CyTOF

Last updated

Cytometry by time of flight, or CyTOF, is an application of mass cytometry used to quantify labeled targets on the surface and interior of single cells. CyTOF allows the quantification of multiple cellular components simultaneously using an ICP-MS detector.

Contents

CyTOF takes advantage of immunolabeling to quantify proteins, carbohydrates or lipids in a cell. Targets are selected to answer a specific research question and are labeled with lanthanide metal tagged antibodies. Labeled cells are nebulized and mixed with heated argon gas to dry the cell containing particles. The sample-gas mixture is focused and ignited with an argon plasma torch.  This breaks the cells into their individual atoms and creates an ion cloud. Abundant low weight ions generated from environmental air and biological molecules are removed using a quadrupole mass analyzer. The remaining heavy ions from the antibody tags are quantified by Time-of-flight mass spectrometry. [1] Ion abundances correlate with the amount of target per cell and can be used to infer cellular qualities. [2]

Mass spectrometry's sensitivity to detect different ions allows measurements of upwards of 50 targets per cell while avoiding issues with spectral overlap seen when using fluorescent probes. [3] [4] However, this sensitivity also means trace heavy metal contamination is a concern. [5] Using large numbers of probes creates new problems in analyzing the high dimensional data generated. [6]

Figure 1: Major steps of a CyTOF procedure. Isotope chelation, antibody tagging, cellular staining, and aerosol injection into the ICP-MS, and data analysis. CyTOF Workflow.png
Figure 1: Major steps of a CyTOF procedure. Isotope chelation, antibody tagging, cellular staining, and aerosol injection into the ICP-MS, and data analysis.

History

In 1994 Tsutomu Nomizu and colleagues at Nagoya University performed the first mass spectrometry experiments of single cells. Nomizu realized that single cells could be nebulized, dried, and ignited in plasma to generate clouds of ions which could be detected by emission spectrometry. [7] In this type of experiment elements such as calcium within the cell could be quantified. Inspired by Flow cytometry, in 2007 Scott D. Tanner built upon this ICP-MS with the first multiplexed assay using lanthanide metals to label DNA and cell surface markers. [8] In 2008 Tanner described the tandem attachment of a flow cytometer to an ICP-MS instrument as well as new antibody tags that would allow massively multiplexed analysis of cell markers. [9] By further optimizing the detection speed and sensitivity of this flow coupled to ICP-MS they built the first CyTOF instrument. [1]

The CyTOF instrument was originally owned by the Canadian company DVS Sciences but is now the exclusive product of Fluidigm after their acquisition in 2014 of DVS sciences. In 2022 Fluidigm received a capitol infusion and changed its name to Standard BioTools. [10] There have been 4 iterations of the CyTOF apparatus named CyTOF, CyTOF2, Helios™ [5] and CyTOF XT. [11] The successive improvements were largely in increased detection range and software parameters with the Helios instrument able to detect from metals ranging from yttrium-89 to bismuth-209 and throughput and analyze 2000 events per minute.

Workflow

The Lanthanide group of elements are used for tagging antibodies, as the background in biological samples is very low. [12] When choosing the appropriate isotope for the biomarker, low expression biomarkers should be paired with an isotope that has high signal intensity. [13] If a less pure isotope must be used, it should be paired with a low expression biomarker, to minimize any non specific binding or background.

Isotope polymers are constructed using diethylenetriaminepentaacetic acid (DTPA) chelator to bind ions together. [13] The polymer terminates with a thiol or a maleimide that links it to reduced disulfides in the Fc region of the antibody. [14] Four to five polymers are bound to an antibody, resulting in about 100 isotope atoms per antibody. [14] Tagged antibodies may be in solution, conjugated to beads, or surface immobilized. The cell staining follows the same procedures as in fluorescent staining for flow cytometry. [14]

To distinguish between live and dead cells, cells can be probed with rhodium, an intercalator which can only penetrate dead cells. Then all cells are fixed and stained with iridium, which penetrates all cells, to be able to visualize which are alive. [14]

The cell introduction method of the mass cytometer is an aerosol splitter injection. [13] The cells are then captured in a stream of argon gas, then transported to the plasma where they are vaporized, atomized, and ionized. The cell is now a cloud of ions, which passes into the ion optics center. Then a time of flight analyzer is used to measure the mass of the ions.

