Understanding Variation in the Human 4 Functional Profiling: An Introduction 17 Immune System to the Technology Dr. Petter Brodin | Karolinska Institutet Dmitry Bandura | Fluidigm Tackling Dynamic Complexity Unlocking the Potential of 20 to Understand Immunotherapy 8 Mass Cytometry Responses in Multiple Sclerosis Sean Bendall | Stanford University Sonia Gavasso | University of Bergen Discovering Normal to Better Single-Cell Insights into AML 11 Understand Abnormal: Searching 23 Cancer Biology for an Improved ALL Diagnosis Dr. Zohar Sachs | University of Minnesota Dr. Kara Davis | Stanford University Mass Cytometry Data Processing Profiling T Cell Diversity in Solid Tumors 26 with Cytobank 14 with Mass Cytometry Hannah Polikowsky | Cytobank Dr. Kavita Dhodapkar | Yale University School of Medicine These articles describe key work by eight leaders in the field of mass cytometry. 41 42 43 44 45 47 48 49 Nb Mo Tc Ru Rh Ag Cd In MASS CYTOMETRY PROVIDES A ROBUST, reproducible way to measure over 40 parameters,77 78 79 dramatically increasing the information producedIr Pt Au and yielding new insights into systems biology at a single-cell level. 57 58 59 La Ce Pr DMITRY BANDURA, PHD General Manager and Senior Vice President 58 59 Canadian60 Operations61 62 63 64 65 Ce Pr FluidigmNd Pm Sm Eu Gd Tb 64 65 66 67 68 69 70 71 Gd Tb Dy Ho Er Tm Yb Lu Mass Cytometry for Discovery and Functional Profiling: An Introduction to the Technology Dmitry Bandura, PhD General Manager and Senior Vice President Canadian Operations Fluidigm Mass cytometry enables simultaneous measurement The Beginnings of Mass Cytometry of more than 40 parameters on millions of individual As with many other technologies, early mass cytometer prototypes cells by using stable metal isotope tags as probes. were somewhat unseemly machines. The earliest working model, covering 9 square meters, was built at the end of 2006 by engineers Focusing and resolving these probes by time-of-flight (TOF) technology and scientists in the Scott Tanner Lab in the basement of the Lash into discrete and finely resolved mass peaks allows for essentially Miller Chemical Laboratories building at the University of Toronto. compensation-free signal at the single-cell level. Many more probes The first prototypes of antibody labeling reagents were developed can be simultaneously measured than is possible with traditional in parallel in collaboration with the labs of Professors Mitch Winnik fluorescence-based techniques. The various tags also provide similar and Mark Nitz. The project was supported by Genome Canada via signal intensities, removing a critical source of variation seen with the Ontario Genomics Institute, with close collaboration and support fluorescence. Finally, since the rare earth metals in Fluidigm probes are from Professor John Dick, Canada Research Chair in Stem Cell not naturally found in cells, there is no intrinsic background. All of these Biology. In 2008, an updated prototype—drastically improved in size attributes make it surprisingly easy to design large panels of metal and abilities and developed by DVS Sciences Inc., founded by Scott isotope-tagged antibodies, nucleic acid intercalators and other probes Tanner, Dmitry Bandura, Olga Ornatsky and Vladimir Baranov—was used to measure a variety of intracellular and extracellular moieties. purchased by the University Health Network of Toronto. The details of that pivotal instrument were described in a 2009 Analytical Chemistry In addition, the third-generation Fluidigm mass cytometer, Helios™, publication (Bandura et al.) that by the end of 2016 was among the includes software that automatically calibrates and optimizes the top 1% most highly cited chemistry publications. system for sample acquisition. No additional assay-specific instrument optimization is required. Because mass cytometers have no difference In 2009, Professor Garry Nolan of Stanford University bought the in system configuration, comparison of data across different instruments instrument. His pioneering work with the technology across a range can be done in a very straightforward manner, only requiring a simple of biological applications highlighted its vast potential and drove its normalization procedure that is also included in the software. wider adoption and use. A pivotal article on the use of mass cytometry for highly multiplexed characterization of bone marrow cells was All in all, the system provides a robust, reproducible, reliable way to published by Nolan’s group in collaboration with DVS in Science in measure over 40 parameters, dramatically increasing the amount of 2011. The mass cytometers now placed in major research institutions information produced and yielding new insights into systems biology and biopharma sites worldwide continue to drive innovation and have at a single-cell level. resulted in hundreds of peer-reviewed publications. 