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Reproducibility of lung volume measurements.

Test reproducibility is an important consideration when interpreting results and should be set as a goal during data collection. Reproducibility criteria may need to be different for different subject groups and are instrument and procedure-dependent. Ideally, the within-subject variability for each lung volume and measurement technique used should be established for each laboratory. These values also need to be established for each different subject group (age and disease). At a minimum, test reproducibility should be monitored and controlled and each laboratory should define their between-day reproducibility of measurements on at least one "reference" subject from ongoing periodic (e.g., weekly or monthly) measurements as part of their laboratory's quality control programme. For plethysmographic measurements functional residual capacity (FRC)pleth multiple determinations and a corresponding test reproducibility criteria is probably justified.

Adult↗

Toward quantitative simulation of germinal center dynamics: biological and modeling insights from experimental validation.

As models of immune system dynamics are developed, it is important to validate them with specific experimental data in order to understand their shortcomings and guide them toward becoming predictive. In this paper, we examine whether a particular mathematical model of germinal center dynamics, proposed by Oprea and Perelson, can reproduce experimental data from two specific primary responses, namely those directed against the haptens 2-phenyl-5-oxazolone and (4-hydroxy-3-nitrophenyl)acetyl. We develop formulas for estimating response-specific model parameters, as well as constraints for validating the model. In addition, we outline a general methodology for translating a continuous/deterministic model, expressed as a set of ordinary differential equations, into a discrete/stochastic framework. This methodology is used to create a new implementation of the Oprea and Perelson model that enables comparison with data on individual germinal centers. We conclude that while the model can reproduce the average dynamics of splenic germinal centers, it is at best incomplete and does not reproduce the distribution of individual germinal center behaviors. In addition to suggesting possible extensions to the model which can reconcile the dynamics with some aspects of the experimental data, we make a number of specific predictions that can be tested by in vivo experiments to obtain further insights and validation.

Antibody Affinity↗

The Euro-Growth Study: why, who, and how.

BACKGROUND: Assessment of growth is important in health management of infants and children. Evaluation of growth performance requires anthropometric measurements, with proper interpretation depending on the use of appropriate references. Europe-specific growth references have not been available. The purpose of the present study was to generate such references for infants and children from birth to 3 years of age. The study was further intended to assess the influence of nutrition and lifestyle factors on growth. METHODS: The Euro-Growth Study was designed as a multicenter longitudinal cohort study. This report describes the study design, the sample (cohort), and the methods used. Quality control measures included standardized measurement techniques with ongoing cross-sectional and longitudinal consistency checks. Selectivity in participation and discontinuation and reproducibility of data over time were evaluated. RESULTS: Of 2,245 infants who were enrolled in the study at 22 study sites in 11 countries, 1,746 (78%) provided longitudinal data until 12 months of age, 1,205 (57%) until 24 months of age, and 1071 (48%) until 36 months of age. Anthropometric measurements were performed on 21,773 occasions. As a result of cross-sectional and longitudinal data checks, 209 data points (0.09% of all data points) were rejected. Comparison of participants's demographic data with those of nonparticipating local subjects indicated that the cohort was selective in maternal age (higher), maternal education level (higher), household location, and family structure. The withdrawal rate during the study was low (24%), and withdrawals occurred at random. Analysis of reproducibility over time resulted in the elimination of the data from one study site. CONCLUSION: The Euro-Growth Study provided longitudinal growth data from a large cohort of normal European children. Because of the chosen sampling method, the study cohort ws somewhat selective relative to the local background population. Internal validity was satisfactory in that the reproducibility of anthropometric measurements was high in 21 sites and the withdrawal rate was low and random.

Anthropometry↗

ASFV DNA polymerse X is extremely error-prone under diverse assay conditions and within multiple DNA sequence contexts.

We previously demonstrated that the DNA repair system encoded by the African swine fever virus (ASFV) is both extremely error-prone during the single-nucleotide gap-filling step (catalyzed by ASFV DNA polymerase X) and extremely error-tolerant during the nick-sealing step (catalyzed by ASFV DNA ligase). On the basis of these findings we have suggested that at least some of the diversity known to exist among ASFV isolates may be a consequence of mutagenic DNA repair, wherein damaged nucleotides are replaced with undamaged but incorrect nucleotides by Pol X and the resultant mismatched nicks are sealed by ASFV DNA ligase. Recently, this hypothesis appeared to be discredited by Salas and co-workers [(2003) J. Mol. Biol. 326, 1403-1412], who reported the fidelity of Pol X to be, on average, 2 orders of magnitude higher than what we previously published. In an effort to address this discrepancy and provide a definitive conclusion about the fidelity of Pol X, herein we examine the fidelity of Pol X-catalyzed single-nucleotide gap-filling in both the steady state and the pre-steady state under a diverse array of assay conditions (varying pH and ionic strength) and within different DNA sequence contexts. These studies corroborate our previously published data (demonstrating the low fidelity of Pol X to be independent of assay condition/sequence context), do not reproduce the data of Salas et al., and therefore confirm Pol X to be one of the most error-prone polymerases known. These results are discussed in light of ASFV biology and the mutagenic DNA repair hypothesis described above.

