Development and validation of a model for radiofrequency ablation]

Since 1986, cardiac arrhythmias have been successfully treated by destroying the underlying arrhythmogenic substrate with radiofrequency energy (radiofrequency ablation). The aim of this study was to develop a model for radiofrequency ablation enabling evaluation of the temperature distribution within cardiac tissue and the influence of electrode tissue contact. The model describes a 7F electrode, 4 mm in length and positioned perpendicular to the tissue. Heat convection within the tissue and heat lost via the blood was taken into account. Simulation of constant tissue exposure to 4 W resulted in a temperature increase of 35 degrees C after 10 sec. The temperature increase in the depths was less steep, but constant, and exceeded the electrode temperature at depths of 1 mm after 40 sec, 2 mm after 100 sec, and 3 mm after 200 sec. Electrode tissue contact proved to have a great influence on tissue temperature. Poor contact resulted in a temperature rise of only 0.68 degree C with a maximum of 50 W, whereas with ideal contact, 4 W sufficed to achieve a chosen setpoint of 70 degrees C. The model was validated in an in vitro setup using ventricular tissue from the pig. A strong correlation was found between simulated heating efficiency during temperature-controlled ablations under different contact conditions, and the respective measured values in the in vitro setup with a correlation coefficient of 0.97.     

A new graphic method for determining the rate constant for affinity of a ligand for its receptor]

A new method of determination of the equilibrium constant for a ligand binding to acceptor and evaluation of the number of binding sites on the acceptor molecules (or cells) is suggested. The method is simpler, more convenient, and more precise than Klotz's or Scatchard's method.     

Development and validation of a HPLC method for the quantification of baculovirus particles

A HPLC method using an anion exchange column was developed for the quantification of baculovirus particles. To properly detect the virus eluting from the column, a nucleic acid dye was used to amplify the signal projected by the virus. The viral genome was labeled by incubating the virus with SYBR Green I at 37°C for a minimum of 1h. The virus was specifically eluted from the contaminants in 8.9 min at a NaCl concentration of 480 mM NaCl (in 20 mM Tris-HCl, pH 7.5). The total run time of the method was 25 min. The method resulted in a linear response from 1×10(8) to 5.0×10(10)viral particles (VP/ml). The detection limit was 3.0×10(7) and the quantification limit was 1×10(8)VP/ml. The intra-assay precision was <10% for both purified and crude virus preparations whereas the inter-assay precisions were <5% and <10% for purified and crude virus preparations, respectively. The recovery/accuracy of the method ranged from 78 to 101%. This method is a robust monitoring tool to facilitate research activities with baculovirus vector and accelerate development of baculovirus-based processes for manufacturing of biologics.     

Development and validation of a simple method for the extraction of human skin melanocytes

Primary melanocytes in culture are useful models for studying epidermal pigmentation and efficacy of melanogenic compounds, or developing advanced therapy medicinal products. Cell extraction is an inevitable and critical step in the establishment of cell cultures. Many enzymatic methods for extracting and growing cells derived from human skin, such as melanocytes, are described in literature. They are usually based on two enzymatic steps, Trypsin in combination with Dispase, in order to separate dermis from epidermis and subsequently to provide a suspension of epidermal cells. The objective of this work was to develop and validate an extraction method of human skin melanocytes being simple, effective and applicable to smaller skin samples, and avoiding animal reagents. TrypLE? product was tested on very limited size of human skin, equivalent of multiple 3-mm punch biopsies, and was compared to Trypsin/Dispase enzymes. Functionality of extracted cells was evaluated by analysis of viability, morphology and melanin production. In comparison with Trypsin/Dispase incubation method, the main advantages of TrypLE? incubation method were the easier of separation between dermis and epidermis and the higher population of melanocytes after extraction. Both protocols preserved morphological and biological characteristics of melanocytes. The minimum size of skin sample that allowed the extraction of functional cells was 6 × 3-mm punch biopsies (e.g., 42 mm²) whatever the method used. In conclusion, this new procedure based on TrypLE? incubation would be suitable for establishment of optimal primary melanocytes cultures for clinical applications and research.     

