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Preparation and in vitro evaluation of sustained release tablet formulations of diclofenac sodium.

The effects of formulation variables on the release profile of diclofenac sodium (DS) from hydroxypropylmethyl cellulose (HPMC) and chitosan matrix tablets were studied. DS tablets were prepared by wet granulation and direct compression methods and different ratios of HPMC and chitosan were used. Physical properties of the prepared tablets and targeted commercial sustained release (SR) tablet and the drug release were studied in tablets that were placed in 0.1 M HCl for 1 h and phosphate buffer solution was added to reach pH value of 7.5. In vitro studies showed that 20% HPMC contained SR formulation with direct (dry) compression method is the optimum formulation due to its better targeting profile in terms of release. This formulation also exhibited the best-fitted formulation into the zero order kinetics. The precision and accuracy of the analytical method were also checked. The repeatability and reproducibility of the method were also determined.

Chemistry, Pharmaceutical↗

Estimation of accordance and concordance in inter-laboratory trials of analytical methods with qualitative results.

In this short communication, we discuss alternative ways to estimate accordance and concordance, which are, for qualitative methods, the analogues of the well-known concepts repeatability and reproducibility. We argue that estimators in a random framework appear to be more appropriate than the estimators in a fixed framework as they were originally introduced by Langton et al. [International Journal of Food Microbiology 79 (2002) 171]. A simple example of their application is given.

Food Microbiology↗

Reduction and enhancement of Plasmodium falciparum transmission by endemic human sera.

Transmission of Plasmodium falciparum from man to mosquito can be affected by human sera. Whereas serum-dependent reduction of transmission has been shown to be reproducible, there is limited evidence for enhancement of transmission. We aimed to assess the prevalence and reproducibility of transmission enhancement (TE) by human sera from different geographic areas (n = 642), in comparison with the capacity for transmission reduction (TR). The overall prevalence of TE (7%) was lower than that of TR (48%) and its effect generally weaker but reproducible in repeated measurements. TR but not TE showed a significant association with the presence of serum antibodies against Pfs48/45 and a non-significant trend to the presence of anti-Pfs230 antibodies.

Adolescent↗

Indirect MR lymphangiography of the head and neck using conventional gadolinium contrast: a pilot study in humans.

PURPOSE: To evaluate indirect magnetic resonance lymphangiography (MR-LAG) using interstitial injection of conventional gadolinium contrast (gadoteridol and gadopentetate dimeglumine) for delineating the primary lymphatic drainage of head-and-neck sites. METHODS AND MATERIALS: We performed head-and-neck MR-LAG in 5 healthy volunteers, with injection of dermal and mucosal sites. We evaluated the safety of the procedure, the patterns of enhancement categorized by injection site and nodal level, the time course of enhancement, the optimal concentration and volume of contrast, and the optimal imaging sequence. RESULTS: The worst side effects of interstitial contrast injection were brief, mild pain and swelling at the injected sites that were self-limited. MR-LAG resulted in consistent visualization of the primary lymphatic drainage pattern specific to each injected site, which was reproducible on repeated examinations. The best enhancement was obtained with injection of small volumes (0.3-0.5 mL) of either agent diluted, imaging within 5-15 min of injection, and a three-dimensional fast spoiled gradient echo sequence with magnetization transfer. CONCLUSIONS: We found head-and-neck MR-LAG to be a safe, convenient imaging method that provides functional information about the lymphatic drainage of injected sites. Applied to head-and-neck cancer, it has the potential to identify sites at highest risk of occult metastatic spread for radiotherapy or surgical planning, and possibly to visualize micrometastases.

Adult↗

Liquid chromatography-tandem mass spectrometry determination of loperamide and its main metabolite desmethylloperamide in biological specimens and application to forensic cases.

A liquid chromatographic mass spectrometric (LC/MS/MS) method has been developed for the determination of loperamide in whole blood and other biological specimens. The procedure involves liquid-liquid extraction of loperamide, desmethylloperamide and methadone-D3 (internal standard) with butyl acetate. Confirmation and quantification was done by positive electrospray ionisation with a triple quadrupole mass spectrometer operating in multiple reaction-monitoring (MRM) mode. Two MRM transitions of each compound were established and identification criteria were set up based on the ratio of the responses between the two MRM transitions of each compound. The standard curves were linear over a working range of 0.1-500 microg/kg for all transitions. The limit of quantification was 0.1 microg/kg in whole blood. The repeatability and reproducibility within the laboratory expressed by relative standard deviation were less than 5 and 11%, respectively, and the accuracy was better than 9%. The method was developed to examine a feces sample from a child whose mother was suspected of Münchausen syndrome by proxy and it proved to be suitable for forensic cases being simple, selective and reproducible. The method was also applied for a case investigation involving a overdose of loperamide.