Data analysis

Ions are accelerated through the spectrometer in pulses. The electron cloud generated from a single cell is typically 10-150 pulses. The output of a Helios™ run is a binary integrated mass data (IMD) file that contains electron intensities measured from the ions for each mass channel. The continuous pulses must be resolved into individual cell events corresponding to the ion cloud generated from one cell. Each bin of between 10-150 pulses that passes the user set lower convolution threshold, is considered a cell event by the Helios™ software. [1] [5] The lower convolution threshold is the minimal ion count that must be reached across all ion channels to be considered a cell event. The value for this parameter increases with the number of ions being measured and thus more counts are required to define a cell event when more labels are used. [5]  

For data analysis, the IMD file is converted into the flow cytometry standard (FCS) format. This file contains the total ion counts for each channel for every cell arranged in a matrix and is the same file generated during flow cytometry. [5] Manual gating of this data can be performed as is done for flow cytometry and most of the tools available for flow cytometry analysis have been ported to CyTOF (See flow cytometry bioinformatics). [6] CyTOF data is typically high dimensional. To delineate relationships between cell populations dimensionality reduction algorithms are often used. Several multidimensional analysis clustering algorithms are common. Popular tools include tSNE, FlowSOM, and the diffusion pseudo time (DPT). [6] The downstream analysis methods depend on the research goals.

Applications

CyTOF provides important information at a single cell level about protein expression, immunophenotype, and functional characterization. It is a valuable tool in immunology, where the large number of parameters has helped to elucidate the workings of this complex system. [15] [13] For example, natural killer cells have diverse properties affected by numerous markers in various combinations, which could not be analyzed with ease prior to this technology. [16] Simultaneously measuring many biomarkers makes it possible to identify over 30 distinct immunophenotype subsets within one complex group of cells. [15] This can help to more fully characterize immune function, infectious disease, and cancers, and understand cells response to therapy.

Advantages and disadvantages

The major advantage of CYTOF is the ability to investigate a larger number of parameters per panel than other cytometry methods. This allows a greater understanding of complex and heterogeneous cell populations, without the need for many complex and overlapping panels. Panels can include up to 45 antibodies, as opposed to the 10 that can be done in conventional flow cytometry but require great expertise to design. [15] However, development of spectral flow cytometry has closed the gap between flow and mass cytometry in terms of the maximum number of antibodies that can be used. More antibodies per panel saves on time, allows understanding of a larger picture, and requires fewer numbers of cells per experiment, which is particularly advantageous when samples are limited such as with tumour studies. [4]

The use of the heavy metal isotopes also lowers background when compared to using fluorescent antibodies. Some cell types, such as myeloid cells, have high rates of autofluorescence that create a lot of background noise in flow cytometry. [4] However, the rare heavy metal isotopes used are not present in biological systems, therefore there is very little or no background seen, and overall sensitivity is increased. [4] The detection overlap between the different heavy metals is also very low compared to the overlap seen in fluorescent cytometry, which makes it much simpler to design a panel of many markers.

Fluorescent dyes are subject to photobleaching, requiring the entire process to happen within a few hours after staining. Metal tagged antibodies however are viable for up to two weeks without losing signal, adding more flexibility to experiments. The stained samples can also be cryopreserved, which may be particularly useful for clinical trials when samples are collected over a longer period of time. [4]

Costs of CyTOF are high, as the metal-tagged antibodies and antibody conjunction kits are expensive.  A major downside of CyTOF is that acquisition flow rate is quite slow compared to flow cytometry, by almost an order of magnitude. [13] [4] Because heavy metals are common in laboratory reagents, avoiding contamination during sample preparation is very important.

Related Research Articles

<span class="mw-page-title-main">Inductively coupled plasma mass spectrometry</span> Type of mass spectrometry that uses an inductively coupled plasma to ionize the sample

Inductively coupled plasma mass spectrometry (ICP-MS) is a type of mass spectrometry that uses an inductively coupled plasma to ionize the sample. It atomizes the sample and creates atomic and small polyatomic ions, which are then detected. It is known and used for its ability to detect metals and several non-metals in liquid samples at very low concentrations. It can detect different isotopes of the same element, which makes it a versatile tool in isotopic labeling.

<span class="mw-page-title-main">Proteomics</span> Large-scale study of proteins

Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In addition, other kinds of proteins include antibodies that protect an organism from infection, and hormones that send important signals throughout the body.

Glycomics is the comprehensive study of glycomes, including genetic, physiologic, pathologic, and other aspects. Glycomics "is the systematic study of all glycan structures of a given cell type or organism" and is a subset of glycobiology. The term glycomics is derived from the chemical prefix for sweetness or a sugar, "glyco-", and was formed to follow the omics naming convention established by genomics and proteomics.