Asfarviridae↗

Cortical and cancellous bone in the human femoral neck: evaluation of an interactive image analysis system.

An interactive image analysis package was developed to examine whole cross-sections from the femoral neck. The package quantifies cortical width (Ct.Wi), cortical porosity (Ct.Po), and proportions of cortical, cancellous bone as a percentage of bone plus marrow area. Segmental analysis was used to quantify circumferential variations in bone distribution within the femoral cross-section. To evaluate reproducibility of data four independent operators analyzed previously prepared femoral neck sections from a 2000 BC population. Differences in total and circumferential distributions of cortical and cancellous bone with respect to gender and age of samples were demonstrated. Reproducibility was assessed using coefficients of variation (CV). Analysis of sections using a variable magnification, giving largest possible image size, rather than a set magnification reduced variation between operators for all measurements. Use of a calculated threshold significantly decreased variation between operators for the proportions of cortical and cancellous bone (p < or = 0.026). Dividing the image into 8 rather than 16 segments also improved reproducibility. There was little agreement between operators in the determination of cortical porosity. The mean CV for the other quantitative indices such as cortical width and proportions of cortical and cancellous bone ranged from 4.87% to 13.52%. The genders showed similar patterns in circumferential distribution of bone. Cortical width was significantly greater in the inferior region compared to the other areas, whereas percent cortical bone was lowest at the superior region. The center of mass (COM) for the younger age group was located anteriorly, whereas in the older samples the COM was located posteriorly of the center of area (p = 0.041). Basic data relating to cortical and cancellous bone of acceptable reproducibility in comparison with current standards in iliac histomorphometry can now be provided at modest cost in operator time and resources.

Adult↗

Reproducibility of 24-hour serum growth hormone profiles in man.

OBJECTIVE: To study the reproducibility of 24-h serum growth hormone (GH) concentration profiles in adults. DESIGN: 24-h serum GH concentrations were constructed by drawing blood samples at 20-min intervals. Four study occasions over a period of 1 year were chosen to assess the reproducibility. SUBJECTS: Six healthy adult male volunteers of normal height and weight and aged between 20 and 22 years. MEASURES: The resulting GH data arrays were analysed by Fourier transformation. Between and within individual variations were calculated and expressed in terms of coefficients of variation and data plotted to show variations between groups and individuals. RESULTS: The frequency component of GH secretion occurred with a dominant periodicity of between 160 and 240 min. Precise estimates of spectral power (strength of oscillatory activity) and period were obtained for group data, but such estimates cannot be inferred from a single profile. There was no significant difference in 24-h mean serum GH concentration over the year of study: occasion 1 (February) mean 2.0 mU/l (SD 0.5); occasion 2 (March) mean 3.7 mU/l (SD 2.8); occasion 3 (July) mean 2.7 mU/l (SD 1.4); occasion 4 (February) 2.5 mU/l (SD 1.5) (mean within individual coefficient of variation 35%, range 9-58). The concentrations of factors known to influence GH synthesis and secretion, insulin-like growth factor I, thyroxine, testosterone and oestradiol, varied little over the year of study. CONCLUSIONS: These data demonstrate that group data are reproducible in terms of oscillatory activity and the amount of GH secreted and that 24-h GH profiles should be used predominantly for analysing group data. The variability between individual profiles limits their value in the investigation of children with growth failure and suspected GH insufficiency.

Adult↗

A standardized ZAP-70 assay--lessons learned in the trenches.