Antidiarrheals↗

Analysis of strawberry volatiles using comprehensive two-dimensional gas chromatography with headspace solid-phase microextraction.

The aims of the current study were to develop an enantioselective multi-dimensional gas chromatography (GC x GC) method for the examination of strawberry volatiles and to use this method to make comparisons between the volatile profiles of different cultivars and between fresh picked and post-harvest berries of the same cultivar. Strawberry volatiles were sampled using solid-phase microextraction (SPME), and the repeatability and reproducibility of this method was examined. Semi-quantitative analysis of the volatiles was conducted using the relatively new technique of comprehensive multi-dimensional gas chromatography, using enantioselective (chiral) columns for the differentiation of analyte enantiomers. Chiral GC x GC facilitated the detection of key enantiomers in strawberry flavour. The (-)-enantiomer of 2,5-dimethyl-4-hydroxy-(2H)-furan-3-one (DMHF) and the S-enantiomer of linalool were tentatively identified as the predominant forms in both the cultivars Selva and Adina. The compounds benzaldehyde and methyl hexanoate were shown to decrease in post-harvest berries, whilst DMHF and nerolidol increased upon storage.

Chromatography, Gas↗

Rapid high-performance liquid chromatographic method for Vitamin C determination in human milk versus an enzymatic method.

Vitamin C is an antioxidant that can be considered a possible biomarker of oxidative stability in human milk. A high-performance liquid chromatographic method was developed and validated for determining the total Vitamin C (ascorbic acid and dehydroascorbic acid) and ascorbic acid levels in human milk. This method was then compared with an enzymatic method (a Colorimetric technique) for quantifying ascorbic acid levels. Repeatability and reproducibility were acceptable for all methods. However, the high-performance liquid chromatography (HPLC) technique provided more satisfactory results than the enzymatic method due to this last method detected 37% less ascorbic acid and does not determine the total Vitamin C because of the enzymatic method cannot reduce the dehydroascorbic acid (DHA) to ascorbic acid. Furthermore, the HPLC method has the added advantages that it requires less reagents and material, and is simpler and less time consuming than the enzymatic method. In conclusion, the drawbacks of this enzymatic method would justify its substitution for a HPLC method.

Ascorbic Acid↗

Chiral separation and quantification of R/S-amphetamine, R/S-methamphetamine, R/S-MDA, R/S-MDMA, and R/S-MDEA in whole blood by GC-EI-MS.

The enantioselective composition of the amphetamines is of interest, as the enantiomers show differences in their pharmacological effects and several methods for chiral separation of amphetamines have been described. Only a few methods have used whole blood as matrix and none of these separates both classic amphetamines (amphetamine and methamphetamine) and designer amphetamines (MDA, MDMA and MDEA). The aim of this study was, therefore, to develop a method for enantioselective analysis of AM, MA, MDA, MDMA, and MDEA in whole blood. The amphetamines were extracted from 0.5 g of whole blood by liquid-liquid extraction. After derivatization with R-MTPCl, the resulting diastereomers were separated by GC on a HP-5MS column and detected by SIM-MS. R-MTPCl was used as derivatization reagent because of the stability of this reagent and good separation of these analytes. Through the method, development time and temperature of the derivatization were optimized, and by admixture of 0.02% triethylamine it became possible to detect the amphetamines in adequately low concentrations as more analytes were derivatized. The method was validated and it was linear from 0.004 to 3 microg/g per enantiomer. The accuracy was within 91-115%, while the repeatability and reproducibility were < or =15% R.S.D. A method suitable for enantioselective separation and analysis of the amphetamines has been achieved, and the method was applied to analysis of whole blood samples originating from traffic and criminal cases and post mortem cases.

3,4-Methylenedioxyamphetamine↗

In vivo data of epidermal thickness evaluated by optical coherence tomography: effects of age, gender, skin type, and anatomic site.

