Multivariate Analysis for Animal Selection in Experimental Research

Background: Several researchers seek methods for the selection of homogeneous groups of animals in experimental studies, a fact justified because homogeneity is an indispensable prerequisite for casualization of treatments. The lack of robust methods that comply with statistical and biological principles is the reason why researchers use empirical or subjective methods, influencing their results. Objective: To develop a multivariate statistical model for the selection of a homogeneous group of animals for experimental research and to elaborate a computational package to use it. Methods: The set of echocardiographic data of 115 male Wistar rats with supravalvular aortic stenosis (AoS) was used as an example of model development. Initially, the data were standardized, and became dimensionless. Then, the variance matrix of the set was submitted to principal components analysis (PCA), aiming at reducing the parametric space and at retaining the relevant variability. That technique established a new Cartesian system into which the animals were allocated, and finally the confidence region (ellipsoid) was built for the profile of the animals’ homogeneous responses. The animals located inside the ellipsoid were considered as belonging to the homogeneous batch; those outside the ellipsoid were considered spurious. Results: The PCA established eight descriptive axes that represented the accumulated variance of the data set in 88.71%. The allocation of the animals in the new system and the construction of the confidence region revealed six spurious animals as compared to the homogeneous batch of 109 animals. Conclusion: The biometric criterion presented proved to be effective, because it considers the animal as a whole, analyzing jointly all parameters measured, in addition to having a small discard rate.

Serial High-Sensitivity Troponin T in Post-Primary Angioplasty Exercise Test

Abstract Background: The kinetics of high-sensitivity troponin T (hscTnT) release should be studied in different situations, including functional tests with transient ischemic abnormalities. Objective: To evaluate the release of hscTnT by serial measurements after exercise testing (ET), and to correlate hscTnT elevations with abnormalities suggestive of ischemia. Methods: Patients with acute ST-segment elevation myocardial infarction (STEMI) undergoing primary angioplasty were referred for ET 3 months after infarction. Blood samples were collected to measure basal hscTnT immediately before (TnT0h), 2 (TnT2h), 5 (TnT5h), and 8 hours (TnT8h) after ET. The outcomes were peak hscTnT, TnT5h/TnT0h ratio, and the area under the blood concentration-time curve (AUC) for hscTnT levels. Log-transformation was performed on hscTnT values, and comparisons were assessed with the geometric mean ratio, along with their 95% confidence intervals. Statistical significance was assessed by analysis of covariance with no adjustment, and then, adjusted for TnT0h, age and sex, followed by additional variables (metabolic equivalents, maximum heart rate achieved, anterior wall STEMI, and creatinine clearance). Results: This study included 95 patients. The highest geometric means were observed at 5 hours (TnT5h). After adjustments, peak hscTnT, TnT5h/TnT0h and AUC were 59% (p = 0.002), 59% (p = 0.003) and 45% (p = 0.003) higher, respectively, in patients with an abnormal ET as compared to those with normal tests. Conclusion: Higher elevations of hscTnT may occur after an abnormal ET as compared to a normal ET in patients with STEMI.

Exploration of the visual system : Part 2. In vivo analysis methods : virtual-reality optomotor system, fundus examination and fluorescent angiography

The mouse has emerged as an animal model for many diseases. At IRO, we have used this animal to understand the development of many eye diseases and treatment of some of them. Precise evaluation of vision is a prerequisite for both these approaches. In this unit we describe three ways to measure vision: testing the optokinetic response, and evaluating the fundus by direct observation and by fluorescent angiography.

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples.

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.

