Preanalytical Pitfalls in Untargeted Plasma Nuclear Magnetic Resonance Metabolomics of Endocrine Hypertension

Despite considerable morbidity and mortality, numerous cases of endocrine hypertension (EHT) forms, including primary aldosteronism (PA), pheochromocytoma and functional paraganglioma (PPGL), and Cushing's syndrome (CS), remain undetected. We aimed to establish signatures for the different forms of EHT, investigate potentially confounding effects and establish unbiased disease biomarkers. Plasma samples were obtained from 13 biobanks across seven countries and analyzed using untargeted NMR metabolomics. We compared unstratified samples of 106 PHT patients to 231 EHT patients, including 104 PA, 94 PPGL and 33 CS patients. Spectra were subjected to a multivariate statistical comparison of PHT to EHT forms and the associated signatures were obtained. Three approaches were applied to investigate and correct confounding effects. Though we found signatures that could separate PHT from EHT forms, there were also key similarities with the signatures of sample center of origin and sample age. The study design restricted the applicability of the corrections employed. With the samples that were available, no biomarkers for PHT vs. EHT could be identified. The complexity of the confounding effects, evidenced by their robustness to correction approaches, highlighted the need for a consensus on how to deal with variabilities probably attributed to preanalytical factors in retrospective, multicenter metabolomics studies

Mono/Multi-material Characterization Using Hyperspectral Images and Multi-Block Non-Negative Matrix Factorization

Plastic sorting is a very essential step in waste management, especially due to the presence of multilayer plastics. These monomaterial and multimaterial plastics are widely employed to enhance the functional properties of packaging, combining beneficial properties in thickness, mechanical strength, and heat tolerance. However, materials containing multiple polymer species need to be pretreated before they can be recycled as monomaterials and therefore should not end up in monomaterial streams. Industry 4.0 has significantly improved materials sorting of plastic packaging in speed and accuracy compared to manual sorting, specifically through Near Infrared Hyperspectral Imaging (NIRHSI) that provides an automated, fast, and accurate material characterization, without sample preparation. Identification of multimaterials with HSI however requires novel dedicated approaches for chemical pattern recognition. Non negative Matrix Factorization, NMF, is widely used for the chemical resolution of hyperspectral images. Chemically relevant model constraints may make it specifically valuable to identify multilayer plastics through HSI. Specifically, Multi Block Non Negative Matrix Factorization (MBNMF) with correspondence among different chemical species constraint may be used to evaluate the presence or absence of particular polymer species. To translate the MBNMF model into an evidence based sorting decision, we extended the model with an F test to distinguish between monomaterial and multimaterial objects. The benefits of our new approach, MBNMF, were illustrated by the identification of several plastic waste objects

Systematic reduction of Hyperspectral Images for high-throughput Plastic Characterization

Hyperspectral Imaging (HSI) combines microscopy and spectroscopy to assess the spatial distribution of spectroscopically active compounds in objects, and has diverse applications in food quality control, pharmaceutical processes, and waste sorting. However, due to the large size of HSI datasets, it can be challenging to analyze and store them within a reasonable digital infrastructure, especially in waste sorting where speed and data storage resources are limited. Additionally, as with most spectroscopic data, there is significant redundancy, making pixel and variable selection crucial for retaining chemical information. Recent high-tech developments in chemometrics enable automated and evidence-based data reduction, which can substantially enhance the speed and performance of Non-Negative Matrix Factorization (NMF), a widely used algorithm for chemical resolution of HSI data. By recovering the pure contribution maps and spectral profiles of distributed compounds, NMF can provide evidence-based sorting decisions for efficient waste management. To improve the quality and efficiency of data analysis on hyperspectral imaging (HSI) data, we apply a convex-hull method to select essential pixels and wavelengths and remove uninformative and redundant information. This process minimizes computational strain and effectively eliminates highly mixed pixels. By reducing data redundancy, data investigation and analysis become more straightforward, as demonstrated in both simulated and real HSI data for plastic sorting

Comprehensive multivariate evaluation of the effects on cell phenotypes in multicolor flow cytometry data using ANOVA simultaneous component analysis

This work proposes an approach to assess the effects observed in multicolor flow cytometry (MFC) experiments, for all markers and experimental factors simultaneously. It achieves this end by extending ANOVA simultaneous component analysis (ASCA), a multivariate version of ANOVA, to flow cytometry data. It is based on an initial multiset PCA model to describe the main variation patterns of cell marker expression, followed by an ASCA model on the histograms built from these PCA scores. This approach allows for determining the variations in cell phenotype distribution that are related to the experimental design. On a data set from a study of the immune response to prolonged physical exercise, the proposed method computed the effect size and statistical significance of all the experimental factors and their interactions. Most notably, it provided easily interpretable submodels for the overall effect of the walking exercise and for the interaction between exercise and the responsiveness to a bacterial stimulus. The application of a time-guided sequential clustering algorithm to the ASCA scores revealed a stratification of the studied individuals based on their neutrophil activation dynamics. These effects were not clearly detectable using PCA alone. In comparison with pairwise classification models by DAMACY (a discriminant analysis method for MFC data), ASCA results were less detailed in describing differences between specific samples, but had the advantage of modeling several factors and levels simultaneously. Such characteristics make the proposed implementation of ASCA an effective and complementary addition to the chemometric methodologies for the analysis of MFC data

Flow cytometric evaluation of the neutrophil compartment in COVID-19 at hospital presentation: A normal response to an abnormal situation