BACKGROUND: The knowledge of epidermal thickness (ET) is of great significance in many areas of medical and biological research. OBJECTIVES: We aimed to assess optical coherence tomography (OCT) in terms of precision, and to investigate the influence of several constitutional factors, such as age, gender, skin type, and anatomic site, on the mean ET using OCT in vivo. METHODS: Eighty-three subjects were studied using OCT in vivo. Intra- and inter-day repeatability measurements were performed. The mean ET was assessed in six different body sites of young (20-40 years old) and old (60-80 years old) Caucasians, respectively. An ethnic group was included into the study. RESULTS: OCT proved to be a precise technique in terms of repeatability and reproducibility as expressed in low coefficients of variation. Comparison of young and old Caucasians demonstrated a significant decrease of ET with age in all anatomic sites investigated. ET assessed in males and females did not significantly differ, except for forehead skin which is significantly thinner in old females than in males. ET observed in Caucasians did not significantly differ from ET measured in ethnic individuals. Anatomic sites insignificantly influenced ET on an inter-individual level. However, differences of ET between body sites on an intra-individual level are significant. CONCLUSIONS: This was the first systematic in vivo study on ET investigating several influencing parameters of the epidermal dimension in a reasonable study sample by means of OCT. The results presented here may serve as ET reference data in a variety of clinical and experimental matters.

Adult↗

A validated cold vapour-AAS method for determining mercury in human red blood cells.

A cold vapour-atomic absorption spectrometry (CV-AAS) method for determining mercury in dental students and clinical teaching staff red blood cells at a dental school using amalgam as a restorative material has been validated. A number of blood samples (n = 122) from dental students in years I to V, clinical teachers in restorative dentistry and controls were collected and analysed. Accuracy, linearity, precision (repeatability and reproducibility) and robustness of the method have been determined, and detection and quantification limits have been calculated. Linearity of response was verified for concentrations ranging from 5 to 40 microgL(-1) of mercury. Correlation coefficient of the calibration straight lines was always >/=0.99. Intra-day precision of the method gave coefficient of variation (CV) of 5.51%. Inter-day precision of the method calculated after analysis of five different concentrations of mercury standard solutions by the same analyst in different days and by two different analysts in different days gave coefficient of variation 4.89 and 5.44%, respectively. The accuracy of the method was calculated a CRM NIST 966 (toxic metals in bovine blood) total amount of mercury was found a concentration of 28.83 +/- 2.2 microgL(-1). Recovery was 89.27%. Robustness of the method evaluated by changing different experimental conditions under which analyses performed, fractional factorial design was done for assessing robustness of the method. Root mean square error was found out as 1.56. Limits of detection and quantification were 1.84 and 4.03 microg of Hg per litre of sample, respectively. Results show the suitability of the method for direct measurement of mercury in red blood cells and the importance of the working conditions for people dealing with amalgam at a dental school.

Cold Temperature↗

Application of an improved column characterisation system to evaluate the within and between batch variability.

The selection of a reversed-phase liquid chromatographic column with suitable selectivity for a particular separation is difficult if the brand name of the column is not known. A project to develop a chromatographic test procedure to characterize reversed-phase liquid chromatography C18 columns was started earlier and resulted in a fast, simple, repeatable and reproducible test procedure using four column parameters. Here, this procedure is used to evaluate the diversity of columns originating from the same batch as well as from different batches. The determination of one of the parameters, the retention factor of 2,2'-dipyridyl, was improved and a simplified test procedure is proposed.

2,2'-Dipyridyl↗

Validation of a foot-and-mouth disease antibody screening solid-phase competition ELISA (SPCE).

This paper describes the validation of a solid-phase competition enzyme-linked immunosorbent assay (SPCE) for the serological detection of antibody to serotype O foot-and-mouth disease (FMD) in sheep, cattle and pigs. The specificity of the SPCE was calculated from the results of testing known negative sera from sheep, cattle and pigs (n=3030, 1418 and 1495, respectively). The mean percentage inhibition (PI) for known negative sheep, cattle and pig sera were 19.3, 24.1 and 20.8%, respectively. The specificity of the SPCE at a cut-off point (COP) of 60 PI was 99.50% for sheep sera, 99.44% for cattle sera and 100% for pig sera. The analytical sensitivity of the SPCE was examined by testing sera from sheep, cattle and pigs. Based on the testing of serial bleeds from experimentally infected animals, seroconversion at the 60 PI COP occurred between 4 and 9 days post-infection or -exposure, similar to that observed using the virus neutralisation test (VNT) with a COP of 1/45. When applied to 267 sheep and 143 pig samples, that were obtained in Great Britain (GB) during the 2001 FMD UK outbreak, the SPCE identified more positive samples than did the VNT. Estimates of the accuracy, repeatability and reproducibility of the SPCE were verified during the large-scale serosurveillance necessitated by the 2001 outbreak. Results from field and experimental sera showed that when compared against the VNT, the sensitivity of the SPCE was less affected by the choice of virus strain used in the test. Using the O(1) UKG 2001 FMD virus in the VNT with samples representative of the uninfected GB sheep population, the test specificity was 100% at a COP of 1/45.