Development and validation of a liquid chromatographic method for Casiopeina IIgly in rat plasma

A sensitive and specific liquid chromatographic method using solid-phase extraction with Sep-pak cartridges has been developed for the determination of Casiopeina IIgly and validated over the linear range 2.5-50 microg/ml in rat plasma. The analysis was performed on a Symetry C(18) (5 microm) column with a Phenomenex C(18) precolumn. The mobile phase was methanol-water (58:42, v/v). The column effluent was monitored at 273 nm. The results showed that the assay is sensitive at 2.5 microg/ml. Maximum intra-day coefficient of variation was 11.47%. The recovery based upon addition of internal standard to rat plasma was 80.98%. The method was used to perform preclinical pharmacokinetic studies in rat plasma and was found to be satisfactory.     

Development and implementation of a spatial unit non-overlapping water stress index for water scarcity evaluation with a moderate spatial resolution

Water scarcity is a serious global problem, and accurate estimations are urgently needed. The Water stress index (WSI) is one of the most commonly used methods for global or large scale water scarcity evaluation, but this method lacks of consideration of water demand and water supply positions non-overlapped spatial distribution, tends to overestimate water stress when applied to a moderate resolution grid (e.g., 1 km). In this study, we used a non-overlapping water supply-and-demand unit approach to improve the calculation scheme and constructed a spatial unit non-overlapping WSI model (Sun-WSI model). We then applied the new model to the Yarlung Tsangpo-Brahmaputra River (TBR) and estimated monthly water stress from 2006 to 2012 with a 1 km spatial resolution. The results showed that the determination coefficient (R²) between the normalized Drought Index (DI) and water stress was mainly in the range of 0.2–0.7, accounting for 77.4% of the study area. The spatial pattern of water stress estimated by the Sun-WSI model was consistent with the DI. Further analysis showed that both overall and grid water stress estimated by the Sun-WSI model were close to the results from existing studies; however the Sun-WSI model had a higher spatial resolution. With a 1 km resolution, the Sun-WSI model performed better than the conventional WSI with respect to both overall results and spatial details. This suggests that the Sun-WSI model is suitable for evaluating regional or moderate-resolution grid water scarcity.     

Development and validation of a liquid chromatographic method for Casiopeina IIIi in rat plasma

In order to measure changes in physiological CO concentrations in blood with good accuracy, a method was developed using gas chromatography with flame ionisation detection (250 degrees C). A nickel catalyst system was fitted to convert CO to methane at 375 degrees C after separation with a molecular sieve column at 35 degrees C. Helium was used as carrier at 30 ml/min. Porcine or human blood (400 microl) was sampled in gastight tubes and treated with sulfuric acid and saponin (800 microl). Accuracy was 1.4% and 1.5% (RSD), respectively. Precision was 2.8% (porcine blood). Limit of detection was 0.01 nmol/ml gas and limit of quantification 12 nmol/ml blood. Calibration was made in the interval 12-514 nmol/ml blood (corresponding to 0.1-6% COHb). Samples were stable for at least a month at +4 degrees C. This paper describes a method with high sensitivity and good accuracy, suitable for analysis of low CO concentrations.     

Key aspects of analytical method validation and linearity evaluation

Method validation may be regarded as one of the most well-known areas in analytical chemistry as is reflected in the substantial number of articles submitted and published in peer review journals every year. However, some of the relevant parameters recommended by regulatory bodies are often used interchangeably and incorrectly or are miscalculated, due to few references to evaluate some of the terms as well as wrong application of the mathematical and statistical approaches used in their estimation. These mistakes have led to misinterpretation and ambiguity in the terminology and in some instances to wrong scientific conclusions. In this article, the definitions of various relevant performance indicators such as selectivity, specificity, accuracy, precision, linearity, range, limit of detection, limit of quantitation, ruggedness, and robustness are critically discussed with a view to prevent their erroneous usage and ensure scientific correctness and consistency among publications.     

