Highly multiparametric analysis by mass cytometry

Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Nitz, Mark; Winnik, Mitchell A.; Tanner, Scott D.

doi:10.1016/j.jim.2010.07.002

Cited by 340 publications

(331 citation statements)

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“…CyTOF (Time‐of‐Flight cytometer) mass cytometer is able to combine single cell analysis typical of flow cytometry, with the high resolution and sensitivity of TOF‐mass spectrometry 55, 56 exploiting heavy metal isotopes conjugated to molecules bind to antibodies. Overcoming some of the existing challenges in flow cytometry, CyTOF has been exploited to characterize diverse populations of immune cells 56.…”

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confidence: 99%

Multiparametric Whole Blood Dissection: A one‐shot comprehensive picture of the human hematopoietic system

et al. 2017

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Human hematopoiesis is a complex and dynamic system where morphologically and functionally diverse mature cell types are generated and maintained throughout life by bone marrow (BM) Hematopoietic Stem/Progenitor Cells (HSPC). Congenital and acquired hematopoietic disorders are often diagnosed through the detection of aberrant frequency or composition of hematopoietic cell populations. We here describe a novel protocol, called “Whole Blood Dissection” (WBD), capable of analyzing in a single test‐tube, hematopoietic progenitors and all major mature cell lineages composing either BM or peripheral blood (PB) through a multiparametric flow‐cytometry analysis. WBD allows unambiguously identifying in the same tube up to 23 different blood cell types including HSPC subtypes and all the major myeloid and lymphoid lineage compartments at different stages of maturation, through a combination of 17 surface and 1 viability cell markers. We assessed the efficacy of WBD by analyzing BM and PB samples from adult (n = 8) and pediatric (n = 9) healthy donors highlighting age‐related shift in cell composition. We also tested the capability of WBD on detecting aberrant hematopoietic cell composition in clinical samples of patients with primary immunodeficiency or leukemia unveiling expected and novel hematopoietic unbalances. Overall, WBD allows unambiguously identifying >99% of the cell subpopulations composing a blood sample in a reproducible, standardized, cost‐, and time‐efficient manner. This tool has a wide range of potential pre‐clinical and clinical applications going from the characterization of hematopoietic disorders to the monitoring of hematopoietic reconstitution in patients after transplant or gene therapy. © 2017 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

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Multiparametric Whole Blood Dissection: A one‐shot comprehensive picture of the human hematopoietic system

et al. 2017

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“…This is the primary reason for the use of metal-chelating DNA intercalators to ensure correct percent-of-parent statistics, as all cells will be detected regardless of whether any antibody probes bind. 3,[6][7] Finally, the current cell transmission efficiency of the machine is ~25%, compared to an efficiency of >90% for a fluorescent flow cytometer. Therefore, a larger starting number of cells is often required, particularly for rare cell populations.…”

Section: Discussionmentioning

confidence: 99%

“…[1][2][3] Due to the purity of the metals and the mass resolution of ICP-MS, there is effectively no "spectral overlap." Since most of the elements used are not biologically relevant, there is also little background to cause "autofluoresence."…”

Section: Discussionmentioning

confidence: 99%

“…3,[5][6][7] The purpose of this article is to provide information on the proper use of a CyTOF mass cytometer for running samples. Maintenance of the nebulizer by backflushing with and storage in 5% citranox will minimized build-up of cell debris and metals, thereby reducing the likelihood of clogs.…”

Section: Discussionmentioning

confidence: 99%

“…[2][3][4][5] Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence.…”

Section: Introductionmentioning

confidence: 99%

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Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

Maecker

2012

JoVE

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In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes. [2][3][4][5] Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples. [6][7] Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal. Video LinkThe video component of this article can be found at http://www.jove.com/video/4398/ ProtocolAll cell samples for the CyTOF must be fixed and permeabilized. This enables greater entry of the iridium-containing DNA intercalator, and also prevents cell lysis during the MilliQ water wash and resuspension steps immediately before injecting into the mass cytometer.

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Principle of Mass Cytometry

Korin

Ghorbanihaghjo

2018

Encyclopedia of Analytical Chemistry

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Mass cytometry is an evolving technology combining flow cytometry methodology with inductively coupled plasma mass spectrometry (ICP‐MS). It overcomes flow cytometry's inherent limitation, by conjugated metal isotopes as reporter tags to antibodies instead of fluorophores. Its great advantage lies in its capacity to analyze more than 45 parameters simultaneously, at a single‐cell level, with very low signal spillover between adjacent masses. Mass cytometry high parameterization offers unparalleled characterization of single cells within heterogeneous populations, in a system‐wide view manner. Accordingly, it has been applied for a deeper understanding of specific cell subsets and network signaling. These new phenotypic characterization capabilities have provided a significant advancement in our ability to resolve biological questions. The high dimensionality of the data necessitates the development of new analytical tools, using data reduction and clustering of cells, in order to visualize and interpret the data. In this article, we will discuss the main principles of mass cytometry, its applications, main advancements, and the tools available for analysis.

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Highly multiparametric analysis by mass cytometry

Cited by 340 publications

References 50 publications

Multiparametric Whole Blood Dissection: A one‐shot comprehensive picture of the human hematopoietic system

Multiparametric Whole Blood Dissection: A one‐shot comprehensive picture of the human hematopoietic system

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

Principle of Mass Cytometry

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