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Systemic Lupus Erythematosus
Published in Jason Liebowitz, Philip Seo, David Hellmann, Michael Zeide, Clinical Innovation in Rheumatology, 2023
Vaneet K. Sandhu, Neha V. Chiruvolu, Daniel J. Wallace
Additional technological advances include an array-based multiplex ELISA system that has been used to analyze urine for 1,000 proteins. Phase 1 trials were able to identify multiple proteins in the urine of patients with LN that were not found in the urine of healthy controls.14 Separately, CYTOF mass cytometry (CyTOF) is used for comprehensive multidimensional single-cell phenotyping that uses rare earth metal isotope tags instead of fluorescent-tagged antibodies. One study using this technology showed significant differences between circulating immune cells in blood compared to healthy controls. They found high numbers of activated CD57+ CD8 T cells and reduced NK cell percentages in SLE patients.14
Time to ‘Couple’ Redox Biology with Exercise Immunology
Published in James N. Cobley, Gareth W. Davison, Oxidative Eustress in Exercise Physiology, 2022
Alex J. Wadley, Steven J. Coles
The development of CyTOF™ mass cytometry may be a solution to current experimental limitations. This technology has permitted high-resolution assessment of natural killer/T cell immunophenotype and functional analysis at the single-cell level (Kay, Strauss-Albee and Blish, 2016; Brodie and Tosevski, 2018). Like traditional flow cytometry, CyTOF™ mass cytometry utilises specific antibodies (labelled with metals in this instance) to detect the expression of various cellular antigens (Heck, Bishop and Ellis, 2019). Because the technology is based on time-of-flight mass spectrometry as a detection method, the limitations/complications associated with standard flow cytometry (e.g., spectral overlap) are ameliorated. Therefore, upwards of 50 different immunophenotypic and(or) functional molecules for a single cell may be detected simultaneously. So far protocols have been established to concurrently evaluate cytokines, transcription factors and immunophenotypic markers (Lin, Gupta and Maecker, 2015; Simoni et al., 2018). In theory, the analysis of protein/peptide markers of the intracellular redox environment (e.g., peroxiredoxin, thioredoxin, glutaredoxin, glutathione) as well as redox-sensitive transcription factors implicated with oxidative and reductive stress (Bellezza et al., 2018) could easily be incorporated into CyTOF™ mass cytometry workflows.
An overview of multiplexed analyses of CAR T-cell therapies: insights and potential
Published in Expert Review of Proteomics, 2021
Brittany Paige DePriest, Noah Vieira, Alan Bidgoli, Sophie Paczesny
Like spectral flow cytometry, mass cytometry, also called CyTOF, can assess high numbers of parameters (typically 40+). CyTOF is a technology that uses antibodies tagged with stable heavy metal ion isotopes (rather than fluorochromes) and provides readout using time of flight mass spectrometry [70]. Therefore, isotope labels have created high dimensional profiles to decipher proteins involved in cellular functioning using CyTOF at the single cell level [71,72]. The main of advantage of CyTOF is that it allows for the combination of more antibody specificities in a single sample without significant spill over between channels [52]. This technology is then combined with computational tools such as Citrix, PhenoGraph, SPADE, viSNE and X-shift that use various algorithms to analyze the complex datasets. CyTOF has been used to evaluate expression of surface or intracellular proteins related to T-cell function demonstrated at pre and post CAR-T infusion times resulting in identification of specific populations of CD4+ CAR-T cells associated with clinical outcome at 6 months [73]. CAR-T therapies can also be monitored via CyTOF technology as evident in diffuse large B-cell lymphoma (DLBCL) and ALL patients treated with Tisagenlecleucel [74] and multiple myeloma patients treated with novel BCMA CAR-T therapy [75]. Future direction includes functional profiling of genetically engineered hematopoietic progenitor stem cells (HPSCs) to allow for antigen-specific immunotherapy with avoidance of on-target, off-tumor toxicity, though this has currently only been shown in animal models [76].
The role of proteomics in the multiplexed analysis of gene alterations in human cancer
Published in Expert Review of Proteomics, 2021
Niraj Babu, Mohd Younis Bhat, Arivusudar Everad John, Aditi Chatterjee
Fluorescence-based cytometry techniques have been crucial for the study of immune systems at a cellular resolution. Although the complexity in compensation for spectral overlap and the vast parameters to be considered limit the application of fluorescence-based mass cytometry, the development of this technique has been driven by technological advancement. Particularly, the novel combination of mass spectrometry and flow cytometry have resulted in the technology of cytometry by time-of-flight (CyTOF), otherwise known as mass cytometry, which has been utilized in multiparametric analysis of single cells [144]. Using the combination of antibodies tagged with rare-earth metals, coupled with plasma ionization and TOF detector, CyTOF provides simultaneous profiling of close to 50 molecular parameters in each cell. Hence, CyTOF and its utility can significantly propel the quantification of disease characteristics in cancer cells and can also be used for tissue-based identification of molecular alterations in frozen and paraffin-embedded tumor samples [145].
Approaching complexity: systems biology and ms-based techniques to address immune signaling
Published in Expert Review of Proteomics, 2020
Joseph Gillen, Caleb Bridgwater, Aleksandra Nita-Lazar
One of the earliest methods that uses mass spectrometry to analyze single cells is mass cytometry, also known as cytometry by time-of-flight or CyTOF. There are many excellent reviews of CyTOF that explain the mechanisms, benefits, and concerns involved in these experiments (reviewed in [54–56]). Briefly, specific targets are labeled with heavy metal tags then the single cells isolated and atomized using argon plasma. Then using the sensitivity and accuracy of an elemental time-of-flight mass spectrometer to detect the heavy metal tags, CyTOF can quantify up to 40 possible elements at a single cell level. While the measurement of 40 individual elements is an improvement over the number of elements that can be simultaneously measured with fluorophores (a process limited to less than 20 simultaneous measurements), the method of does have drawbacks. First, CyTOF uses atomization of the cells with argon plasma to free the metal markers for detection and cannot be used to collect live cells and the sensitivity for detection of the mass reporters prevents the detection of extremely rare molecules.