Absorbance enhancement in microplate wells for improved-sensitivity biosensors

A generic optical biosensing strategy was developed that relies on the absorbance enhancement phenomenon occurring in a multiple scattering matrix. Experimentally, inserts made of glass fiber membrane were placed into microplate wells in order to significantly lengthen the trajectory of the incident light through the sample and therefore increase the corresponding absorbance. Enhancement factor was calculated by comparing the absorbance values measured for a given amount of dye with and without the absorbance-enhancing inserts in the wells. Moreover, the dilution of dye in solutions with different refractive indices (RI) clearly revealed that the enhancement factor increased with the ΔRI between the membrane and the surrounding medium, reaching a maximum value (EF>25) when the membranes were dried. On this basis, two H2O2-biosensing systems were developed based on the biofunctionalization of the glass fiber inserts either with cytochrome c or horseradish peroxidase (HRP) and the analytical performances were systematically compared with the corresponding bioassay in solution. The efficiency of the absorbance-enhancement approach was particularly clear in the case of the cytochrome c-based biosensor with a sensitivity gain of 40 folds and wider dynamic range. Therefore, the developed strategy represents a promising way to convert standard colorimetric bioassays into optical biosensors with improved sensitivity.

Chirality in the new generation of antidepressants: stereoselective analysis of the enantiomers of mirtazapine, N-demethylmirtazapine, and 8-hydroxymirtazapine by LC-MS.

Mirtazapine is an antidepressant that acts specifically on noradrenergic and sertonergic receptors. A LC-MS method was developed that allows the simultaneous analysis of the R-(-)- and S-(+)-enantiomers of mirtazapine (MIR), demethylmirtazapine (DMIR), and 8-hydroxymirtazapine (8-OH-MIR) in plasma of MIR-treated patients. The method involves a 3-step liquid-liquid extraction, an HPLC separation on a Chirobiotic V column, and MS detection in electrospray mode. The limit of quantification (LOQ) for all enantiomers was 0.5 ng/mL, and the intra- and interday CVs were within 3.3% to 11.7% (concentration ranges 5-50 ng/mL). A method is also presented for the quantitative analysis of glucuroconjugated MIR and 8-OH-MIR. S-(+)-8-OH-MIR is present in plasma mainly as its glucuronide. Preliminary data suggest that in all patients, except in those comedicated with CYP2D6 inhibitors such as fluoxetine and thioridazine, R-(-)-MIR concentrations were higher than those of S-(+)MIR. Moreover, fluvoxamine seems also to inhibit the metabolism of MIR. Therefore, this method seems to be suitable for the stereoselective assay of MIR and its metabolites in plasma of patients comedicated with MIR and other drugs for routine and research purposes.

Prognostication of neurologic outcome in cardiac arrest patients after mild therapeutic hypothermia: a meta-analysis of the current literature.

PURPOSE: To assess the sensitivity and false positive rate (FPR) of neurological examination and somatosensory evoked potentials (SSEPs) to predict poor outcome in adult patients treated with therapeutic hypothermia after cardiopulmonary resuscitation (CPR). METHODS: MEDLINE and EMBASE were searched for cohort studies describing the association of clinical neurological examination or SSEPs after return of spontaneous circulation with neurological outcome. Poor outcome was defined as severe disability, vegetative state and death. Sensitivity and FPR were determined. RESULTS: A total of 1,153 patients from ten studies were included. The FPR of a bilaterally absent cortical N20 response of the SSEP could be calculated from nine studies including 492 patients. The SSEP had an FPR of 0.007 (confidence interval, CI, 0.001-0.047) to predict poor outcome. The Glasgow coma score (GCS) motor response was assessed in 811 patients from nine studies. A GCS motor score of 1-2 at 72 h had a high FPR of 0.21 (CI 0.08-0.43). Corneal reflex and pupillary reactivity at 72 h after the arrest were available in 429 and 566 patients, respectively. Bilaterally absent corneal reflexes had an FPR of 0.02 (CI 0.002-0.13). Bilaterally absent pupillary reflexes had an FPR of 0.004 (CI 0.001-0.03). CONCLUSIONS: At 72 h after the arrest the motor response to painful stimuli and the corneal reflexes are not a reliable tool for the early prediction of poor outcome in patients treated with hypothermia. The reliability of the pupillary response to light and the SSEP is comparable to that in patients not treated with hypothermia.

