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International audience; The experimental study of material or structural behaviours often consists of a multiparametric analysis. To describe the physical phenomenon in a limited region, it is valuable to identify the coefficients of a multilinear model with a regression. But the precision on these coefficients depends on the arrangement of the test matrix which represents the parameter values taken for each test. The optimal matrix form is reached by an orthogonal test matrix, but these orthogonal designs are restricting and sometimes impossible to realise because of physical constraints. Nevertheless, according to a criterion, optimal designs can be defined by respecting certain constraints: D-optimal designs are presented.

Numerous flow cytometric analyses are based on DNA content studies. We have considered firstly monoparametric cell cycle analyses, which only take DNA content into account, but are sometimes of limited interest. Then, we have presented multiparametric analyses, which can be used to improve cycle phase identification by taking simultaneously into account DNA and other cellular components, or by considering some events occurring during cell cycle. Finally, we have discussed monoparametric and multiparametric cell cycle analysis interest in various application fields, particularly in pharmacology, toxicology, tumoral pathology and higher plant system studies.

The authors have recently shown that cell cycle characteristics of in situ cell populations can be determined using the SAMBA 200 cell image processor by computing 15 densitometric and texture parameters on each Feulgen-stained nucleus and multiparametric analysis of data. The present paper displays the importance of chromatin pattern assessment and detection of conformational changes in DNA structure, based on nine nuclear texture parameters measured from the grey level cooccurrence and the run-length section matrices. Reference files were constructed by merging respective reference files (G0/G1, S, G2 and M) of MDA AG and MCF-7, two mammary epithelial cell lines presenting different morphological aspects and hormone responses, these files were found to be valid in the reclassification of any mammary epithelial cell in culture with a diploid or near diploid pattern. Moreover, the authors demonstrate that chromatin texture changes, following direct interaction of chemotherapeutic drugs with DNA, may be assessed owing to nuclear texture parameters. Consecutive to daunomycin addition (0.5 microgram/ml) and concomitant to the appearance of nuclear morphological alterations in MDA AG sensitive cells as viewed by microscopic observation, discriminant factorial analysis showed progressively increasing erroneous reclassification from 15 to 72 h of treatment. These experimental results were exploited with a kinetic mathematical model to quantify the daunomycin blocking effect: 20% in S phase and 80% in G2 phase. Interestingly, no textural change was observed on MDA A1 anthracycline resistant cells, indicating that these texture parameters may permit distinction of drug sensitive cells. This methodology 1) can be applied to test in vitro resistance-reversal molecules, 2) may be extended to other therapeutic agents giving rise to conformational changes in DNA structure, and 3) can be applied to cytopunctions or imprints of tumor biopsies with diploid-like DNA content to follow evolution of drug sensitivity or resistance during course of therapy.

Lymphocyte subset analysis was performed on 114 healthy children and 84 healthy adults. Samples were prepared by a whole blood lysis technique and analyzed by flow cytometry. The percentage and total number of CD3+, CD4+, CD8+, and CD19+ lymphocytes were calculated for each of six age groups. A direct correlation with age was seen in the percentages of CD3+, CD4+, and CD8+ lymphocytes. The absolute number and percentage of total lymphocytes, the percentage and absolute number of CD19+ lymphocytes, and the absolute number of CD3+ lymphocytes decreased with age. No significant correlation with age was observed for white blood cells, the absolute number of CD4+ and CD8+ lymphocytes, and the CD4+/CD8+ ratio.

An autostage cytofluorometry system for multiparametric analysis is described. The system consists of a compact epi-illumination cytofluorometer equipped with an autostage device, a small data processing unit, an autostage controller and a microcomputer. In this system, repeated cytofluorometry and morphological observations for the same series of cells can be made at predetermined cell positions (X, Y-values) on a slideglass, permitting accurate multiparametric cytofluorometry in the same cells, even for a mixed cell population. Fluorochromes can be applied in any combination, if they can be utilized sequentially by staining and destaining cells. Application experiments demonstrated that the present system allows detailed cell cycle analysis.

