Multiparameter analysis of stimulated human peripheral blood mononuclear cells: A comparison of mass and fluorescence cytometry

Nicholas, Katherine J.; Greenplate, Allison R.; Flaherty, David K.; Matlock, Brittany K.; San-Juan, Juan F.; Smith, Rita M.; Irish, Jonathan M.; Kalams, Spyros A.

doi:10.1002/cyto.a.22799

Cited by 55 publications

(70 citation statements)

References 37 publications

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“…The results from the mass cytometry analysis could to a certain extent be confirmed using a more conventional flow cytometry approach, an approach that has also been done by other studies (39, 54). The adjusted flow cytometry panel could provide a more direct clinical utility than a mass cytometry panel.…”

Section: Discussionsupporting

confidence: 63%

Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease

et al. 2017

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Chronic graft-versus-host disease (cGVHD) is a debilitating complication arising in around half of all patients treated with an allogeneic hematopoietic stem cell transplantation. Even though treatment of severe cGVHD has improved during recent years, it remains one of the main causes of morbidity and mortality in affected patients. Biomarkers in blood that could aid in the diagnosis and classification of cGVHD severity are needed for the development of novel treatment strategies that can alleviate symptoms and reduce the need for painful and sometimes complicated tissue biopsies. Methods that comprehensively profile complex biological systems such as the immune system can reveal unanticipated markers when used with the appropriate methods of data analysis. Here, we used mass cytometry, flow cytometry, enzyme-linked immunosorbent assay, and multiplex assays to systematically profile immune cell populations in 68 patients with varying grades of cGVHD. We identified multiple subpopulations across T, B, and NK-cell lineages that distinguished patients with cGVHD from those without cGVHD and which were associated in varying ways with severity of cGVHD. Specifically, initial flow cytometry demonstrated that patients with more severe cGVHD had lower mucosal-associated T cell frequencies, with a concomitant higher level of CD38 expression on T cells. Mass cytometry could identify unique subpopulations specific for cGVHD severity albeit with some seemingly conflicting results. For instance, patients with severe cGVHD had an increased frequency of activated B cells compared to patients with moderate cGVHD while activated B cells were found at a reduced frequency in patients with mild cGVHD compared to patients without cGVHD. Moreover, results indicate it may be possible to validate mass cytometry results with clinically viable, smaller flow cytometry panels. Finally, no differences in levels of blood soluble markers could be identified, with the exception for the semi-soluble combined marker B-cell activating factor/B cell ratio, which was increased in patients with mild cGVHD compared to patients without cGVHD. These findings suggest that interdependencies between such perturbed subpopulations of cells play a role in cGVHD pathogenesis and can serve as future diagnostic and therapeutic targets.

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Section: Discussionsupporting

confidence: 63%

Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease

et al. 2017

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“…These included immune cells in tonsil, infiltrating immune cells in glioma and melanoma, and tissue-specific cell subsets, such as cancer cell subsets, endothelial cells, glial cells, pericytes, and stem-like cells in gliomas. A difference in abundance of T cells observed between fluorescence and mass cytometry was determined to be due to use of different anti-CD3 antibody clones, as has been previously reported (25,26).…”

Section: Discussionmentioning

confidence: 55%

Single Cell Analysis of Human Tissues and Solid Tumors with Mass Cytometry

et al. 2017

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“…For both fluorescence cytometry and mass cytometry, cells were initially gated using DNA content (Hoescht – fluorescence cytometry) or intercalator (Iridium – mass cytometry) following established procedures to identify intact single cells and eliminate cell doublets and clusters from analysis (16, 76, 77). Single cells were then analyzed for intensity of antibody conjugates.…”

Section: Methodsmentioning

confidence: 99%

Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks

et al. 2016

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Cellular heterogeneity poses a significant challenge to understanding tissue level phenotypes and confounds conventional bulk analyses. To facilitate the analysis of signaling at the single-cell level in human tissues, we applied mass cytometry using CyTOF (Cytometry Time-of-Flight) to formalin-fixed paraffin-embedded (FFPE) normal and diseased intestinal specimens. We developed and validated a technique called FFPE-DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue), a single-cell approach for characterizing native signaling states from embedded solid tissue samples. We applied FFPE-DISSECT coupled to mass cytometry and found differential signaling by tumor necrosis factor α (TNF-α) in intestinal enterocytes, goblet cells and enteroendocrine cells, implicating the role of the downstream RAS-RAF-MEK-ERK signaling pathway in dictating goblet cell identity. In addition, application of FFPE-DISSECT, mass cytometry, and data-driven computational analyses to human colon specimens confirmed reduced differentiation in colorectal cancer (CRC) compared to normal colon, and revealed quantitative increases in inter- and intra-tissue heterogeneity in CRC with regards to the modular regulation of signaling pathways. Specifically, modular co-regulation of the kinases P38 and ERK, the translation regulator 4EBP1, and the transcription factor CREB in the proliferative compartment of the normal colon was loss in CRC, as evidenced by their impaired coordination over samplings of single cells in tissue. Our data suggest that this single-cell approach, applied in conjunction with genomic annotation, such as microsatellite instability and mutations in KRAS and BRAF, allows rapid and detailed characterization of cellular heterogeneity from clinical repositories of embedded human tissues. FFPE-DISSECT coupled of mass cytometry can be used for deriving cellular landscapes from archived patient samples, beyond CRC, and as a high resolution tool for disease characterization and subtyping.

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Multiparameter analysis of stimulated human peripheral blood mononuclear cells: A comparison of mass and fluorescence cytometry

Cited by 55 publications

References 37 publications

Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease

Combining Flow and Mass Cytometry in the Search for Biomarkers in Chronic Graft-versus-Host Disease

Single Cell Analysis of Human Tissues and Solid Tumors with Mass Cytometry

Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks

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