Standardized Multi-Color Flow Cytometry and Computational Biomarker Discovery

Schlickeiser, Stephan; Streitz, Mathias; Sawitzki, Birgit

doi:10.1007/978-1-4939-3139-2_15

Cited by 12 publications

(9 citation statements)

References 48 publications

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“…For initial clustering during SPADE analysis (to generate the tree), expression of HLA-DR, CD11c, CD1c, CD141, slan (MDC8), CD14, and CD123 was used. An adapted script provided by Stephan Schlickeiser (Institute of Medical Immunology, Charité University Medicine, Berlin, Germany) (23) was used to perform the clustering analysis in R (24). The expression data of three independent donors were used to generate 120 clusters.…”

Section: Methodsmentioning

confidence: 99%

Anaphylatoxin C5a Regulates 6-Sulfo-LacNAc Dendritic Cell Function in Human through Crosstalk with Toll-Like Receptor-Induced CREB Signaling

et al. 2017

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Activation of antigen-presenting dendritic cells (DCs) and the complement system are essential early events in the immune defense against invading pathogens. Recently, we and others demonstrated immunological crosstalk between signaling from receptors recognizing complement activation products and PAMPs on DCs. This affects DC effector function, as demonstrated by the finding that C5a prevents induction of pro-inflammatory cytokines by toll-like receptor (TLR) ligands in human monocyte-derived DCs (moDCs). Here, we demonstrate that this regulatory crosstalk is specifically important in 6-sulfo LacNAc dendritic cells (slanDCs), the most pro-inflammatory DC subset found in human. C5aR and TLR signaling show profound interference in the ERK/p38/CREB1 signaling pathways. C5aR signaling accelerates TLR-induced CREB1 phosphorylation both in moDC and slanDC. This is key in the regulatory effect of C5a on pro-inflammatory DC maturation by mediating induction of IL-10, which subsequently inhibits pro-inflammatory cytokine production via negative feedback signaling. Importantly, the regulatory effect of C5a affects T-cell immunity by decreasing Th1 and cytotoxic CD8 T-cell responses. The finding that the pro-inflammatory effector function of slanDC can be down modulated by activation products of the complement system highlights the existence of intricate regulatory interactions between various arms of the immune system. Intensive immune monitoring of patients suffering from complement-mediated diseases or patients receiving complement modulating compounds can give more inside in the contribution of complement receptor and TLR crosstalk in APCs in disease.

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Section: Methodsmentioning

confidence: 99%

Anaphylatoxin C5a Regulates 6-Sulfo-LacNAc Dendritic Cell Function in Human through Crosstalk with Toll-Like Receptor-Induced CREB Signaling

et al. 2017

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“…Reproducible flow cytometry starts at the sample preparation stage, with clearly defined standard operating procedures (SOPs) for sample handling and staining, followed by an appropriate setup of the flow cytometer 20 . The use of setup beads, a proper panel design and standardized nomenclature for markers and channels 21 all contribute to increasing the quality and reproducibility of flow cytometry experiments.…”

Section: Standardization and Reproducibilitymentioning

confidence: 99%

Computational flow cytometry: helping to make sense of high-dimensional immunology data

2016

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Recent advances in flow cytometry allow scientists to measure an increasing number of parameters per cell, generating huge and high-dimensional datasets. To analyse, visualize and interpret these data, newly available computational techniques should be adopted, evaluated and improved upon by the immunological community. Computational flow cytometry is emerging as an important new field at the intersection of immunology and computational biology; it allows new biological knowledge to be extracted from high-throughput single-cell data. This Review provides non-experts with a broad and practical overview of the many recent developments in computational flow cytometry.