BACKGROUND: The disease of chronic lymphocytic leukemia (CLL) has been shown to exhibit varying clinical outcomes based on reported laboratory parameters. One of these parameters involves the measurement of the protein levels of zeta-associated protein (ZAP-70) in CLL cells. A standardized assay has not yet reached consensus in the clinical cytometry community. METHODS: We developed a system using the 8-peak Rainbow beads as our fluorescence calibrator along with a fixed cell control. Using a panel of CD19-PE, CD5-FITC, and ZAP-70-Alexa 647, we stained normal whole blood, and blood and bone marrow from patients with CLL to determine the level of ZAP-70 expression in T-cell, B-cell, and CLL-cell populations. ZAP-70 expression was reported in molecules equivalent fluorescence (MEFL) based on the calibration of the flow cytometer with the 8-peak Rainbow beads. RESULTS: Daily assay performance as well as operating MEFL defined ranges for ZAP-70 detection in CLL were developed. A rank-order, nonparametric approach to reference ranges was used to assign a cutoff for "negative" as well as ranges for "intermediate" and "positive" staining using T and B cells from a pool of 50 normal subjects, and CLL cells from 395 patients. The assay was validated in a multi-institutional study and has demonstrated correlation with published techniques. Since its initial development, the assay has been implemented at two additional laboratory sites and has been shown to produce reproducible, correlated data at all sites. CONCLUSIONS: Strict adherence to standardization can yield an assay that is predictable, reliable, and reproducible as well as capable of multisite implementation. The Rainbow beads provide a common platform for system calibration. The fixed cell culture controls provide a common target to test antibody. The final level of control tests the sensitivity of ZAP-70 detection in a normal peripheral blood sample stained along with the submitted CLL patients. The acceptance/rejection of test results must meet all three levels of control before patient results are reported.

Antibodies, Monoclonal↗

Quantitative approaches for assessing chromosome loss in Saccharomyces cerevisiae: general methods for analyzing downturns in dose response.

Statistical methods are considered for analysis of data arising from a mitotic chromosome loss assay in Saccharomyces cerevisiae strain D61.M. The methods make use of reproducibility trial data from the assay (presented herein) and previous data, which suggest a unimodal, 'umbrella-patterned' dose response. Computer simulations are employed to illustrate the operating characteristics of the umbrella response methods. These methods are generally applicable to any toxicity assay that exhibits a downturn in dose response. Experimental design considerations are also discussed. These include applications of 2-stage sampling rules to first gauge the dose window of peak response, then test if the response deviates significantly from untreated levels.

Alleles↗

Uniform processing and analysis of IGVF massively parallel reporter assay data with MPRAsnakeflow.

As researchers and clinicians seek to identify human genomic alterations relevant to traits and disorders, identifying and aggregating evidence providing mechanistic support for associations between alterations and phenotypes remains challenging. In particular, the study of noncoding genomic variation remains a major challenge because of the lack of accurate functional annotation for activity in a given context and across alleles. Experimental evidence is critical for prioritizing and interpreting functional effects of genetic alterations. Massively parallel reporter assays (MPRAs) have emerged as a powerful high-throughput approach, enabling quantification of regulatory element activity and allelic effects, as well as systematic dissection of gene regulatory logic and variant effects across different contexts. However, the diversity of MPRA designs, lack of standardized formats, and many potential processing parameters hamper data integration, reproducibility, and meta-analyses across studies. To address these challenges, the Impact of Genomic Variation on Function (IGVF) Consortium established an MPRA focus group to develop community standards, including harmonized file formats, and robust analysis pipelines for a wide range of library types and experimental designs. Here, we present these formats and comprehensive computational tools, MPRAlib and MPRAsnakeflow, for uniform processing from raw sequencing reads to counts, processing, and visualization. Using diverse MPRA data sets, we investigated technical variability sources including barcode sequence bias, outlier barcodes, and delivery method (episomal vs. lentiviral). Our results establish best practices for MPRA data generation and analysis, facilitating robust, reproducible research and large-scale integration. The presented tools and standards are publicly available, providing a foundation for future collaborative efforts in regulatory genomics.

Humans↗

Development of clinical standards for flow cytometry.

Flow cytometers are instruments that can determine multiparameter data simultaneously and have a great potential in providing unique information about cells. To transfer this potential to the clinical laboratory, the development and the proper use of standards and calibrators are required to ensure comparability and reproducibility of data from different flow cytometers. Although there are a number of types of standards for flow cytometry, each has its specifically designed purpose to ensure that data from this complex instrument are accurate, precise, and reproducible among instruments over time.

CD3 Complex↗

Validation of the Japanese version of the Quality of Life-Assessment of Growth Hormone Deficiency in Adults (QoL-AGHDA).