Animals↗

A critical investigation of ISO 11948-2 and ISO 11948-1 for predicting the leakage performance of small disposable incontinence pads for lightly incontinent women.

ISO 11948-2--an international standard laboratory method developed to predict the leakage performance of small disposable pads for lightly incontinent women--was investigated. The repeatability and reproducibility (precision within and between laboratories, respectively) of two variants on the method were found to be poor. The coefficient of variation for each method variant in each laboratory (two laboratories ran each variant) was higher than 40% for about half the 12 products evaluated. Results differed by up to 94% between laboratories for a given product. The ability of the method to predict the leakage performance of pads was investigated by measuring correlations between the clinical evaluations of the 12 products, and technical evaluations using ISO 11948-2. Correlations were very weak (r < or= 0.487). Accordingly, it is recommended that 11948-2 is withdrawn. A second international standard method (ISO 11948-1)--developed for evaluating large pads, but sometimes used on small ones--was also investigated. Correlations between the clinical evaluations of the 12 products and technical evaluations using ISO 11948-1 were weak (r < or = 0.560). Accordingly, it is recommended that ISO 11948-1 is not used for evaluating small disposable bodyworn pads for women.

Absorption↗

Use of a spectrophotometric bioassay for determination of microbial sensitivity to manuka honey.

The antimicrobial activity of manuka honey has been well documented (Molan, 1992a,b,c, 1997) [Molan, P.C., 1992. The antibacterial activity of honey. 1: the nature of the antibacterial activity. Bee World 73 (1) 5-28; Molan, P.C., 1992. The antibacterial activity of honey. 2: variation in the potency of the antibacterial activity. Bee World 73 (2) 59-76; Molan, P.C., 1992. Medicinal uses for honey. Beekeepers Quarterly 26; Molan, P.C., 1997. Finding New Zealand honeys with outstanding antibacterial and antifungal activity. New Zealand Beekeeper 4 (10) 20-26]. The current bioassays for determining this antimicrobial effect employ a well diffusion (Ahn and Stiles, 1990) [Ahn, C., Stiles, M.E., 1990. Antibacterial activity of lactic acid bacteria isolated from vacuum-packed meats. Journal of Applied Bacteriology 69, 302-310], (Weston et al., 1999) [Weston, R.J., Mitchell, K.R., Allen, K.L., 1999. Antibacterial phenolic components of New Zealand manuka honey. J. Food Chem. 64, 295-301] or disc diffusion (Taormina et al., 2001) [Taormina, Peter J., Niemira, Brendan A., Beuchat, Larry R., 2001. Inhibitory activity of honey against food borne pathogens as influenced by the presence of hydrogen peroxide and level of antioxidant power. Int. J. Food Microbiol. 69, 217-225] assay using zones of inhibition as indicators of bacterial susceptibility. The development of a 24-h spectrophotometric assay employing 96-well microtiter plates, that is more sensitive and more amenable to statistical analysis than the assays currently employed, was undertaken. This simple and rapid assay permits extensive kinetic studies even in the presence of low honey concentrations, and is capable of detecting inhibitory levels below those recorded for well or disc diffusion assays. In this paper, we compare the assay to both well and disc diffusion assays. The results we obtained for the spectrophotometric method MIC values show that this method has greater sensitivity than the standard well and disc diffusion assays. In addition, inter- and intra-assay variance for this method was investigated, demonstrating the methods reproducibility and repeatability.

Analysis of Variance↗

Use of capillaries in the construction of an MRI phantom for the assessment of diffusion tensor imaging: demonstration of performance.