5 Mass Cytometry: A Detailed Overview ion clouds enter the TOF chamber and are accelerated at constant intervals toward the ion mirror, which redirects the ions For any mass cytometry user, going from metal-labeled cells to toward the detector. The electric fields in the accelerator and the flow cytometry standard (FCS) file is a relatively straightforward ion mirror are configured to focus ions into tight time resolved process, very similar to fluorescence flow cytometry. It includes bands. Each packet of ions, representing a single cell, then three sequential steps (see the figure below): 1) sample introduction, resolves into a series of bands, with the lightest probes reaching atomization and ionization, 2) TOF ion separation and detection, and the detector first and each successively heavier mass reaching the 3) data capture and preprocessing. detector in turn. 1. Sample Introduction and Ionization 3. Data Capture and Preprocessing Cells are stained with a panel of metal-conjugated antibodies Data for each band is digitized sequentially and integrated to and probes using methods very similar to flow cytometry. The obtain ion counts for the channels selected. As a result, the data cell suspension is then introduced into the nebulizer, where acquired contains the integrated number of total ion counts for it is aerosolized and delivered to the plasma torch through each selected analyte on a per-cell basis. This data is saved the heated spray. The single-cell aerosol droplets that exit as text (.txt) and FCS 3.0 format for convenient data analysis the spray chamber are transmitted to the inductively coupled in compatible software programs. plasma (ICP) source, where they are vaporized, atomized and ionized in the plasma for subsequent mass analysis. This Time-of-flight ion separation and detection Data capture and preprocessing process results in the formation of an ion cloud containing the ions derived from metal-conjugated probes, endogenous cell components and argon. The ion cloud then passes through the Detector Amplifier plasma-vacuum interface, which efficiently transports ions from the plasma at atmospheric pressure to the chambers that house Digitizer the ion optics at less than 10–3 Torr. TOF 2. Time-of-Flight Ion Separation and Detection High-pass optic Spray chamber ICP torch The ion clouds that exit the high-pass ion optics consist of a Deflector mixture of high molecular weight probes in a randomly distributed Nebulizer array. These ions are sent to the TOF mass analyzer, which Vacuum interface separates the ions on the basis of mass-to-charge ratio. The Sample introduction and ionization 6 The Future of Mass Cytometry When the technology was being developed, the founders conducted “The mass cytometers now placed in a market research study using a grant co-funded by Ontario Centres major research institutions and biopharma of Excellence and MDS Sciex. This study asked flow cytometry users whether they could see potential for application of this technique. sites worldwide continue to drive innovation Although responses were mixed, it is notable that many users asked, and have resulted in hundreds of peer- “Why would I need to analyze 20 markers simultaneously?” or “How reviewed publications.” could I ever need so many parameters?” Today, a typical mass cytometry user would ask for many more parameters than 20. What was unimaginable to the average medical researcher 10 years ago is REFERENCES becoming commonplace and driving cutting-edge research to address complex biological problems. Bandura, D.R., Baranov, V.I., Ornatsky, O.I., Antonov, A., Kinach, R., Lou, X., Pavlov, S., Vorobiev, S., Dick, J.E., Tanner, S.D. “Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry.” Analytical Chemistry 81 (2009): 6,813–6,822. Bendall, S.C., Simonds, E.F., Qiu, P., Amir el-A.D., Kritzik, P.O., Finck, R., Bruggner, R.V., Melamed, R., Trejo, A., Ornatsky, O.I., Balderas, R.S., Plevritis, S.K., Sachs, K., Pe’er, D., Tanner, S.D., Nolan, G.P.
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