Development and validation of a reversed-phase HPLC method for determination of lesatropane and enantiomeric impurity

Lesatropane is a novel muscarinic receptor agonist and is currently being under preclinical development in China as a single enantiomer drug for the treatment of primary glaucoma. A reversed-phase chiral HPLC method for determination of lesatropane and enantiomeric impurity was developed. Enantiomeric separation of lesatropane from its enantiomer (desatropane) was achieved in normal-phase mode with Chiralpak AD-H and in reversed-phase mode with Chiralpak AS-RH. The conditions using a Chiralpak AS-RH column and mobile phase of K(2) HPO(4) -KH(2) PO(4) (pH 7.0; 0.02 M)-acetonitrile (69:31, v/v) at a flow rate of 0.5 ml/min have been fully validated with satisfactory specificity, linearity, accuracy, and precision. The method was found to be suitable for the simultaneous quantitation of lesatropane and enantiomeric impurity desatropane.     

Development and validation of a novel method for evaluating behavior and temperament in guide dogs

Most guide and service dog organizations would benefit from the development of accurate methods for the early evaluation of canine temperament traits. This paper describes the development and validation of a novel questionnaire method for assessing behavior and temperament in 1-year-old guide dogs. Volunteer puppy-raisers scored a total of 1097 prospective guide dogs on a series of 40 semantic differential-type, behavioral rating scales. Principle components factor analysis of these scores extracted eight stable and interpretable common factors: stranger-directed fear/aggression, non-social fear, energy level, owner-directed aggression, chasing, trainability, attachment, and dog-directed fear/aggression. Three of these eight factors exhibited moderate internal consistency (Cronbach's alpha>/=0.72), while the reliabilities of the remaining factors were relatively low (Cronbach's alpha=0.53-0.61). The eight factors were then validated against the guide dog school's own criteria for rejecting dogs for behavioral reasons. The results of this analysis confirmed the construct validity of the puppy raisers' questionnaire assessments of their dogs, and suggested that such methods can provide a useful and accurate means of predicting the suitability of dogs for guiding work. Various modifications to the original questionnaire are proposed in order to enhance its overall reliability.     

Development and validation of a molecular method for the diagnosis of medically important fungal infections

The increasing incidence of severe fungal infections highlights the need for rapid and precise identification methods in clinical mycology. The aim of this study was to develop and validate a culture-indipendent molecular approach that could allow the detection of fungal pathogens in clinical samples, with particular attention to the identification of drug-resistant Candida and Aspergillus species. A real-time multiplex PCR assay was developed using TaqMan probes specific for highly discriminating ITS sequences. In its multiplex format the assay showed a high specificity, clearly discriminating among different species, as well as a high sensitivity (20 CFU/1 mL sample), making it a potentially useful starting point for the development of a more complete molecular diagnostic assay.     

Development and validation of a computational method for assessment of missense variants in hypertrophic cardiomyopathy

Assessing the significance of novel genetic variants revealed by DNA sequencing is a major challenge to the integration of genomic techniques with medical practice. Many variants remain difficult to classify by traditional genetic methods. Computational methods have been developed that could contribute to classifying these variants, but they have not been properly validated and are generally not considered mature enough to be used effectively in a clinical setting. We developed a computational method for predicting the effects of missense variants detected in patients with hypertrophic cardiomyopathy (HCM). We used a curated clinical data set of 74 missense variants in six genes associated with HCM to train and validate an automated predictor. The predictor is based on support vector regression and uses phylogenetic and structural features specific to genes involved in HCM. Ten-fold cross validation estimated our predictor's sensitivity at 94% (95% confidence interval: 83%-98%) and specificity at 89% (95% confidence interval: 72%-100%). This corresponds to an odds ratio of 10 for a prediction of pathogenic (95% confidence interval: 4.0-infinity), or an odds ratio of 9.9 for a prediction of benign (95% confidence interval: 4.6-21). Coverage (proportion of variants for which a prediction was made) was 57% (95% confidence interval: 49%-64%). This performance exceeds that of existing methods that are not specifically designed for HCM. The accuracy of this predictor provides support for the clinical use of automated predictions alongside family segregation and population frequency data in the interpretation of new missense variants and suggests future development of similar tools for other diseases.     