Although diffusion tensor imaging (DTI) shows great potential for the diagnosis of a variety of pathologies, no consensus for an appropriate assessment standard of DTI exists. This study examined the feasibility of using water-filled arrays of glass capillaries to construct a DTI phantom suitable for making repeated and reproducible measurements required in a quality assessment program. Three phantoms were constructed using arrays of capillaries with three inner diameters (23, 48, and 82 microm). Data were acquired using DTI protocols; the fractional anisotropy (FA), mean apparent diffusion coefficient (ADC) and principal eigenvectors of the diffusion tensors were calculated. This study demonstrated four results: (1) echo-planar images show that susceptibility within the capillary arrays does not lead to substantial differences in precessional frequency in regions containing the arrays and neither do the regions show noticeable image distortion; (2) principal eigenvectors of the diffusion tensors agree to within<10.3 degrees of the array orientations; (3) mean FA values (0.18-0.50) and ADC values (1.40-1.93x10-(3) mm2/s) within specified regions of interest are in general agreement with simulations after a simple noise correction; and (4) these array performance characteristics are observable using a typical clinical DTI protocol.

Anisotropy↗

Quantitative comparison of four brain extraction algorithms.

In a companion paper (Rehm et al., 2004), we introduced Minneapolis Consensus Strip (McStrip), a hybrid algorithm for brain/non-brain segmentation. In this paper, we compare the performance of McStrip and three brain extraction algorithms (BEAs) in widespread use within the neuroimaging community--Statistical Parametric Mapping v.2 (SPM2), Brain Extraction Tool (BET), and Brain Surface Extractor (BSE)--to the "gold standard" of manually stripped T1-weighted MRI brain volumes. Our comparison was based on quantitative boundary and volume metrics, reproducibility across repeat scans of a single subject, and assessments of performance consistency across datasets acquired on different scanners at different institutions. McStrip, a hybrid method incorporating warping to a template, intensity thresholding, and edge detection, consistently outperformed SPM2, BET, and BSE, all of which rely on a single algorithmic strategy.

Algorithms↗

Anterior chamber width measurement by high-speed optical coherence tomography.

OBJECTIVE: To measure anterior chamber (AC) width and other dimensions relevant to the sizing of phakic intraocular lenses (IOLs) with a high-speed optical coherence tomography (OCT) system. DESIGN: Cross-sectional observational study. PARTICIPANTS: Both eyes of 20 normal volunteers. METHODS: A novel high-speed (4000 axial scans/second) OCT prototype was developed for anterior segment scanning. The system uses long wavelength (1310 nm) for deeper angle penetration, rectangular scanning for undistorted imaging, and short image acquisition time (0.125 seconds) to reduce motion error. Three horizontal cross-sectional OCT images (15.5 mm wide and 6 mm deep) of the anterior segment were obtained from each eye with real-time image display to guide centration on the corneal apex. Image processing software was developed to correct for image warping resulting from index transitions. Anterior chamber dimensions were measured using computer calipers by 3 expert raters (ophthalmologists). Analysis of variance was used to determine interrater, interimage, right versus left eye, and intersubject standard deviation (SD) of OCT measurements. MAIN OUTCOME MEASURES: Anterior chamber width (recess to recess), AC depth, and crystalline lens vault as measured by OCT; external white-to-white (WTW) corneal diameter (CD) as measured by Holladay-Godwin gauge. RESULTS: The mean AC width was 12.53+/-0.47 mm (intereye SD), and the mean corneal diameter was 11.78+/-0.57 mm. Optical coherence tomography measurement of AC width has good repeatability from image to image (SD, 0.134 mm), but there was significant difference between raters (SD, 0.215 mm). Estimation of AC width from WTW CD by linear regression was relatively inaccurate (residual SD, 0.41 mm). The mean AC depth was 2.99+/-0.323 mm (intereye SD), with repeatability of less than 0.001 mm (interimage SD), and the mean crystalline lens vault was 0.39+/-0.27 mm with 0.023 mm repeatability. CONCLUSIONS: Reproducible OCT AC biometry was demonstrated using a high-speed OCT prototype. Further improvement in reproducibility may be achieved by automating the measurements with a computer. Direct OCT AC width measurement may improve sizing of angle-supported AC IOLs over conventional estimation by WTW CD. The measurement of AC depth and lens vault also may be useful for other types of phakic AC IOLs.

Adult↗

Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.

We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

Animals↗