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Characterizing dog breed differentiation with microsatellite markers.

Estimates of dog breed differentiation at microsatellite loci were obtained: (1) by contrasting data from German shepherds with a composite breed mixture sample; (2) by examining the Wahlund effect in new and published breed mixture data sets; and (3) from a comparison of published retriever and dachshund allele frequencies. In terms of F-statistics, the estimates ranged FBT = 0.22-0.30, and were therefore considerably greater than a previous estimate from allozyme data (FBT = 0.10). The new estimates are consistent with a view that the genetic basis of an average breed effectively corresponds to four genomes sampled from the total, or ancestral, dog gene pool.

Alleles↗

Recombinant, B-domain deleted factor VIII (r-VIII SQ): pharmacokinetics and initial safety aspects in hemophilia A patients.

The pharmacokinetics of a second-generation recombinant B-domain deleted factor VIII (FVIII) preparation (r-VIII SQ) were studied in 36 patients with severe hemophilia A. In contrast to full-length recombinant FVIII, no albumin needs to be added to stabilize the final formulation of this B-domain deleted FVIII preparation. The in vivo recovery and half-life of r-VIII SQ were similar to those of plasma-derived (pd) FVIII (mean half-life of r-VIII SQ, 11.7 h). The volume of distribution and clearance were slightly, but significantly, higher for r-VIII SQ than for pdFVIII (p < 0.05). Peak plasma levels of FVIII were consistently related to the administered dose of r-VIII SQ (r = 0.94, p < 0.0001). The pharmacokinetic profile of r-VIII SQ remained essentially unchanged in a dose range of 25-100 IU/kg body weight and could be reproduced after repeated doses. r-VIII SQ was well tolerated. In conclusion, deletion of the B-domain of FVIII does not influence its in vivo pharmacokinetics.

Adolescent↗

Assessment of clonality in cutaneous lymphoid infiltrates by polymerase chain reaction analysis of immunoglobulin heavy chain gene rearrangement.

Determination of the biologic potential of cutaneous lymphoid infiltrates may be difficult by standard histologic or immunohistologic examination. The polymerase chain reaction (PCR) has been used to document clonal rearrangements of the immunoglobulin heavy chain gene in paraffin-embedded fixed tissues. To explore the value of PCR in evaluation of cutaneous lymphoid infiltrates, 93 archival nonmycotic lymphoid lesions (28 small or mixed lymphocytic lymphomas, 15 large cell lymphomas, 40 benign infiltrates, and 10 with atypical features) were analyzed. These cases had been previously immunophenotyped on paraffin sections, and clinical follow-up from 7 to 20 years was gathered. DNA products were generated using a seminested PCR technique, separated by 5% polyacrylamide gel electrophoresis, stained with ethidium bromide, and visualized under UV light. Cases with a 100- to 120-base pair band were scored as positive. Of the 28 small or mixed cell lymphomas, 23 had a B-cell immunophenotype or consisted of a mixture of B and T cells; of these, seven (30%) demonstrated a monoclonal pattern, and three (13%) were indeterminate. Twelve large cell cases were B-cell or mixed; five (42%) of these were positive for a monoclonal band, while four (33%) were indeterminate. None of five T-cell small cell or three T-cell large cell lymphomas demonstrated a monoclonal band. In contrast, 39 of the 40 benign cases were T-cell predominant or mixed lesions. Nevertheless, 18 of these 40 cases on initial testing suggested possible monoclonality. Six were indeterminate, and 12 demonstrated apparent monoclonal bands, of which four were reproducible on repeat testing. No histologic or clinical features of lymphoma were present in 17 of these 18 cases, suggesting that they represent false-positive results. Most of the latter lesions showed sparse perivascular infiltrates, with very few B cells. This suggests that amplification of the immunoglobulin heavy chain gene from a small number of lymphocytes may produce a monoclonal band. In summary, PCR may provide adjunct information about clonality in selected lymphoid skin lesions, but is rather insensitive in this setting. Such data must be carefully considered in the context of the histologic, immunohistologic, and clinical findings, particularly when assessing sparse infiltrates, because of the potential for false-positive results.