Pareto or log-normal? A recursive-truncation approach to the distribution of (all) cities

Traditionally, it is assumed that the population size of cities in a country follows a Pareto distribution. This assumption is typically supported by nding evidence of Zipf's Law. Recent studies question this nding, highlighting that, while the Pareto distribution may t reasonably well when the data is truncated at the upper tail, i.e. for the largest cities of a country, the log-normal distribution may apply when all cities are considered. Moreover, conclusions may be sensitive to the choice of a particular truncation threshold, a yet overlooked issue in the literature. In this paper, then, we reassess the city size distribution in relation to its sensitivity to the choice of truncation point. In particular, we look at US Census data and apply a recursive-truncation approach to estimate Zipf's Law and a non-parametric alternative test where we consider each possible truncation point of the distribution of all cities. Results con rm the sensitivity of results to the truncation point. Moreover, repeating the analysis over simulated data con rms the di culty of distinguishing a Pareto tail from the tail of a log-normal and, in turn, identifying the city size distribution as a false or a weak Pareto law.

β-Glucan antigenemia assay for the diagnosis of invasive fungal infections in patients with hematological malignancies: a systematic review and meta-analysis of cohort studies from the Third European Conference on Infections in Leukemia (ECIL-3).

BACKGROUND: Invasive fungal infections (IFIs) are life-threatening complications in patients with hemato-oncological malignancies, and early diagnosis is crucial for outcome. The compound 1,3-β-D-glucan (BG), a cell wall component of most fungal species, can be detected in blood during IFI. Four commercial BG antigenemia assays are available (Fungitell, Fungitec-G, Wako, and Maruha). This meta-analysis from the Third European Conference on Infections in Leukemia (ECIL-3) assessed the performance of BG assays for the diagnosis of IFI in hemato-oncological patients. METHODS: Studies reporting the performance of BG antigenemia assays for the diagnosis of IFI (European Organization for Research and Treatment of Cancer and Mycoses Study Group criteria) in hemato-oncological patients were identified. The analysis was focused on high-quality cohort studies with exclusion of case-control studies. Meta-analysis was performed by conventional meta-analytical pooling and bivariate analysis. RESULTS: Six cohort studies were included (1771 adult patients with 414 IFIs of which 215 were proven or probable). Similar performance was observed among the different BG assays. For the cutoff recommended by the manufacturer, the diagnostic performance of the BG assay in proven or probable IFI was better with 2 consecutive positive test results (diagnostic odds ratio for 2 consecutive vs one single positive results, 111.8 [95% confidence interval {CI}, 38.6-324.1] vs 16.3 [95% CI, 6.5-40.8], respectively; heterogeneity index for 2 consecutive vs one single positive results, 0% vs 72.6%, respectively). For 2 consecutive tests, sensitivity and specificity were 49.6% (95% CI, 34.0%-65.3%) and 98.9% (95% CI, 97.4%-99.5%), respectively. Estimated positive and negative predictive values for an IFI prevalence of 10% were 83.5% and 94.6%, respectively. CONCLUSIONS: Different BG assays have similar accuracy for the diagnosis of IFI in hemato-oncological patients. Two consecutive positive antigenemia assays have very high specificity, positive predictive value, and negative predictive value. Because sensitivity is low, the test needs to be combined with clinical, radiological, and microbiological findings.

Decision analysis for the genotype designation in low-template-DNA profiles

What genotype should the scientist specify for conducting a database search to try to find the source of a low-template-DNA (lt-DNA) trace? When the scientist answers this question, he or she makes a decision. Here, we approach this decision problem from a normative point of view by defining a decision-theoretic framework for answering this question for one locus. This framework combines the probability distribution describing the uncertainty over the trace's donor's possible genotypes with a loss function describing the scientist's preferences concerning false exclusions and false inclusions that may result from the database search. According to this approach, the scientist should choose the genotype designation that minimizes the expected loss. To illustrate the results produced by this approach, we apply it to two hypothetical cases: (1) the case of observing one peak for allele xi on a single electropherogram, and (2) the case of observing one peak for allele xi on one replicate, and a pair of peaks for alleles xi and xj, i ≠ j, on a second replicate. Given that the probabilities of allele drop-out are defined as functions of the observed peak heights, the threshold values marking the turning points when the scientist should switch from one designation to another are derived in terms of the observed peak heights. For each case, sensitivity analyses show the impact of the model's parameters on these threshold values. The results support the conclusion that the procedure should not focus on a single threshold value for making this decision for all alleles, all loci and in all laboratories.