<span class="mw-page-title-main">Flow cytometry</span> Lab technique in biology and chemistry

Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.

<span class="mw-page-title-main">Plasma cell</span> White blood cell that secretes large volumes of antibodies

Plasma cells, also called plasma B cells or effector B cells, are white blood cells that originate in the lymphoid organs as B lymphocytes and secrete large quantities of proteins called antibodies in response to being presented specific substances called antigens. These antibodies are transported from the plasma cells by the blood plasma and the lymphatic system to the site of the target antigen, where they initiate its neutralization or destruction. B cells differentiate into plasma cells that produce antibody molecules closely modeled after the receptors of the precursor B cell.

Surface-enhanced laser desorption/ionization (SELDI) is a soft ionization method in mass spectrometry (MS) used for the analysis of protein mixtures. It is a variation of matrix-assisted laser desorption/ionization (MALDI). In MALDI, the sample is mixed with a matrix material and applied to a metal plate before irradiation by a laser, whereas in SELDI, proteins of interest in a sample become bound to a surface before MS analysis. The sample surface is a key component in the purification, desorption, and ionization of the sample. SELDI is typically used with time-of-flight (TOF) mass spectrometers and is used to detect proteins in tissue samples, blood, urine, or other clinical samples, however, SELDI technology can potentially be used in any application by simply modifying the sample surface.

A tandem mass tag (TMT) is a chemical label that facilitates sample multiplexing in mass spectrometry (MS)-based quantification and identification of biological macromolecules such as proteins, peptides and nucleic acids. TMT belongs to a family of reagents referred to as isobaric mass tags which are a set of molecules with the same mass, but yield reporter ions of differing mass after fragmentation. The relative ratio of the measured reporter ions represents the relative abundance of the tagged molecule, although ion suppression has a detrimental effect on accuracy. Despite these complications, TMT-based proteomics has been shown to afford higher precision than Label-free quantification. In addition to aiding in protein quantification, TMT tags can also increase the detection sensitivity of certain highly hydrophilic analytes, such as phosphopeptides, in RPLC-MS analyses.

<span class="mw-page-title-main">Protein mass spectrometry</span> Application of mass spectrometry

Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Mass spectrometry is an important method for the accurate mass determination and characterization of proteins, and a variety of methods and instrumentations have been developed for its many uses. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. It can also be used to localize proteins to the various organelles, and determine the interactions between different proteins as well as with membrane lipids.

<span class="mw-page-title-main">Quantitative proteomics</span> Analytical chemistry technique

Quantitative proteomics is an analytical chemistry technique for determining the amount of proteins in a sample. The methods for protein identification are identical to those used in general proteomics, but include quantification as an additional dimension. Rather than just providing lists of proteins identified in a certain sample, quantitative proteomics yields information about the physiological differences between two biological samples. For example, this approach can be used to compare samples from healthy and diseased patients. Quantitative proteomics is mainly performed by two-dimensional gel electrophoresis (2-DE), preparative one-dimensional gel electrophoresis, or mass spectrometry (MS). However, a recent developed method of quantitative dot blot (QDB) analysis is able to measure both the absolute and relative quantity of an individual proteins in the sample in high throughput format, thus open a new direction for proteomic research. In contrast to 2-DE, which requires MS for the downstream protein identification, MS technology can identify and quantify the changes.

<span class="mw-page-title-main">Ion-mobility spectrometry–mass spectrometry</span>

Ion mobility spectrometry–mass spectrometry (IMS-MS) is an analytical chemistry method that separates gas phase ions based on their interaction with a collision gas and their masses. In the first step, the ions are separated according to their mobility through a buffer gas on a millisecond timescale using an ion mobility spectrometer. The separated ions are then introduced into a mass analyzer in a second step where their mass-to-charge ratios can be determined on a microsecond timescale. The effective separation of analytes achieved with this method makes it widely applicable in the analysis of complex samples such as in proteomics and metabolomics.

A collision/reaction cell is a device used in inductively coupled plasma mass spectrometry to remove interfering ions through ion/neutral reactions.

<span class="mw-page-title-main">Selected reaction monitoring</span> Tandem mass spectrometry method

Selected reaction monitoring (SRM), also called Multiple reaction monitoring, (MRM), is a method used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ions is selected in the second mass spectrometer stage for detection.

<span class="mw-page-title-main">Microfluorimetry</span>

Microfluorimetry is an adaption of fluorimetry for studying the biochemical and biophysical properties of cells by using microscopy to image cell components tagged with fluorescent molecules. It is a type of microphotometry that gives a quantitative measure of the qualitative nature of fluorescent measurement and therefore, allows for definitive results that would have been previously indiscernible to the naked eye.