Development and validation of a UPLC/MS method for a nutritional metabolomic study of human plasma

In order to study the effect of a diet on metabolites found in body fluids such as plasma, we have developed and validated a UPLC/MS method. While methods using NMR have been well established to analyse different biological tissues, recent studies have described robust untargeted UPLC-MS methods for plasma analysis. One major concern when profiling plasma is the presence of an important quantity of proteins which have to be precipitated without any loss of metabolites prior to LC/MS analysis. The utilization of untargeted approaches in nutritional metabolomics still suffers from the lack of identification of specific biomarkers. We therefore suggest an alternative method still using a global approach but focusing at the same time on metabolites previously described in human plasma in order to detect biomarkers of metabolic dysregulations. Thus, to fulfil our objectives, analytical parameters were tested (i) the anticoagulant type for sample collection, (ii) the protein precipitation method and (iii) UPLC/MS analytical conditions. Three protein precipitation methods and two anticoagulants were tested and compared. The method utilizing blood collection on heparin and methanol precipitation was chosen for giving the most reproducible results while keeping the complexity of the sample. Finally, a validation was proposed to evaluate the stability of this analytical method applied to a large batch of samples for nutritional metabolomic studies.     

Development and validation of a method for purification of mallein for the diagnosis of glanders in equines

ABSTRACT: BACKGROUND: The allergic test of mallein is one of the most frequently used tests, together with the Complement Fixation Test (CFT), for the diagnosis of glanders in endemic areas. Mallein, a purified protein derivative (PPD), is produced similarly to PPD tuberculin and the end product is a primarily proteic antigen, which is only poorly purified. The immuno-allergic activity of mallein is believed to be due to a high molecular weight group of proteins present in the antigen. To improve the quality of the antigen, in terms of sensitivity and specificity, a new method of mallein production was developed, in which purification was accomplished by ultrafiltration in a Tangential Flow Filtration system (TFF). RESULTS: The TFF methodology efficiently separated the high and low molecular weight protein groups of mallein. The five TFF-purified malleins, produced from Burkholderia mallei strains isolated from clinical cases of glanders in Brazil, proved to be more potent than standard mallein in the induction of an allergic reaction in sensitized animals. Regarding specificity, two of the purified malleins were equivalent to the standard and three were less specific. CONCLUSION: Some of the TFF-purified malleins showed considerable potential to be used as an auxiliary test in the diagnosis of glanders.     

Development,validation and application of a multi-mycotoxin method for the analysis of whole wheat plants

Mycotoxins are known to affect the health of humans and husbandry animals. In contrast to wheat grains used for food and feed, whole wheat plants are rarely analysed for mycotoxins, although contaminated straw could additionally expose animals to these toxic compounds. Since the entire wheat plant may also act as source of mycotoxins emitted into the environment, an analytical method was developed, optimised and validated for the analysis of 28 different mycotoxins in above-ground material from whole wheat plants. The method comprises solid-liquid extraction and a clean-up step using a Varian Bond Elut Mycotoxin^® cartridge, followed by liquid chromatography with electrospray ionisation and triple quadrupole mass spectrometry. Total method recoveries for 26 out of 28 compounds were between 69 and 122% and showed limits of detection from 1 to 26 ng/g_{dry weight (dw)}. The overall repeatability for all validated compounds was on average 7%, and their mean ion suppression 65%. Those rather high matrix effects made it necessary to use matrix-matched calibrations to quantify mycotoxins within whole wheat plants. The applicability of this method is illustrated with data from a winter wheat test field to examine the risks of environmental contamination by toxins following artificial inoculation separately with four different Fusarium species. The selected data originate from samples of a part of the field which was inoculated with Fusarium crookwellense. In the wheat samples, various trichothecenes (3-acetyl-deoxynivalenol, deoxynivalenol, diacetoxyscirpenol, fusarenone-X, nivalenol, HT-2 toxin, and T-2 toxin) as well as beauvericin and zearalenone were identified with concentrations ranging from 32 ng/g_dw to 12 × 10³ ng/g_dw.     

Application of a graphic method for analyzing the kinetics of multienzyme reactions

A new graphic method is proposed to solve kinetic equations for polyenzymic reactions. Each graph apex is corresponded by the transmitting function deduced from kinetic equations by means of Laplas transformation. Application of this procedure allows to simplify the solution of kinetic equations and its analysis. The procedure suggested makes it possible to use the methods of automatic control when solving theoretical problems of enzymology.     

Development and validation of a nested-PCR-denaturing gradient gel electrophoresis method for taxonomic characterization of bifidobacterial communities

The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a "complete" community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.