OBJECTIVE: To evaluate validity and reliability of the Japanese version of the Quality of Life-Assessment of Growth Hormone Deficiency in Adults (QoL-AGHDA). DESIGN: Observational study; cross-sectional, longitudinal. METHODS: Seventy-five adults with growth hormone deficiency completed the SF-36 (a generic health-related QOL scale) and the QoL-AGHDA before growth hormone replacement therapy and approximately 3 weeks later (when the therapy began). A sample (n=1000) of controls from the general population was also studied. We computed rates of missing data, measured reproducibility and internal consistency reliability, and tested for known-groups validity, concurrent validity, unidimensionality (by principle component analysis), and content validity. RESULTS: Rates of missing data were low (0-1.4%). The mean of QoL-AGHDA scores in the patients was 8.2 (SD, 6.4). The scores were reproducible (k=0.41-0.78), and internally consistent (alpha=0.91) and the scale was unidimensional. QoL-AGHDA scores were associated with SF-36 scores as hypothesized. Scores were significantly higher in the patients than in controls (8.1+/-0.7, and 5.6+/-0.2, P<0.001). Discrimination between patients and controls was slightly better using scores on the "General Health" and "Role Physical" subscale of the SF-36 as explanatory variables than using QoL-AGHDA scores. CONCLUSIONS: The QoL-AGHDA's reliability, validity, and rates of missing data were satisfactory, and the scale was confirmed to be unidimensional. However, because some subscales of the SF-36 were better for discriminating patients from controls, the content validity of the QoL-AGHDA may need to be re-evaluated.

Adolescent↗

Rapid enrichment of bioactive milk proteins and iterative, consolidated protein identification by multidimensional protein identification technology.

Direct injection of complex protein mixtures, e.g. those derived from crude biological fluids, is often incompatible with conventional LC supports, because of column clogging and rapid deterioration of chromatographic performance. In this paper, we report the use of restricted access media to rapidly enrich and fractionate human breast milk. This resin, combining size exclusion and anion exchange functionalities, yielded a fraction enriched in soluble CD14 and showing specific sCD14-dependant activity. This fraction was split into five aliquots, which were individually characterized using multidimensional protein identification technology. Reproducibility of the results was addressed by analysing and comparing five datasets using different protein identification tools available within the Sequest software. Furthermore, a comparison of three major releases of the Ensembl human protein database was performed to examine the effect of database updates on our results. We report here the benefit of repeated analysis of aliquots of the same fraction: first to increase the confidence in peptide identification by repeated confirmation in several aliquots; and second to assess experimental reproducibility. We demonstrate furthermore the effect of database modifications on the results and the importance of constantly re-analysing data with new releases to keep them consistent and up to date with the latest protein identities and predictions available.

Amino Acid Sequence↗

Processes leading to a spatial aggregation of Echinococcus multilocularis in its natural intermediate host Microtus arvalis.

The small fox tapeworm (Echinococcus multilocularis) shows a heterogeneous spatial distribution in the intermediate host (Microtus arvalis). To identify the ecological processes responsible for this heterogeneity, we developed a spatially explicit simulation model. The model combines individual-based (foxes, Vulpes vulpes) and grid-based (voles) techniques to simulate the infections in both intermediate and definite host. If host populations are homogeneously mixed, the model reproduces field data for parasite prevalence only for a limited number of parameter combinations. As ecological parameters inevitably vary to a certain degree, we discarded the homogeneous mixing model as insufficient to gain insight into the ecology of the fox tapeworm cycle. We analysed five different model scenarios, each focussing on an ecological process that might be responsible for the heterogeneous spatial distribution of E. mulitlocularis in the intermediate host. Field studies revealed that the prevalence ratio between intermediate and definite host remains stable over a wide range of ecological conditions. Thus, by varying the parameters in simulation experiments, we used the robustness of the agreement between field data and model output as quality criterion for the five scenarios. Only one of the five scenarios was found to reproduce the prevalence ratio over a sufficient range of parameter combinations. In the accentuated scenario most tapeworm eggs die due to bad environmental conditions before they cause infections in the intermediate host. This scenario is supported by the known sensitivity of tapeworm eggs to high temperatures and dry conditions. The identified process is likely to lead to a heterogeneous availability of infective eggs and thus to a clumped distribution of infected intermediate hosts. In conclusion, areas with humid conditions and low temperatures must be pointed out as high risk areas for human exposure to E. multilocularis eggs as well.

Animals↗

Clustering methods for analyzing large data sets: gonad development, a study case.