Mass spectrometric immunoassay (MSIA) is a rapid method is used to detect and/ or quantify antigens and or antibody analytes. This method uses an analyte affinity isolation to extract targeted molecules and internal standards from biological fluid in preparation for matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). This method allows for "top down" and "bottom up" analysis. This sensitive method allows for a new and improved process for detecting multiple antigens and antibodies in a single assay. This assay is also capable of distinguishing mass shifted forms of the same molecule via a panantibody, as well as distinguish point mutations in proteins. Each specific form is detected uniquely based on their characteristic molecular mass. MSIA has dual specificity because of the antibody-antigen reaction coupled with the power of a mass spectrometer.

<span class="mw-page-title-main">Single-cell analysis</span> Testbg biochemical processes and reactions in an individual cell

In the field of cellular biology, single-cell analysis is the study of genomics, transcriptomics, proteomics, metabolomics and cell–cell interactions at the single cell level. The concept of single-cell analysis originated in the 1970s. Before the discovery of heterogeneity, single-cell analysis mainly referred to the analysis or manipulation of an individual cell in a bulk population of cells at a particular condition using optical or electronic microscope. To date, due to the heterogeneity seen in both eukaryotic and prokaryotic cell populations, analyzing a single cell makes it possible to discover mechanisms not seen when studying a bulk population of cells. Technologies such as fluorescence-activated cell sorting (FACS) allow the precise isolation of selected single cells from complex samples, while high throughput single cell partitioning technologies, enable the simultaneous molecular analysis of hundreds or thousands of single unsorted cells; this is particularly useful for the analysis of transcriptome variation in genotypically identical cells, allowing the definition of otherwise undetectable cell subtypes. The development of new technologies is increasing our ability to analyze the genome and transcriptome of single cells, as well as to quantify their proteome and metabolome. Mass spectrometry techniques have become important analytical tools for proteomic and metabolomic analysis of single cells. Recent advances have enabled quantifying thousands of protein across hundreds of single cells, and thus make possible new types of analysis. In situ sequencing and fluorescence in situ hybridization (FISH) do not require that cells be isolated and are increasingly being used for analysis of tissues.

<span class="mw-page-title-main">Mass cytometry</span> Laboratory technique

Mass cytometry is a mass spectrometry technique based on inductively coupled plasma mass spectrometry and time of flight mass spectrometry used for the determination of the properties of cells (cytometry). In this approach, antibodies are conjugated with isotopically pure elements, and these antibodies are used to label cellular proteins. Cells are nebulized and sent through an argon plasma, which ionizes the metal-conjugated antibodies. The metal signals are then analyzed by a time-of-flight mass spectrometer. The approach overcomes limitations of spectral overlap in flow cytometry by utilizing discrete isotopes as a reporter system instead of traditional fluorophores which have broad emission spectra.

<span class="mw-page-title-main">Nanoscale secondary ion mass spectrometry</span>

NanoSIMS is an analytical instrument manufactured by CAMECA which operates on the principle of secondary ion mass spectrometry. The NanoSIMS is used to acquire nanoscale resolution measurements of the elemental and isotopic composition of a sample. The NanoSIMS is able to create nanoscale maps of elemental or isotopic distribution, parallel acquisition of up to seven masses, isotopic identification, high mass resolution, subparts-per-million sensitivity with spatial resolution down to 50 nm.

Complement-dependent cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When they are bound to surface antigen on target cell, the classical complement pathway is triggered by bonding protein C1q to these antibodies, resulting in formation of a membrane attack complex (MAC) and target cell lysis.

<span class="mw-page-title-main">Olga Ornatsky</span> Canadian Scientist

Olga Ornatsky is a Soviet born, Canadian scientist. Ornatsky co-founded DVS Sciences in 2004 along with Dmitry Bandura, Vladimir Baranov and Scott D. Tanner.

