FLOOD: FLow cytometric Orthogonal Orientation for Diagnosis

Jansen, Jeroen J.; Hilvering, Bart; Doel, André van den; Pickkers, Peter; Koenderman, Leo; Buydens, L.M.C.; Brink, Oscar F. van den

doi:10.1016/j.chemolab.2015.12.001

Cited by 10 publications

(22 citation statements)

References 45 publications

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“…For multi-dimensional analysis of the flow cytometry data the PCA based method FLOOD was used. [ 25 ] The neutrophils were identified from the original fcs files based on FSC/SSC and CD16+CD66b+ expression ( S1 Fig ) (FlowJo analysis software, Tree Star Inc., Ashland, Oregon). Only cells within this gate were exported as fcs3.0 files.…”

Section: Methodsmentioning

confidence: 99%

“…This has led to the development of data analysis tools that provide a multi-dimensional view on all markers expressed on individual cells. We have developed such a tool, FLOOD [ 25 ], that enables the recognition of multi-dimensional differences in neutrophil phenotypes by staining with many markers in addition to CD62L and CD16. Using this analysis method, we have previously reported that only normal neutrophils (CD16 bright CD62L bright ) are mobilized from the marginated pool upon a single anaerobic exercise bout.…”

Section: Introductionmentioning

confidence: 99%

“…Using this analysis method, we have previously reported that only normal neutrophils (CD16 bright CD62L bright ) are mobilized from the marginated pool upon a single anaerobic exercise bout. [ 25 ]…”

Section: Introductionmentioning

confidence: 99%

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Multi-dimensional flow cytometry analysis reveals increasing changes in the systemic neutrophil compartment during seven consecutive days of endurance exercise

et al. 2018

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Endurance exercise is associated with a transient increase in neutrophil counts in the peripheral blood. Here we investigate the impact of intensified endurance exercise on the neutrophil compartment. We hypothesized that intensified endurance exercise leads to mobilization of neutrophil subsets, which are normally absent in the blood. Furthermore, we followed the potential build-up of neutrophil activation and the impact on overnight recovery of the neutrophil compartment during a seven-day cycling tour. The neutrophil compartment was studied in 28 healthy amateur cyclists participating in an eight-day strenuous cycling tour. Blood samples were taken at baseline, after 4 days and after 7 days of cycling. The neutrophil compartment was analyzed in terms of numbers and its phenotype by deep phenotyping of flow cytometry data with the multi-dimensional analysis method FLOOD. Repeated endurance exercise led to a gradual increase in total neutrophil counts over the days leading to a 1.26 fold-increase (95%CI 1.01–1.51 p = 0.0431) in the morning of day 8. Flow cytometric measurements revealed the appearance of 2 additional neutrophil subsets: CD16brightCD62Ldim and CD16dimCD62Lbright. A complex change in neutrophil phenotypes was present characterized by decreased expression of both CD11b and CD62L and marked increased expression of LAIR-1, VLA-4 and CBRM1/5. The changes in expression were found on all neutrophils present in the blood. Strikingly, in strong contrast to our findings during acute inflammation evoked by LPS challenge, these neutrophils did not upregulate classical degranulation markers. In fact, our FLOOD analysis revealed that the exercise induced neutrophil phenotype did not overlap with the neutrophil subsets arising upon acute inflammation. In conclusion, during multiple days of endurance exercise the neutrophil compartment does not regain homeostasis overnight. Thereby our study supports the concept of a build-up of inflammatory cues during repeated endurance exercise training, causing a prolonged change of the systemic neutrophil compartment.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Multi-dimensional flow cytometry analysis reveals increasing changes in the systemic neutrophil compartment during seven consecutive days of endurance exercise

et al. 2018

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“…Multidimensional single-cell analysis was performed by principal component analysis (PCA) on log-normalized MFIs of CD3/ CD19/CD56-negative cells in the monocyte FSC/SSC gate as described by Jansen et al [17] with a few modifications. In short, single-cell fluorescence intensities were extracted from fcs files using FCS Express V5 (De Novo Software, Glendale, CA, USA).…”

