Multiple stained samples are not appropriate compensation controls

Roederer, Mario

doi:10.1002/cyto.a.21093

Cited by 5 publications

(9 citation statements)

References 1 publication

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“…These matrix elements, the so called spillover coefficients, guarantee the perfect compensation of single stained controls but in our two examples do not result in correct compensation of the multiple stained controls. In spite of Roederer’s expectation (2) we could demonstrate that the quality of the compensation of multi stained controls does not practically change by changing the size of the data sets of single stained controls.…”

contrasting

confidence: 61%

“…Roederer questions one aspect of our paper, our empirical finding that using multiple stained and single stained controls together give better overall compensation results (2). In his critique, he ignores our principal result, which consists of an optimization method for compensation that allows the use of additional samples beyond the single stained samples that are used for compensation in the commercial software developed by Roederer.…”

mentioning

confidence: 89%

“…We demonstrate that our method is superior to the standard approach to compensation supported by commercial flow cytometry software which uses only single stained control samples. Two investigators have published critiques (2, 3) of our manuscript to which we now respond.…”

mentioning

confidence: 99%

“…The utilized five fluorochromes are: (1) FITC (CD45-FITC Beckman Coulter, Cat# PN IM0782U), (2) PE (anti-human CD45-PE BioLegend, Cat# 304008), (3) Pacific Blue anti-human CD45-Pacific Blue BioLegend, Cat#304022), (4) PE-Cy7 (anti-human CD45-PE-Cy7 BioLegend, Cat#304016), (5) APC (CD45-APC Beckman Coulter, Cat# PN IM2473U), while the characteristics of the respective five band pass filters (center wavelength/band width in nm ) are: (1) 530/30, (2) 575/30, (3) 450/50, (4) 780/60, (5) 660/20.…”

mentioning

confidence: 99%

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Authors response to correspondence about an improved compensation method

Sugár¹,

González-Lergier²,

Sealfon³

2011

Cytometry

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contrasting

confidence: 61%

mentioning

confidence: 89%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Authors response to correspondence about an improved compensation method

Sugár¹,

González-Lergier²,

Sealfon³

2011

Cytometry

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“…Following identification of these subsets, each respective function was analyzed by creating a single gate. All functional responses shown were subjected to background subtraction on the basis of the unstimulated control for each patient, as described previously (37); all response data equal to or less than the 75th percentile for the background-subtracted values were removed. Coexpression analysis of IFN-␥, TNF-␣, IL-21, CD40L, and CD107a was performed with a Boolean gating strategy and the PESTLE and SPICE software suite (NIH/M.…”

Section: Methodsmentioning

confidence: 99%

Modulation of Vaccine-Induced CD4 T Cell Functional Profiles by Changes in Components of HIV Vaccine Regimens in Humans

Pissani

Schulte

Eller

et al. 2018

J Virol

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To date, six vaccine strategies have been evaluated in clinical trials for their efficacy at inducing protective immune responses against HIV infection. However, only the ALVAC-HIV/AIDSVAX B/E vaccine (RV144 trial) has demonstrated protection, albeit modestly (31%; = 0.03). One potential correlate of protection was a low-frequency HIV-specific CD4 T cell population with diverse functionality. Although CD4 T cells, particularly T follicular helper (Tfh) cells, are critical for effective antibody responses, most studies involving HIV vaccines have focused on humoral immunity or CD8 T cell effector responses, and little is known about the functionality and frequency of vaccine-induced CD4 T cells. We therefore assessed responses from several phase I/II clinical trials and compared them to responses to natural HIV-1 infection. We found that all vaccines induced a lower magnitude of HIV-specific CD4 T cell responses than that observed for chronic infection. Responses differed in functionality, with a CD40 ligand (CD40L)-dominated response and more Tfh cells after vaccination, whereas chronic HIV infection provoked tumor necrosis factor alpha (TNF-α)-dominated responses. The vaccine delivery route further impacted CD4 T cells, showing a stronger Th1 polarization after dendritic cell delivery than after intramuscular vaccination. In prime/boost regimens, the choice of prime and boost influenced the functional profile of CD4 T cells to induce more or less polyfunctionality. In summary, vaccine-induced CD4 T cell responses differ remarkably between vaccination strategies, modes of delivery, and boosts and do not resemble those induced by chronic HIV infection. Understanding the functional profiles of CD4 T cells that best facilitate protective antibody responses will be critical if CD4 T cell responses are to be considered a clinical trial go/no-go criterion. Only one HIV-1 candidate vaccine strategy has shown protection, albeit marginally (31%), against HIV-1 acquisition, and correlates of protection suggested that a multifunctional CD4 T cell immune response may be important for this protective effect. Therefore, the functional phenotypes of HIV-specific CD4 T cell responses induced by different phase I and phase II clinical trials were assessed to better show how different vaccine strategies influence the phenotype and function of HIV-specific CD4 T cell immune responses. The significance of this research lies in our comprehensive comparison of the compositions of the T cell immune responses to different HIV vaccine modalities. Specifically, our work allows for the evaluation of vaccination strategies in terms of their success at inducing Tfh cell populations.

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Immune Cell Phenotyping Using Flow Cytometry

Oughton

Kerkvliet

2015

CP Toxicology

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Fluorescent immunophenotyping uses fluorescently-conjugated antibodies to identify, characterize and quantify distinct subpopulations of cells within heterogeneous single-cell populations, either in the context of tissue (using fluorescence and imaging microscopy) or in a single-cell suspension (using multiparameter imaging microscopy, imaging cytometry, and/or flow cytometry). Flow cytometry is an optical, laser-based technology which analyzes the physical and fluorescent properties of cells in suspension in real-time as they flow through the instrument. This approach has a number of advantages over other techniques that can be used for characterizing cell populations in single-cell suspensions, in that it can nonsubjectively interrogate up to millions of cells and acquire data on the presence of different cell subpopulations and phenotypical changes within these populations in seconds. This unit describes basic procedures for the direct and indirect immunofluorescent staining of surface and intracellular proteins that are expressed by lymphoid cells which have been isolated from tissues or blood. Protocols for the resolution of dead cells and for the fixation of cells are also included. This unit also provides essential information relating to the selection and titration of antibodies, fluorochrome choice, spectral overlap and compensation, the use of controls, and the standardization of data acquisition and analysis. It also highlights new technologies and platforms that can be used to interrogate the presence of cell subpopulations and their phenotype to an even greater depth.

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Multiple stained samples are not appropriate compensation controls

Cited by 5 publications

References 1 publication

Authors response to correspondence about an improved compensation method

Authors response to correspondence about an improved compensation method

Modulation of Vaccine-Induced CD4 T Cell Functional Profiles by Changes in Components of HIV Vaccine Regimens in Humans

Immune Cell Phenotyping Using Flow Cytometry

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