B-Lymphocytes↗

The effect of solasodine on the body temperature.

The action of solasodine, the steroid-structure alcaloid of Solanum laciniatum Ait., was studied on normal and pyretic body temperature of rats and mice. In rats a single dosage of 3 mg/kg depressed by an average of --1.5+/-0.3 degrees C the normal temperature for 24 h. With larger dosages or longer treatment the effect could not be intensified and tolerance was also not observed. In mice the temperature decrease was even more explicit, --2.0+/-0.2 degrees C lasting 48 h. The effect was reproducible by repeated treatment. In mice fever provoked by a suspension of killed bacteria, Pyrago¿, or by 2,4-dinitrophenol (DNP) could be counteracted with 1 mmole/kg solasodine. Body temperature depressed with solasodine was not raised by Pyrago but became higher after administration of DNP, in part of the animals even comparable to normal initial averages. Following solasodine treatment the body temperature of normothermic rats and mice decreased to subnormal values. Pyrexia produced either with centrally acting Pyrago or with the peripherally acting DNP was depressed with small solasodine dosages. Previous administration of the alcaloid counteracted Pyrago effect but could not inhibit the development of DNP action. These data are pertaining to the possible central effect of solasodine.

Alkaloids↗

Direct 24-hour presumptive enumeration of Escherichia coli O157:H7 in foods using hydrophobic grid membrane filter followed by serological confirmation: collaborative study.

Fifteen laboratories took part in a collaborative study to validate a method for enumerating Escherichia coli O157:H7. The method is based on use of a hydrophobic grid membrane filter and consists of 24 h presumptive enumeration on SD-39 Agar and serological confirmation to yield a confirmed E. coli O157:H7 count. Six food products were analyzed: pasteurized apple cider, pasteurized 2% milk, cottage cheese, cooked ground pork, raw ground beef, and frozen whole egg. The test method produced significantly higher confirmed count results than did the reference method for milk, pork, and beef. Test method results were numerically higher than but statistically equivalent to reference method results for cheese, cider, and egg. The test method produced lower repeatability and reproducibility values than did the reference method for most food/inoculation level combinations and values very similar to those of the reference method for the remaining combinations. Overall, 94% of presumptive positive isolates from the test method were confirmed serologically as E coli O157:H7, and 98% of these were also biochemically typical of E. coli O157:H7 (completed test). Corresponding rates for the reference method were 69 and 98%, respectively. On the basis of the results of this collaborative study and the precollaborative study that preceded it, it is recommended that this method be adopted official first action for enumeration of E. coli O157:H7 in meats, poultry, dairy foods, infant formula, liquid eggs, mayonnaise, and apple cider.

Animals↗

Atomic Force microscopy of neuron networks.

We imaged uncoated neuron networks by an atomic force microscope in the repulsive regime of contact mode. Images of granule cells and their axons have been clearly revealed with details smaller than 20 nm. The good stability of the sample and the mechanical reproducibility of the microscope allowed the imaging of a neuron culture area of several square microns. By combining tens of images, we were able to reconstruct a highly defined neuronal network. Furthermore, the images were very reproducible over repeated scanning acquisition, demonstrating the mechanical and thermal stability of the instrument-sample system.

Animal Population Groups↗

Determination of residues of flumequine and 7-hydroxyflumequine in edible sheep tissues by liquid chromatography with fluorimetric and ultraviolet detection.