148 patients with various forms of cerebrovascular disease (CVD) were studied by means of a multiparametric analysis of in vitro platelet aggregation, based on the following six parameters: ADP and epinephrine primary and secondary aggregation thresholds and percent maximum aggregation induced by optimal concentrations of ADP and epinephrine. These patients were assigned to four study groups, according to clinical diagnosis supported by CT scan, of transient ischemic attack and reversible neurological deficit (TIA-RIND), or completed stroke, in the presence or absence respectively of antiplatelet medical treatment at the time of the study. A statistically significant increase of the in vitro platelet aggregation was found in 44.4% of the untreated TIA-RIND patients and in 33.9% of the untreated stroke patients. However this last group showed a higher percentage of very marked hyperaggregation. Differences between the two treated study groups and controls were not significant. No difference was found in collagen- and ristocetin-induced aggregation between the patient groups and the controls.

The functionalization of nanoparticles with functional moieties is a key strategy to achieve cell targeting in nanomedicine. The interplay between size and ligand number is crucial for the formulation performance and needs to be properly characterized to understand nanoparticle structure-activity relations. However, there is a lack of methods able to measure both size and ligand number at the same time and at the single particle level. Here, we address this issue by introducing a correlative light and electron microscopy (CLEM) method combining super-resolution microscopy (SRM) and transmission electron microscopy (TEM) imaging. We apply our super-resCLEM method to characterize the relationship between size and ligand number and density in PLGA-PEG nanoparticles. We highlight how heterogeneity found in size can impact ligand distribution and how a significant part of the nanoparticle population goes completely undetected in the single-technique analysis. Super-resCLEM holds great promise for the multiparametric analysis of other parameters and nanomaterials.

Publisher Summary This chapter discusses analytical instrumentation for analysis, selection, and sorting. It examines the types of noninvasive cellular experimentation that may be performed and subsequently used for selection criteria and presents anchorage-dependent cell-sorting strategies. The construction of an instrument to analyze, select, and sort anchored cells proceeds using various components—namely, (1) microscope, (2) tunable laser, (3) optical detectors, (4) acousto-optical modulator and controllable series of neutral density filters, (5) high-speed two-dimensional stages, and (6) computer. High-resolution computer imaging and video frame grabbers (devices that digitize video images for display on computer terminals) permit simultaneous overlays of fluorescent and phase images of cells. Multiple fluorescent probes may be monitored and then examined for coincidence with intracellular structures. The advantage of single-cell analysis of anchorage-dependent cells is that, for the most part, it removes the demands of rapid data capture as a consideration in experimental design and multiparametric analysis. Cells may be maintained under growth conditions and optically probed in a continuous manner. Removal of time constraints for analysis permits a wide range of dynamic experimentation and enables multiple experiments to be performed on the same cell with the capability for long-term monitoring.

A possibility of both analysis and various presentation of multi-dimensional information, i.e. properties of ZnO varistor system with additives, sintered within temperature range 1323-1423 K, Has been presented it this paper by non-parametric methods of pattern recognition. An optimal linear transformation from six- into two-dimensional space has been performed. Thus, a direct look into mutual relations existing between characteristics of a system that is sintered at different temperatures is possible.

Non-Hodgkin’s hematosarcomas (NHHS) or non-Hodgkin’s “lymphomas” are the objects of so many different morphologic microscopic classifications [12,17, 23, 24, 26, 27, 31, 33, 34, 36, 38, 39] that it is almost impossible for clinicians from different centers to compare their observations and therapeutic results. Until the time that an international classification can be accepted by all pathologists, we propose that a categorization using cell membrane markers is helpful for diagnosis and prognosis in these disorders, as in acute lymphoid leukemias [8, 10]. The object of this paper is to summarize the preliminary data given by such categorization of 100 cases of NHHS, with particular reference to its value for prognosis, correlation with the clinical presentation of the disease, and comparison with the WHO microscopic classification [34]. A further paper will be devoted to a computer multiparametric analysis of those cases [11].