References

  1. 1 2 3 Dmitry Bandura; Vladimir Baranov; Olga Ornatsky; Scott D. Tanner; Antonov A; Kinach R; Lou X; Pavlov S; Vorobiev S; Dick JE (August 2009). "Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry". Analytical Chemistry. 81 (16): 6813–22. doi:10.1021/ac901049w. PMID   19601617.
  2. ↑ Wang W, Su B, Pang L, Qiao L, Feng Y, Ouyang Y, et al. (June 2020). "High-dimensional immune profiling by mass cytometry revealed immunosuppression and dysfunction of immunity in COVID-19 patients". Cellular & Molecular Immunology. 17 (6): 650–652. doi:10.1038/s41423-020-0447-2. PMC   7186533 . PMID   32346099.
  3. ↑ Spitzer MH, Nolan GP (May 2016). "Mass Cytometry: Single Cells, Many Features". Cell. 165 (4): 780–91. doi:10.1016/j.cell.2016.04.019. PMC   4860251 . PMID   27153492.
  4. 1 2 3 4 5 6 Gadalla R, Noamani B, MacLeod BL, Dickson RJ, Guo M, Xu W, et al. (2019). "Validation of CyTOF Against Flow Cytometry for Immunological Studies and Monitoring of Human Cancer Clinical Trials". Frontiers in Oncology. 9: 415. doi: 10.3389/fonc.2019.00415 . PMC   6534060 . PMID   31165047.
  5. 1 2 3 4 5 Olsen LR, Leipold MD, Pedersen CB, Maecker HT (February 2019). "The anatomy of single cell mass cytometry data". Cytometry. Part A. 95 (2): 156–172. doi: 10.1002/cyto.a.23621 . PMID   30277658.
  6. 1 2 3 Palit S, Heuser C, de Almeida GP, Theis FJ, Zielinski CE (2019). "Meeting the Challenges of High-Dimensional Single-Cell Data Analysis in Immunology". Frontiers in Immunology. 10: 1515. doi: 10.3389/fimmu.2019.01515 . PMC   6634245 . PMID   31354705.
  7. ↑ Nomizu T, Kaneco S, Tanaka T, Ito D, Kawaguchi H, Vallee BT (1994-10-01). "Determination of Calcium Content in Individual Biological Cells by Inductively Coupled Plasma Atomic Emission Spectrometry". Analytical Chemistry. 66 (19): 3000–3004. doi:10.1021/ac00091a004.
  8. ↑ Tanner SD, Ornatsky O, Bandura DR, Baranov VI (March 2007). "Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology". Spectrochimica Acta Part B: Atomic Spectroscopy. 62 (3): 188–195. Bibcode:2007AcSpe..62..188T. doi:10.1016/j.sab.2007.01.008.
  9. ↑ Tanner SD, Bandura DR, Ornatsky O, Baranov VI, Nitz M, Winnik MA (2008-01-01). "Flow cytometer with mass spectrometer detection for massively multiplexed single-cell biomarker assay". Pure and Applied Chemistry. 80 (12): 2627–2641. doi: 10.1351/pac200880122627 . S2CID   53364129.
  10. ↑ "Standard BioTools| Press Releases | Fluidigm Capital Infusion". www.globenewswire.com. Retrieved 2022-01-22.
  11. ↑ "CyTOF XT Released" (2021-05-21). Fluidigm.
  12. ↑ Ornatsky OI, Kinach R, Bandura DR, Lou X, Tanner SD, Baranov VI, et al. (2008-03-28). "Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry". Journal of Analytical Atomic Spectrometry. 23 (4): 463–469. doi:10.1039/B710510J. PMC   2600572 . PMID   19122859.
  13. 1 2 3 4 5 Chattopadhyay PK, Roederer M (July 2012). "Cytometry: today's technology and tomorrow's horizons". Methods. Flow Cytometry and Cell Sorting: the Next Generation. 57 (3): 251–8. doi:10.1016/j.ymeth.2012.02.009. PMC   3374038 . PMID   22391486.
  14. 1 2 3 4 Tanner SD, Baranov VI, Ornatsky OI, Bandura DR, George TC (May 2013). "An introduction to mass cytometry: fundamentals and applications". Cancer Immunology, Immunotherapy. 62 (5): 955–65. doi:10.1007/s00262-013-1416-8. PMID   23564178. S2CID   8607382.
  15. 1 2 3 Bjornson ZB, Nolan GP, Fantl WJ (August 2013). "Single-cell mass cytometry for analysis of immune system functional states". Current Opinion in Immunology. Host pathogens / Immune senescence. 25 (4): 484–94. doi:10.1016/j.coi.2013.07.004. PMC   3835664 . PMID   23999316.
  16. ↑ Leipold MD, Newell EW, Maecker HT (2015). "Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry". In Shaw AC (ed.). Immunosenescence. Methods in Molecular Biology. Vol. 1343. New York, NY: Springer. pp. 81–95. doi:10.1007/978-1-4939-2963-4_7. ISBN   978-1-4939-2963-4. PMC   4748856 . PMID   26420710.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.