Section: Flow Cytometric Analysismentioning

confidence: 99%

Monocyte Subsets Are Differentially Lost from the Circulation during Acute Inflammation Induced by Human Experimental Endotoxemia

et al. 2017

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Three human monocyte subsets are recognized with different functions in the immune system: CD14⁺⁺/CD16^- classical monocytes (CM), CD14⁺⁺/CD16⁺ intermediate monocytes (IM) and CD14⁺/CD16⁺⁺ non-classical monocytes (NCM). Increased IM and NCM percentages have been reported under inflammatory conditions, yet little is known about monocyte subsets at the onset of inflammation. The human endotoxemia model is uniquely capable of studying the first phases of acute inflammation induced by intravenous injection of 2 ng/kg bodyweight lipopolysaccharide (LPS) into healthy volunteers. After that, monocyte subset counts, activation/differentiation status and chemokine levels were studied over 24 h. The numbers of all subsets were decreased by >95% after LPS injection. CM numbers recovered first (3- 6 h), followed by IM (6-8 h) and NCM numbers (8-24 h). Similarly, increased monocyte counts were observed first in CM (8 h), followed by IM and NCM (24 h). Monocytes did not display a clear activated phenotype (minor increase in CD11b and CD38 expression). Plasma levels of CCL2, CCL4 and CX₃CL1 closely resembled the cell numbers of CM, IM and NCM, respectively. Our study provides critical insights into the earliest stages of acute inflammation and emphasizes the necessity to stain for different monocyte subsets when studying the role of monocytes in disease, as neither function nor kinetics of the subsets overlap.

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“…Two methods, Automated Population Separator (APS) 13 and Flow cytometric Orthogonal Orientation for Diagnosis (FLOOD) 14 use PCA. The advantage of PCA is that you can represent the single cells together with the interrelated expressions of the markers in a biplot.…”

Section: Introductionmentioning

confidence: 99%

Novel data analysis method for multicolour flow cytometry links variability of multiple markers on single cells to a clinical phenotype

Tinnevelt

Kokla

Hilvering

et al. 2017

Sci Rep

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Multicolour Flow Cytometry (MFC) produces multidimensional analytical data on the quantitative expression of multiple markers on single cells. This data contains invaluable biomedical information on (1) the marker expressions per cell, (2) the variation in such expression across cells, (3) the variability of cell marker expression across samples that (4) may vary systematically between cells collected from donors and patients. Current conventional and even advanced data analysis methods for MFC data explore only a subset of these levels. The Discriminant Analysis of MultiAspect CYtometry (DAMACY) we present here provides a comprehensive view on health and disease responses by integrating all four levels. We validate DAMACY by using three distinct datasets: in vivo response of neutrophils evoked by systemic endotoxin challenge, the clonal response of leukocytes in bone marrow of acute myeloid leukaemia (AML) patients, and the complex immune response in blood of asthmatics. DAMACY provided good accuracy 91–100% in the discrimination between health and disease, on par with literature values. Additionally, the method provides figures that give insight into the marker expression and cell variability for more in-depth interpretation, that can benefit both physicians and biomedical researchers to better diagnose and monitor diseases that are reflected by changes in blood leukocytes.

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FLOOD: FLow cytometric Orthogonal Orientation for Diagnosis

Cited by 10 publications

References 45 publications

Multi-dimensional flow cytometry analysis reveals increasing changes in the systemic neutrophil compartment during seven consecutive days of endurance exercise

Multi-dimensional flow cytometry analysis reveals increasing changes in the systemic neutrophil compartment during seven consecutive days of endurance exercise

Monocyte Subsets Are Differentially Lost from the Circulation during Acute Inflammation Induced by Human Experimental Endotoxemia

Novel data analysis method for multicolour flow cytometry links variability of multiple markers on single cells to a clinical phenotype

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