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“…Different consortia listed below aimed to address diverse levels of reproducibility: (a) the reproducibility of interpretation (all consortia; some argue that more is not necessary for their purpose such as Harmonemia (17), AIEOP-BFM group (3,(18)(19)(20)(21)(22)(23), and ERIC (21-23)), (b) the reproducibility of enumeration (prominently in immunology: The ONE Study (24,25), HIPC (14,26,27), but also in the leukemia residual disease detection: EuroFlow (28)(29)(30), COG group (31), and ERIC (21,22)), (c) the reproducibility of staining pattern is typically achieved when the same panel of reagents is used (The ONE Study (25), HIPC (26), EuroFlow (32,33), and COG (31)), which allows definition of the manual analysis strategy and also automated analysis tools, and (d) the reproducibility of patterns including the staining intensity that allows analysis standardization and database-assisted comparison to previous cases in interlaboratory settings: EuroFlow (34) or The Canadian National Transplant Research Program that analyzed multiple immune cells in multiple centers by automated analysis (35). Different consortia listed below aimed to address diverse levels of reproducibility: (a) the reproducibility of interpretation (all consortia; some argue that more is not necessary for their purpose such as Harmonemia (17), AIEOP-BFM group (3,(18)(19)(20)(21)(22)(23), and ERIC (21-23)), (b) the reproducibility of enumeration (prominently in immunology: The ONE Study (24,25), HIPC (14,2...…”

Section: Existing Efforts To Achieve Reproducibilitymentioning

confidence: 99%

“…They designed a panel of six tubes (seven to nine colors) and had those prepared as dried reagents formulated per single test. A detailed guide to their approach was published in 2016 (24). Later, they performed a single laboratory study reporting age and gender differences on a cohort of 98 healthy adults (49).…”

Section: Existing Efforts To Achieve Reproducibilitymentioning

confidence: 99%

Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges

Kalina

2019

Cytometry Pt A

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There is an agreement in the field that interlaboratory reproducibility of flow cytometry measurements as well as the whole studies might be improved by a consensual use of methodological approach. Typically, a consensus is made on a crucial markers needed in the immunostaining panel, sometimes on the particular fluorochrome conjugates and rarely on a complete set of methods for sample preparation. The term "standardization" is used to describe the complete set of methodical steps, while "harmonization" is used for partial agreement on the method. Standardization can provide a platform for improved reproducibility of cytometry results over prolonged periods of time, across different sites and across different instruments. For the purpose of structured discussion, several desired aims are described: common interpretation of the immunophenotype definition of a target subset, accurate quantification, reproducible pattern of a multicolor immunophenotype, and reproducible intensity of all measured parameters. An overview of how standardization was approached by several large consortia is provided: EuroFlow, The ONE Study, Human Immunology Project Consortium (HIPC), and several other groups. Their particular aims and the tools adopted to reach those aims are noted. How those standardization efforts were adopted in the field and how the resulting outcome was evaluated is reviewed. Multiple challenges in the instrument hardware design, instrument setup tools, reagent design, and quality features need to be addressed to achieve optimal standardization. Furthermore, the aims of different studies vary, and thus, the reasonable requirements for standardization differ. A framework of reference for the reasonable outcomes of different approaches is offered. Finally, it is argued that complete standardization is important not only for the reproducibility of measurements but also for education, for quality assessment and for algorithmic data analysis. The different standardized approaches can and in fact should serve as benchmarking reference tools for the development of future flow cytometry studies.

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Standardized Multi-Color Flow Cytometry and Computational Biomarker Discovery

Cited by 12 publications

References 48 publications

Anaphylatoxin C5a Regulates 6-Sulfo-LacNAc Dendritic Cell Function in Human through Crosstalk with Toll-Like Receptor-Induced CREB Signaling

Anaphylatoxin C5a Regulates 6-Sulfo-LacNAc Dendritic Cell Function in Human through Crosstalk with Toll-Like Receptor-Induced CREB Signaling

Computational flow cytometry: helping to make sense of high-dimensional immunology data

Reproducibility of Flow Cytometry Through Standardization: Opportunities and Challenges

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