Flow cytometry has been extensively used to provide accurate estimates of the relative amounts of various cellular constituents (DNA, RNA, proteins) for cell kinetic studies. Multiparametric analysis also supports the recent concept that cell growth and the DNA division cycle may be under distinct regulatory mechanisms. Moreover, metabolic subcompartments of the cell cycle, distinguished by flow cytometry, have offered a highly sensitive cell classification in comparison with the conventional distinction of the four main phases of the cell cycle. Finally, a new sensitive and powerful technology, BrdU/DNA analysis, represents a remarkable maturing of a very useful alternative for the study of DNA synthesis and cell cycle traverse.

Time-resolved inductively coupled plasma mass spectrometry (ICP-MS) has attracted much attention for elemental and multiparametric analysis of single cells, instead of a classical bulk analysis of large amount of cells after a dissolution. In the time-resolved measurement, cells are directly introduced into the plasma via nebulizing or micro drop dispensing, and then ion plumes corresponding to single cells are individually detected with a high time resolution. The sensitivity and cell throughput in the measurement strongly depend on the time resolution. A high cell introduction efficiency into the plasma supports for a reduction of cell consumption. Biomolecules can also be measured through the attachment of elemental tags, and then the amount distribution of elements and biomolecules in single cells can be evaluated, while providing information concerning cell-to-cell variations. By applying ICP time-of-flight mass spectrometry (ICP-TOFMS), multiparametric analysis of elements and biomolecules can be achieved similar to that by a flow cytometer. This article highlights the technical aspects of the time-resolved ICP-MS measurement technique for elemental and multiparametric analysis of single cells.

Based on classic clonogenic assay, it is accepted by the scientific community that, whatever the energy, the relative biological effectiveness of X-rays is equal to 1. However, although X-ray beams are widely used in diagnosis, interventional medicine and radiotherapy, comparisons of their energies are scarce. We therefore assessed in vitro the effects of low- and high-energy X-rays using Human umbilical vein endothelial cells (HUVECs) by performing clonogenic assay, measuring viability/mortality, counting γ-H2AX foci, studying cell proliferation and cellular senescence by flow cytometry and by performing gene analysis on custom arrays. Taken together, excepted for γ-H2AX foci counts, these experiments systematically show more adverse effects of high energy X-rays, while the relative biological effectiveness of photons is around 1, whatever the quality of the X-ray beam. These results strongly suggest that multiparametric analysis should be considered in support of clonogenic assay.

Prostate adenocarcinoma (PCa) is the most frequent noncutaneous cancer among men in developed countries. Magnetic Resonance (MR) has been used to detect PCa and several clinical trials report on the accuracy of the test. Multiparametric MR imaging (mpMRI) is defined as the integration of information from different morphological and functional datasets. mpMRI could be used to increase the performances of prostate MR, therefore allowing a more accurate assessment of the tumor gland extent, while reducing reporting time and interobserver variability. The first step to perform such a multiparametric analysis is to correct for voluntary and involuntary movements during the acquisitions, as well as for image distortion in the Diffusion Weighted (DWI) images. The aim of this work is to present a fully automatic registration algorithm between T2w and DWI images, able to realign the images and to correct the distortions in the DWI. Results showed a good overlap after registration and a strong decrease of mean surface distance in both the central gland and peripheral zone. These promising results suggest that the algorithm could be integrated in a CAD system which will combine the pharmacokinetic parameters derived from DCE-MRI, T2w MRI and DWI MR to generate one comprehensive value assessing the risk of malignancy. However to perform such a multiparametric analysis, it is necessary to correct for voluntary and involuntary (breathing, heart beating) movements during the DCE-MRI acquisition, and to realign also the DCE-MRI sequence to the T2w sequence.

Quantitative multiparametric imaging is a potential key application for Positron Emission Tomography/Magnetic Resonance (PET/MR) hybrid imaging. To enable objective and automatic voxel-based multiparametric analysis in whole-body applications, the purpose of this study was to develop a multimodality whole-body atlas of functional 18F-fluorodeoxyglucose (FDG) PET and anatomical fat-water MR data of adults. Image registration was used to transform PET/MR images of healthy control subjects into male and female reference spaces, producing a fat-water MR, local tissue volume and FDG PET whole-body normal atlas consisting of 12 male (66.6 ± 6.3 years) and 15 female (69.5 ± 3.6 years) subjects. Manual segmentations of tissues and organs in the male and female reference spaces confirmed that the atlas contained adequate physiological and anatomical values. The atlas was applied in two anomaly detection tasks as proof of concept. The first task automatically detected anomalies in two subjects with suspected malignant disease using FDG data. The second task successfully detected abnormal liver fat infiltration in one subject using fat fraction data.