A simple, sensitive, and rapid method for simultaneous determination of residues of flumequine and its microbiologically active metabolite 7-hydroxyflumequine in 100 mg sheep edible tissues (muscle, liver, kidney, and fat) by liquid chromatography is reported. After liquid-liquid cleanup with ethyl acetate, tissue extracts were injected onto a Select B column. The 2 compounds were determined by ultraviolet and fluorimetric detection. The method was repeatable and reproducible for flumequine and 7-hydroxyflumequine in muscle, liver, kidney, and fat, with limits of detection below 2 and 3 micrograms/kg for flumequine and 7-hydroxyflumequine, respectively. Mean recoveries for flumequine were 90 +/- 7, 82 +/- 7, 89 +/- 5, and 82 +/- 6% in muscle, liver, kidney, and fat respectively. Mean recoveries for 7-hydroxyflumequine were 91 +/- 2, 90 +/- 4, 86 +/- 3, and 84 +/- 4% in muscle, liver, kidney, and fat, respectively.

Adipose Tissue↗

Routine analysis of proteins by Kjeldahl and Dumas methods: review and interlaboratory study using dairy products.

The Kjeldahl and Dumas (combustion) methods were compared in 11 laboratories analyzing samples of milk, skim milk powder, whole milk powder, whey protein concentrate, infant formula, casein, caseinate, 2 reference compounds (glycine and EDTA), and a secondary reference skim milk powder. The comparison was conducted by using international standards where applicable. Overall means were 8.818 g N/100 g by the Kjeldahl method and 8.810 g N/100 g by the Dumas method. No evidence was found for a consistent bias between methods that may be of concern in the trading of dairy produce. A review of more than 10 related trials revealed a lack of consensus in the bias between the 2 methods, suggesting that differences in methodology and sources of systematic error may be contributors. For samples containing > 2 g N/100 g, the Dumas relative repeatability and reproducibility standard deviations were consistently about 0.35 and 0.75%, respectively, whereas the corresponding Kjeldahl values declined generally with N content and were significantly larger. The Dumas precision characteristics may be due to the dominance of Leco analyzers in this trials, and in most other recent trials, rather than an inherent method attribute. Protein determination methods for dairy products need to be reviewed and updated. The Dumas method needs Codex Alimentarius status as a recognized test method.

Animals↗

Fluorometric determination of acid phosphatase in cooked, boneless, nonbreaded broiler breast and thigh meat.

This method is applicable for determining activity of acid phosphatase (ACP), a heat-labile enzyme, in cooked, boneless, nonbreaded broiler marinated (83.65% meat) and nonmarinated (100% meat) breast and thigh and in a 50:50 blend of breast and thigh meat. The assay uses a self-indicating substrate that, when acted upon by ACP, loses a phosphate radical and becomes a highly fluorescent compound. Cooked meat is added to deionized distilled water in a 1:3 ratio, blended with a hand-held homogenizer, and then centrifuged at 2500 relative centrifugal force for 5 min. ACP activity in the filtrate is measured after shaking on a Vortex mixer 75 microL of the extract with a pH 5.00 acetate buffer containing a nonfluorescent aromatic monophosphoric ester substrate. The rate of fluorophore formation is monitored during a 3 min incubation period (38 degrees C) in a fluorometer, and ACP enzyme activity (mU/kg sample) is calculated. Three laboratories analyzed 6 cooked poultry products (marinated and nonmarinated breast, thigh, and 50:50 breast/thigh blend). Five cooking temperatures were used to generate different ACP activity levels, which were replicated twice with duplicate samples and duplicate sample tests representing 720 data points. Log10 ACP activity (mU/kg sample) performance repeatability and reproducibility standard deviations (sr and sR) and relative standard deviations (RSDr and RSDR) over 5 cooking treatments for 6 products were as follows: marinated breast: sr = 0.02, sR = 0.08, RSDr = 0.60%, RSDR = 2.12%; nonmarinated breast: sr = 0.02, sR = 0.04, RSDr = 0.66%, RSDR = 1.29%; marinated thigh: sr = 0.01, = 0.01, RSDr = 0.37%, RSDR = 0.37%; nonmarinated thigh: sr = 0.02, sR = 0.05, RSDr = 0.53%, RSDR = 1.43%; marinated 50:50 breast/thigh blend: sr = 0.01, sR = 0.05, RSDr = 0.36%, RSDR = 1.31%; nonmarinated 50:50 breast/thigh blend: sr = 0.01, sR = 0.04, RSDr = 0.32%, RSDR = 1.12%.

