Removing unwanted variation with CytofRUV to integrate multiple CyTOF datasets

Trussart, Marie; Teh, Charis E.; Tan, Tania; Leong, Lawrence; Gray, Daniel H.D.; Speed, Terence P.

doi:10.7554/elife.59630

Cited by 41 publications

(38 citation statements)

References 27 publications

(67 reference statements)

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“…The increasing use of CyTOF in clinical research requires standardized workflows and protocols for the acquisition and analysis of large-scale, multicentre, immune-monitoring clinical studies. Batch-correcting algorithms 89 and reproducible data generation are the basis for reliable and robust clinical analyses. For this objective, a method that uses a lyophilized core antibody panel to streamline blood sample processing has been implemented to reduce technical variation and standardize operations 90 and includes quality control by removing events caused by loss of stability and compensating for signal spillover resulting from isotopic impurities or oxide formation 91 .…”

Section: Single-cell Proteomicsmentioning

confidence: 99%

Immune cell profiling in atherosclerosis: role in research and precision medicine

Fernandez

Giannarelli

2021

Nat Rev Cardiol

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Inflammation is intimately involved at all stages of atherosclerosis and remains a substantial residual cardiovascular risk factor in optimally treated patients. The proof of concept that targeting inflammation reduces cardiovascular events in patients with a history of myocardial infarction has highlighted the urgent need to identify new immunotherapies to treat patients with atherosclerotic cardiovascular disease. Importantly, emerging data from new clinical trials show that successful immunotherapies for atherosclerosis need to be tailored to the specific immune alterations in distinct groups of patients. In this Review, we discuss how single-cell technologies -such as single-cell mass cytometry, single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing -are ideal for mapping the cellular and molecular composition of human atherosclerotic plaques and how these data can aid in the discovery of new precise immunotherapies. We also argue that single-cell data from studies in humans need to be rigorously validated in relevant experimental models, including rapidly emerging single-cell CRISPR screening technologies and mouse models of atherosclerosis. Finally, we discuss the importance of implementing single-cell immune monitoring tools in early phases of drug development to aid in the precise selection of the target patient population for data-driven translation into randomized clinical trials and the successful translation of new immunotherapies into the clinic.

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Section: Single-cell Proteomicsmentioning

confidence: 99%

Immune cell profiling in atherosclerosis: role in research and precision medicine

Fernandez

Giannarelli

2021

Nat Rev Cardiol

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“…Such an approach could be more appropriate in cases where the references’ expression distributions are less aligned. An alternative method, CytofRUV 30 , exploits the concept of pseudo-replicates to remove unwanted variation (RUV) between proteins and cells. Here, cells are grouped into subpopulations using FlowSOM 31 clustering.…”

Section: Discussionmentioning

confidence: 99%

An R-based reproducible and user-friendly preprocessing pipeline for CyTOF data

Crowell

Chevrier²,

Jacobs³

et al. 2022

F1000Res

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Mass cytometry (CyTOF) has become a method of choice for in-depth characterization of tissue heterogeneity in health and disease, and is currently implemented in multiple clinical trials, where higher quality standards must be met. Currently, preprocessing of raw files is commonly performed in independent standalone tools, which makes it difficult to reproduce. Here, we present an R pipeline based on an updated version of CATALYST that covers all preprocessing steps required for downstream mass cytometry analysis in a fully reproducible way. This new version of CATALYST is based on Bioconductor’s SingleCellExperiment class and fully unit tested. The R-based pipeline includes file concatenation, bead-based normalization, single-cell deconvolution, spillover compensation and live cell gating after debris and doublet removal. Importantly, this pipeline also includes different quality checks to assess machine sensitivity and staining performance while allowing also for batch correction. This pipeline is based on open source R packages and can be easily be adapted to different study designs. It therefore has the potential to significantly facilitate the work of CyTOF users while increasing the quality and reproducibility of data generated with this technology.

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“…This is due to technical differences, called batch effects, that affect the signal intensity (on which commonly used unsupervised analytical methods, such as SPADE, visNE, FlowSOM, CITRUS, and UMAP, are based) and need to be distinguished from true biological variability ( 28 ). Several algorithms have been proposed to normalize signal intensity to reduce batch effects before unsupervised cell cluster identification and to compare multiple datasets, such as CytofRUV ( 29 ), and JSOM ( 30 ). iMUBAC can even compare different datasets in the absence of shared technical replicates, used as reference samples, by overlaying cells from several healthy controls as anchors ( 31 ).…”

Section: Discussionmentioning

confidence: 99%

The Route of Vaccine Administration Determines Whether Blood Neutrophils Undergo Long-Term Phenotypic Modifications

et al. 2022

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Innate immunity modulates adaptive immunity and defines the magnitude, quality, and longevity of antigen-specific T- and B- cell immune memory. Various vaccine and administration factors influence the immune response to vaccination, including the route of vaccine delivery. We studied the dynamics of innate cell responses in blood using a preclinical model of non-human primates immunized with a live attenuated vaccinia virus, a recombinant Modified vaccinia virus Ankara (MVA) expressing a gag-pol-nef fusion of HIV-1, and mass cytometry. We previously showed that it induces a strong, early, and transient innate response, but also late phenotypic modifications of blood myeloid cells after two months when injected subcutaneously. Here, we show that the early innate effector cell responses and plasma inflammatory cytokine profiles differ between subcutaneous and intradermal vaccine injection. Additionally, we show that the intradermal administration fails to induce more highly activated/mature neutrophils long after immunization, in contrast to subcutaneous administration. Different batches of antibodies, staining protocols and generations of mass cytometers were used to generate the two datasets. Mass cytometry data were analyzed in parallel using the same analytical pipeline based on three successive clustering steps, including SPADE, and categorical heatmaps were compared using the Manhattan distance to measure the similarity between cell cluster phenotypes. Overall, we show that the vaccine per se is not sufficient for the late phenotypic modifications of innate myeloid cells, which are evocative of innate immune training. Its route of administration is also crucial, likely by influencing the early innate response, and systemic inflammation, and vaccine biodistribution.

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Removing unwanted variation with CytofRUV to integrate multiple CyTOF datasets

Cited by 41 publications

References 27 publications

Immune cell profiling in atherosclerosis: role in research and precision medicine

Immune cell profiling in atherosclerosis: role in research and precision medicine

An R-based reproducible and user-friendly preprocessing pipeline for CyTOF data

The Route of Vaccine Administration Determines Whether Blood Neutrophils Undergo Long-Term Phenotypic Modifications

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