Mycobacterium tuberculosis; T-cells; Immune response Tuberculosis micobacteriana; Células T; Respuesta inmune Tuberculosi micobacteriana; Cèl·lules T; Resposta immune Background: Current blood-based diagnostic tools for TB are insufficient to properly characterize the distinct stages of TB, from the latent infection (LTBI) to its active form (aTB); nor can they assess treatment efficacy. Several immune cell biomarkers have been proposed as potential candidates for the development of improved diagnostic tools. Objective: To compare the capacity of CD27, HLA-DR, CD38 and Ki-67 markers to characterize LTBI, active TB and patients who ended treatment and resolved TB. Methods: Blood was collected from 45 patients defined according to clinical and microbiological criteria as: LTBI, aTB with less than 1 month of treatment and aTB after completing treatment. Peripheral blood mononuclear cells were stimulated with ESAT-6/CFP-10 or PPD antigens and acquired for flow cytometry after labelling with conjugated antibodies against CD3, CD4, CD8, CD27, IFN-γ, TNF-α, CD38, HLA-DR, and Ki-67. Conventional and multiparametric analyses were done with FlowJo and OMIQ, respectively. Results: The expression of CD27, CD38, HLA-DR and Ki-67 markers was analyzed in CD4+ T-cells producing IFN-γ and/or TNF-α cytokines after ESAT-6/CFP-10 or PPD stimulation. Within antigen-responsive CD4+ T-cells, CD27− and CD38+ (ESAT-6/CFP-10-specific), and HLA-DR+ and Ki-67+ (PPD- and ESAT-6/CFP-10-specific) populations were significantly increased in aTB compared to LTBI. Ki-67 demonstrated the best discriminative performance as evaluated by ROC analyses (AUC > 0.9 after PPD stimulation). Data also points to a significant change in the expression of CD38 (ESAT-6/CFP-10-specific) and Ki-67 (PPD- and ESAT-6/CFP-10-specific) after ending the anti-TB treatment regimen. Furthermore, ratio based on the CD27 median fluorescence intensity in CD4+ T-cells over Mtb-specific CD4+ T-cells showed a positive association with aTB over LTBI (ESAT-6/CFP-10-specific). Additionally, multiparametric FlowSOM analyses revealed an increase in CD27 cell clusters and a decrease in HLA-DR cell clusters within Mtb-specific populations after the end of treatment. Conclusion: Our study independently confirms that CD27−, CD38+, HLA-DR+ and Ki-67+ populations on Mtb-specific CD4+ T-cells are increased during active TB disease. Multiparametric analyses unbiasedly identify clusters based on CD27 or HLA-DR whose abundance can be related to treatment efficacy. Further studies are necessary to pinpoint the convergence between conventional and multiparametric approaches. This research was supported by a grant from the Instituto de Salud Carlos III (PI18/00411, PI19/01408 and CP20/00070), integrated in the Plan Nacional de I + D + I and cofunded by the ISCIII Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER); a grant from the Sociedad Española de Neumología y Cirugía Torácica (project 25/2016; SEPAR; Barcelona, Spain); and from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement no. 823854 (INNOVA4TB). JD and IL are researchers from the Miguel Servet programme. AS-M is supported by a Juan Rodés (JR18/00022) postdoctoral fellowship from ISCIII.