Association of mutations in the NALP3/CIAS1/PYPAF1 gene with a broad phenotype including recurrent fever, cold sensitivity, sensorineural deafness, and AA amyloidosis.

OBJECTIVE: Familial cold urticaria (FCU) and Muckle-Wells syndrome (MWS) are dominantly inherited autoinflammatory disorders that cause rashes, fever, arthralgia, and in some subjects, AA amyloidosis, and have been mapped to chromosome 1q44. Sensorineural deafness in MWS, and provocation of symptoms by cold in FCU, are distinctive features. This study was undertaken to characterize the genetic basis of FCU, MWS, and an overlapping disorder in French Canadian, British, and Indian families, respectively. METHODS: Mutations in the candidate gene NALP3, which has also been named CIAS1 and PYPAF1, were sought in the study families, in a British/Spanish patient with apparent sporadic MWS, and in matched population controls. Identified variants were sought in 50 European subjects with uncharacterized, apparently sporadic periodic fever syndromes, 48 subjects with rheumatoid arthritis (RA), and 19 subjects with juvenile idiopathic arthritis (JIA). RESULTS: Point mutations, encoding putative protein variants R262W and L307P, were present in all affected members of the Indian and French Canadian families, respectively, but not in controls. The R262W variant was also present in the subject with sporadic MWS. The V200M variant was present in all affected members of the British family with MWS, in 2 of the 50 subjects with uncharacterized periodic fevers, and in 1 of 130 Caucasian and 2 of 48 Indian healthy controls. No mutations were identified among the subjects with RA or JIA. CONCLUSION: These findings confirm that mutations in the NALP3/CIAS1/PYPAF1 gene are associated with FCU and MWS, and that disease severity and clinical features may differ substantially within and between families. Analysis of this gene will improve classification of patients with inherited or apparently sporadic periodic fever syndromes.

Detection of promoter activity by flow cytometric analysis of GFP reporter expression.

Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.

Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed

Sensitivity of Leishmania spp. to Glibenclamide and 4-Aminopiridine: A Tool for the Study of Drug Resistance Development

We have demonstrated that Leishmania spp. grown as promastigotes, are sensitive to the K+ channel inhibitors 4-aminopyridine and glibenclamide. Their host cells, the macrophages, are not affected by similar concentrations of the drugs. We have also initiated the molecular characterization of the mechanisms involved in the development of drug resistance to glibenclamide by the parasite. Therefore, we have selected experimentally and begun to characterize the Venezuelan Leishmania (Leishmania) strain, NR resistant to glibenclamide [NR(Gr)]. The analysis of genomic DNA evidenced the existence of a fragment which apparently is amplified in NR(Gr). The fragment recognized by the pgpA probe, related to the Leishmania P-glycoprotein family and which was originally isolated from L. tarentolae, showed a size polymorfism between the sensitive and the resistant strain. These results suggest that the development of resistance to glibenclamide in the strain NR(Gr) might be associated with the amplification of the ltpgpA or related gene(s)

Estimation of parametric and nonparametric models for univariate claim severity distributions - an approach using R

This paper presents an analysis of motor vehicle insurance claims relating to vehicle damage and to associated medical expenses. We use univariate severity distributions estimated with parametric and non-parametric methods. The methods are implemented using the statistical package R. Parametric analysis is limited to estimation of normal and lognormal distributions for each of the two claim types. The nonparametric analysis presented involves kernel density estimation. We illustrate the benefits of applying transformations to data prior to employing kernel based methods. We use a log-transformation and an optimal transformation amongst a class of transformations that produces symmetry in the data. The central aim of this paper is to provide educators with material that can be used in the classroom to teach statistical estimation methods, goodness of fit analysis and importantly statistical computing in the context of insurance and risk management. To this end, we have included in the Appendix of this paper all the R code that has been used in the analysis so that readers, both students and educators, can fully explore the techniques described