Analyses of large flow cytometry datasets

Stuchlý, Jan; Kalina, Tomáš

doi:10.1002/cyto.a.22431

Cited by 7 publications

(6 citation statements)

References 12 publications

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“…FCS files were normalized using the method based on landmark registration2627. A probability binning algorithm was applied on the normalized dataset as described previously182829, using flowFP implementation in R package with the following modification – the algorithm preferentially split the bins above the fluorescence background threshold (the threshold is set for each channel separately) to improve the resolution of non-negative populations.…”

Section: Methodsmentioning

confidence: 99%

Common Variable Immunodeficiency patients with a phenotypic profile of immunosenescence present with thrombocytopenia

Stuchlý

Kanderová

Vlková

et al. 2017

Sci Rep

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Common variable immunodeficiency (CVID) is a heterogeneous group of diseases. Our aim was to define sub-groups of CVID patients with similar phenotypes and clinical characteristics. Using eight-color flow cytometry, we analyzed both B- and T-cell phenotypes in a cohort of 88 CVID patients and 48 healthy donors. A hierarchical clustering of probability binning “bins” yielded a separate cluster of 22 CVID patients with an abnormal phenotype. We showed coordinated proportional changes in naïve CD4+ T-cells (decreased), intermediate CD27− CD28+ CD4+ T-cells (increased) and CD21low B-cells (increased) that were stable for over three years. Moreover, the lymphocytes’ immunophenotype in this patient cluster exhibited features of profound immunosenescence and chronic activation. Thrombocytopenia was only found in this cluster (36% of cases, manifested as Immune Thrombocytopenia (ITP) or Evans syndrome). Clinical complications more frequently found in these patients include lung fibrosis (in 59% of cases) and bronchiectasis (55%). The degree of severity of these symptoms corresponded to more deviation from normal levels with respect to CD21low B-cells, naïve CD4+ and CD27− CD28+ over three years. Moreover, th-cells. Next-generation sequencing did not reveal any common genetic background. We delineate a subgroup of CVID patients with activated and immunosenescent immunophenotype of lymphocytes and distinct set of clinical complications without common genetic background.

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Section: Methodsmentioning

confidence: 99%

Common Variable Immunodeficiency patients with a phenotypic profile of immunosenescence present with thrombocytopenia

Stuchlý

Kanderová

Vlková

et al. 2017

Sci Rep

Self Cite

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“…The latter approach can ultimately be used for software‐guided classification of either individual cells to specific cell populations represented in the database, or specific populations of malignant/aberrant cells to given WHO disease categories . However, to provide robust and fully comparable multicenter results, sample preparation and instrument setup and data acquisition must be performed accurately and in a standardized manner . For this purpose, clear and robust standard operating protocols (SOPs), trained personnel, and QA tools are essential.…”

Section: Discussionmentioning

confidence: 99%

Quality assessment program forEuroFlow protocols: Summary results of four‐year (2010–2013) quality assurance rounds

et al. 2014

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Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry.

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“…Computational analysis. FCS files were normalized using the method based on landmark registration 26,27 .…”

Section: Methodsmentioning

confidence: 99%

Common Variable Immunodeficiency Patients With a Phenotypic Profile of Immunosenescence Present With Thrombocytopenia

Kalina¹

2016

Preprint

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Common variable immunodeficiency (CVID) is a heterogeneous group of diseases. Our aim was to define subgroups of CVID patients with similar phenotypes and clinical characteristics. Using eightcolor flow cytometry, we analyzed both Band T-cell phenotypes in a cohort of 88 CVID patients and 48 healthy donors. A hierarchical clustering of probability binning "bins" yielded a separate cluster of 22 CVID patients with an abnormal phenotype. We showed coordinated proportional changes in naïve CD4+ T-cells (decreased), intermediate CD27− CD28+ CD4+ T-cells (increased) and CD21low B-cells (increased) that were stable for over three years. Moreover, the lymphocytes' immunophenotype in this patient cluster exhibited features of profound immunosenescence and chronic activation. Thrombocytopenia was only found in this cluster (36% of cases, manifested as Immune Thrombocytopenia (ITP) or Evans syndrome). Clinical complications more frequently found in these patients include lung fibrosis (in 59% of cases) and bronchiectasis (55%). The degree of severity of these symptoms corresponded to more deviation from normal levels with respect to CD21low B-cells, naïve CD4+ and CD27− CD28+ over three years. Moreover, th-cells. Next-generation sequencing did not reveal any common genetic background. We delineate a subgroup of CVID patients with activated and immunosenescent immunophenotype of lymphocytes and distinct set of clinical complications without common genetic background. Common variable immunodeficiency (CVID) is a heterogeneous collection of diseases defined as hypogammaglobulinemia of unknown cause (secondary hypogammaglobulinemia excluded) with markedly decreased IgG and IgA levels, with or without low IgM levels, displaying a lack of antibody response to vaccination. It is clinically accompanied by infections, autoimmunity, granulomatous disease and, in some cases, lymphoproliferation. In a large study by Resnick 1 , 94% of the patients had a history of infections, while autoimmunity was found in 28%

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Analyses of large flow cytometry datasets

Cited by 7 publications

References 12 publications

Common Variable Immunodeficiency patients with a phenotypic profile of immunosenescence present with thrombocytopenia

Common Variable Immunodeficiency patients with a phenotypic profile of immunosenescence present with thrombocytopenia

Quality assessment program forEuroFlow protocols: Summary results of four‐year (2010–2013) quality assurance rounds

Common Variable Immunodeficiency Patients With a Phenotypic Profile of Immunosenescence Present With Thrombocytopenia

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