Coronavirus disease 2019 (COVID-19) is a rapidly emerging pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Critical COVID-19 is thought to be associated with a hyper-inflammatory process that can develop into acute respiratory distress syndrome, a critical disease normally mediated by dysfunctional neutrophils. This study tested the hypothesis whether the neutrophil compartment displays characteristics of hyperinflammation in COVID-19 patients. Therefore, a prospective study was performed on all patients with suspected COVID-19 presenting at the emergency room of a large academic hospital. Blood drawn within 2 d after hospital presentation was analyzed by point-of-care automated flow cytometry and compared with blood samples collected at later time points. COVID-19 patients did not exhibit neutrophilia or eosinopenia. Unexpectedly neutrophil activation markers (CD11b, CD16, CD10, and CD62L) did not differ between COVID-19-positive patients and COVID-19-negative patients diagnosed with other bacterial/viral infections, or between COVID-19 severity groups. In all patients, a decrease was found in the neutrophil maturation markers indicating an inflammation-induced left shift of the neutrophil compartment. In COVID-19 this was associated with disease severity

High-throughput single cell data analysis - A tutorial

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A novel data fusion method for the effective analysis of multiple panels of flow cytometry data

Contains fulltext : 204061.pdf (publisher's version ) (Open Access)9 p

A novel data fusion method for the effective analysis of multiple panels of flow cytometry data

Multicolour flow cytometry (MFC) is used to measure multiple cellular markers at the single-cell level. Cellular markers may be coloured with different panels of fluorescently-labelled antibodies to enable cell identification or the detection of activated cells in pre-defined, 'gated' specific cell subsets. The number of markers that can be used per measurement is technologically limited however, requiring every panel to be analysed in a separate aliquot measurement. The combined analyses of these dedicated panels may enhance the predictive ability of these measurements and could enrich the interpretation of the immunological information. Here we introduce a fusion method for MFC data, based on DAMACY (Discriminant Analysis of Multi-Aspect Cytometry data), which can combine information from complementary panels. This approach leads to both enhanced predictions and clearer interpretations in comparison with the analysis of separate measurements. We illustrate this method using two datasets: the response of neutrophils evoked by a systemic endotoxin challenge and the activated immune status of the innate cells, T cells and B cells in obese versus lean individuals. The data fusion approach was able to detect cells that do not individually show a difference between clinical phenotypes but do play a role in combination with other cells

Water quality monitoring based on chemometric analysis of high-resolution phytoplankton data measured with flow cytometry

River water is an important source of Dutch drinking water. For this reason, continuous monitoring of river water quality is needed. However, comprehensive chemical analyses with high-resolution gas chromatography [GC]-mass spectrometry [MS]/liquid chromatography [LC]-MS are quite tedious and time consuming; this makes them poorly fit for routine water quality monitoring and, therefore, many pollution events are missed. Phytoplankton are highly sensitive and responsive to toxicity, which makes them highly usable for effect-based water quality monitoring. Flow cytometry can measure the optical properties of phytoplankton every hour, generating a large amount of information-rich data in one year. However, this requires chemometrics, as the resulting fingerprints need to be processed into information about abnormal phytoplankton behaviour. We developed Discriminant Analysis of Multi-Aspect CYtometry (DAMACY) to model the “normal condition” of the phytoplankton community imposed by diurnal, meteorological, and other exogenous influences. DAMACY first describes the cellular variability and distribution of phytoplankton in each measurement using principal component analysis, and then aims to find subtle differences in these phytoplankton distributions that predict normal environmental conditions. Deviations from these normal environmental conditions indicated abnormal phytoplankton behaviour that happened alongside pollution events measured with the GC/MS and LC/MS systems. Thus, our results demonstrate that flow cytometry in combination with chemometrics may be used for an automated hourly assessment of river water quality and as a near real-time early warning for detecting harmful known or unknown contaminants. Finally, both the flow cytometer and the DAMACY algorithm run completely autonomous and only requires maintenance once or twice per year. The warning system results may be uploaded automatically, so that drinking water companies may temporary stop pumping water whenever abnormal phytoplankton behaviour is detected. In the case of prolonged abnormal phytoplankton behaviour, comprehensive analysis may still be used to identify the chemical compound, its origin, and toxicity

Automated flow cytometric identification of disease-specific cells by the ECLIPSE algorithm

Multicolor Flow Cytometry (MFC)-based gating allows the selection of cellular (pheno)types based on their unique marker expression. Current manual gating practice is highly subjective and may remove relevant information to preclude discovery of cell populations with specific co-expression of multiple markers. Only multivariate approaches can extract such aspects of cell variability from multi-dimensional MFC data. We describe the novel method ECLIPSE (Elimination of Cells Lying in Patterns Similar to Endogeneity) to identify and characterize aberrant cells present in individuals out of homeostasis. ECLIPSE combines dimensionality reduction by Simultaneous Component Analysis with Kernel Density Estimates. A Difference between Densities (DbD) is used to eliminate cells in responder samples that overlap in marker expression with cells of controls. Thereby, subsequent data analyses focus on the immune response-specific cells, leading to more informative and focused models. To prove the power of ECLIPSE, we applied the method to study two distinct datasets: the in vivo neutrophil response induced by systemic endotoxin challenge and in studying the heterogeneous immune-response of asthmatics. ECLIPSE described the well-characterized common response in the LPS challenge insightfully, while identifying slight differences between responders. Also, ECLIPSE enabled characterization of the immune response associated to asthma, where the co-expressions between all markers were used to stratify patients according to disease-specific cell profiles