With the development of data set repositories, it is now possible to collate high numbers of related results by gathering data from experiments carried out in different laboratories and addressing similar questions or using a single type of biological material under different conditions. To address the challenge posed by the heterogeneous nature of multiple data sources, this chapter presents several methods used routinely for assessing the quality of data (i.e., reproducibility of replicates and similarity between experimental points obtained under identical or similar biological conditions). As gene clustering on large data sets is not straightforward, this chapter also presents a rapid gene clustering method that involves translating variation profiles from an ordered set of comparisons into chains of symbols. In addition, it shows that lists of genes assembled based on the presence of a common term in their functional description can be used to find the most informative comparisons and to construct from them exemplar chains of symbols that are useful for clustering similar genes. Finally, this symbolic approach is extended to the overall set of biological conditions under study and shows how the resultant collection of variation profiles can be used to construct transcriptional networks, which in turn can be used as powerful tools for gene clustering.

Animals↗

S-matrix analysis of coincident measurement of two-electron energy distribution for double ionization of He in an intense laser field.

Analyses of two mechanisms of double ionization of He in an intense femtosecond laser pulse are made within the S-matrix theory. Their contributions are compared with each other and with the recent data of coincidence measurements for two-electron energy distributions. It is found that the correlated energy-sharing mechanism reproduces the data remarkably well and also confirms the so-called "excessive production" of hot electrons in the process. In contrast, the shakeoff mechanism is found to fail completely to account for either the strength or the trend of the data and is therefore ruled out.

Journal Article↗

A modeling study of bilirubin kinetics during Molecular Adsorbent Recirculating System sessions.

This work presents a quantitative description, by means of a mathematical model, of bilirubin removal during Molecular Adsorbent Recirculating System sessions. The model includes four compartments: two for the patient, and two for the albumin circuit. Equations in each compartment express mass preservation, mass exchange between compartments, and bilirubin-albumin binding kinetics. Model development and validation are based on in vivo data of bilirubin concentration acquired in eight sessions at different times during the session. The accuracy of the model in reproducing real data is high (error in blood = -0.3 +/- 0.93 mg/dL), if three parameters, representing the depurative efficacy of the system (the dialysance of the blood filter and the initial and final clearance of the depurative elements in the albumin circuit), are estimated on each single session. However, model accuracy is only slightly deteriorated (error in blood = -0.4 +/- 0.99 mg/dL) if a single set of parameters (fixing the three parameters at their mean values) is adopted. These results suggest that the model may be used a priori (i.e., using a single set of parameters) to achieve a satisfactory prediction of the overall bilirubin removal, as well as a posteriori for the estimation of device parameters. The latter use may allow the investigation of the dependence of these parameters on the operative and clinical conditions, in the effort to arrive at a rationalization and optimization of the treatment.

Algorithms↗

Analysis of the first international data bank on in vitro NMR relaxation times: animal liver.

Based on an international "Interlaboratory Comparison of Protocol Trials for In Vitro Studies by NMR," the EEC COMAC-BME concerted action on "Identification and Characterization of Biological Tissue by NMR" started collection of a data bank for in vitro relaxation time data of normal and pathological tissues. Relaxation time measurements were performed in a frequency range from 10-90 MHz and in a temperature range from 3 to 40 degrees C; data analysis was done using single exponential fitting routines. Specimens were taken from different organs or tissues (n = 19) of various species (n = 5) and both sexes. More than 900 relaxation time data from 12 participating groups were collected. However, for quantitative analysis we concentrated on liver tissue (287 entries). Main results are the high reproducibility of data from different centers, allowing quantification of temperature dependence of T1 and T2, respectively, in fresh mouse (3.9 and 0.41 msec/degrees C), rat (2.1 and 0.24 msec/degrees C), and hamster (2.6 and 0.22 msec/degrees C) liver. Further, a highly significant species difference between mouse and rat liver, not depending on water content, has been established. We discuss results quantitatively as well as consequences for in vitro protocols and future multicenter data collection.

Animals↗

Dynamic optimization: inverse analysis for the Yurchenko layout vault in women's artistic gymnastics.

The use of dynamic optimization to compute the trajectory of joint torques is not popular due to the large amount of computation required, the choice of initial "guesstimates" of torque values and the mathematical sophistication required to understand the technique. Modern optimal control algorithms circumvent most of these objections to the method. It is our aim to demonstrate that the dynamic optimization technique is feasible for complex movements, using the Yurchenko layout vault as an example. A dynamic optimization method to compute joint torques so that the histories of the angular orientations of the model segments closely approximate the corresponding observed angular coordinate histories is demonstrated with the Yurchenko layout vault using an optimal control package. The objective function used is a measure of distance of fitted segment angles to the data, plus the distance of the fitted whole body centre of mass (CM), from the whole body CM computed from the data. Including the CM into the objective function, facilitates the optimization process so as to obtain a set of torques which reproduced the data. The paper shows that the approach works well for the task examined, that is, where the dynamics of the system change during a movement (impact to postflight).

Adolescent↗