Proposes new classifiers based on fuzzy inference systems (FISs) for the Fetal Heart Rate (FHR) signal analysis. They include standard cardiotocographic (CTG) parameters together with a set of frequency domain and nonlinear indices. The goal is the identification of two very common fetal pathological conditions: Intra-Uterine Growth Retardation (IUGR) and Diabetes type I. The FHR signals obtained from 104 CTG recordings were analyzed (75 Normal, 11 IUGR and 18 Diabetic). Fuzzy classifiers combine the set. Of 10 input data into the S-output set (Normal, IUGR, Maternal Diabetes) by fuzzy relies. Results show FISs predict normal and pathological fetal states even with 100% of correct classifications. Their performance however is always higher than 80% in the whole population, depending on the rule number. This approach can strongly help the automatic CTG signal analysis improving the early discrimination among normal and pathological fetal conditions.

Multimodal image fusion is an important step in multiparametric analysis from different cardiac images recorded over time from different imaging modalities. In fact, low resolution images, such as PET or SPECT can be integrated with high resolution images as MRI or CTI, to show both perfusion and structure information in the same representation. A fundamental step in this integration process is to bring the modalities involved into spatial alignment, a procedure referred as multimodal image fusion. In the present paper we describe a method for multimodal cardiac images fusion, consisting on segmentation, registration and warping phases.

The paper presents the analysis of the Heart Rate Variability (HRV) signal in 19 subjects who recently had a Myocardial Infarction episode (MI). The study follows a nonlinear approach based on the multiparametric analysis of some invariant properties of the dynamical system generating the time series. First the authors reconstruct the system embedding space from the HRV time series. The False Nearest Neighbors (FNN) criterion provides the real embedding dimension value. Results show that through the FNN method it is possible to identify the correct number of LE in the system. Parameter values significantly separate subjects who after MI keep a good performance of the cardiac pump (normal ventricular ejection function, NEF) vs. the group which after MI shows a reduced ventricular ejection fraction (REF).

We investigate phenomena of multistability and complete chaos synchronization in coupled H\'enon maps, which is an invertible system. Multiparametric analysis of a selected family of periodic orbits for coupled Henon maps shows that a single bifurcational mechanism describes both a loss of chaos synchronization and multistability formation. The process of bubbling transition and riddle basins, and the multistability formation in invertible systems are described in detail.

ObjectivesThe severity of neurocognitive impairment increases with prematurity. However, its mechanisms remain poorly understood. Our aim was firstly to identify multiparametric magnetic resonance imaging (MRI) markers that differ according to the degree of prematurity, and secondly to evaluate the impact of clinical complications on these markers.Materials and MethodsWe prospectively enrolled preterm infants who were divided into two groups according to their degree of prematurity: extremely preterm (ResultsThirty-four preterm infants were included. Extremely preterm infants (n= 13) had significantly higher frontal relative volumes (p= 0.04), frontal GM relative volumes (p= 0.03), and regional CBF than very preterm infants, but they had lower brainstem and insular relative volumes (respectivelyp= 0.008 and 0.04). Preterm infants with WM lesions on MRI had significantly lower overall GM CBF (13.3 ± 2 ml/100 g/min versus 17.7 ± 2.5, < ml/100 g/minp= 0.03).ConclusionMagnetic resonance imaging brain scans performed at term-equivalent age in preterm infants provide quantitative imaging parameters that differ with respect to the degree of prematurity, related to brain maturation.

Many anticancer therapies such as antibody-based therapies, cellular therapeutics (e.g., genetically modified cells, regulators of cytokine signaling, and signal transduction), and other biologically tailored interventions strongly influence the immune system and require tools for research, diagnosis, and monitoring. In flow cytometry, in vitro diagnostic (IVD) test kits that have been compiled and validated by the manufacturer are not available for all requirements. Laboratories are therefore usually dependent on modifying commercially available assays or, most often, developing them to meet clinical needs. However, both variants must then undergo full validation to fulfill the IVD regulatory requirements. Flow cytometric immunophenotyping is a multiparametric analysis of parameters, some of which have to be repeatedly adjusted; that must be considered when developing specific antibody panels. Careful adjustments of general rules are required to meet legal and regulatory requirements in the analysis of these assays. Here, we describe the relevant regulatory framework for flow cytometry-based assays and describe methods for the introduction of new antibody combinations into routine work including development of performance specifications, validation, and statistical methodology for design and analysis of the experiments. The aim is to increase reliability, efficiency, and auditability after the introduction of in-house-developed flow cytometry assays.