Acid Phosphatase↗

High performance liquid chromatographic method for the determination of lobenzarit disodium in a sustained release tablet formulation.

A rapid and simple high performance liquid chromatographic method is described and validated for the determination of lobenzarit disodium (CAS 64808-48-6) in a sustained release tablet formulation. The calibration graph was linear over the range 20-105 micrograms/ml. The sensitivity (discriminator capacity) was 2.079 micrograms/ml. The coefficient of variations for repeatability and reproducibility were less than 1.60% and 1.30%, respectively. The accuracy of the method did not depend on lobenzarit concentration in tablets. The mean recovery was found to be 100.62%. The method was selective, even when degradation products were present.

Antirheumatic Agents↗

Determination of calcium by inductively coupled plasma-atomic emission spectrometry, and lead by graphite furnace atomic absorption spectrometry, in calcium supplements after microwave dissolution or dry-ash digestion: method trial.

A 3-laboratory method trial was conducted to evaluate 2 sample digestion procedures and instrumental determination parameters for analysis of calcium and lead in Ca supplements. Calcium supplements were treated by dry-ash digestion or microwave dissolution prior to spectrometric analysis. In each case, Pb was determined by graphite furnace atomic absorption spectrometry and Ca by inductively coupled plasma-atomic emission spectrometry. Blind duplicates of 6 Ca supplement samples were analyzed after each sample treatment procedure. Matrix pairs contained dissimilar Pb levels to cover the analyte range encountered during method development. Calcium content of the Ca supplement samples also reflected the range seen during method development. Stock solutions of Ca and Pb were supplied to collaborators for preparation of quantitation standards to remove a variable external to the method. National Institute of Standards and Technology Standard Reference Material (NIST SRM) 1486, bone meal, was included to assess method accuracy and recovery at NIST certificate Ca and Pb levels for this material (26.58 +/- 0.24% Ca and 1.335 +/- 0.014 micrograms Pb/g). Analyses of the NIST SRM yielded 25.9 +/- 1.1 and 27.2 +/- 2.3% Ca and 1.53 +/- 0.19 and 1.26 +/- 0.19 micrograms Pb/g for dry-ash and microwave procedures, respectively. Statistical analyses of data indicated acceptable repeatability and reproducibility for determination of Pb and Ca in various Ca supplements. With either sample preparation technique, the method is appropriate for determining Pb or Ca in Ca supplements.

Biological Products↗

[Reproducibility of heart rate variability measurements in repeated 24-hour long-term ECG registration].

Heart rate variability (HRV) is considered to indicate cardiac autonomic reflex activity. The reproducibility of HRV determinations from repeated Holter recordings has not been vigorously evaluated. This represents, however, an important question since HRV determinations are increasingly used to examine disease-related changes in the autonomic tone or the effects of therapeutic interventions. Thus, the present study examined the reproducibility of 4 commonly used time-domain parameters (SDNN, SDANN, rMSSD, pNN50) and the spectral components of HRV in 17 healthy volunteers and in patients with coronary artery disease (n = 9) or hypertensive heart disease (n = 9). In all subjects, 2 Holter recordings were obtained 7 days apart. In both, healthy volunteers and in patients, overall reproducibility of HRV parameters was good exhibiting correlation coefficients between 0.60 and 0.98. However, in individual subjects particularly in those without heart disease a higher degree of variability of HRV measurements was observed. In those patients with markedly reduced HRV, reproducibility was best. It is concluded that repeated HRV determinations may be utilized to examine the effects of underlying disease progress or of therapeutic interventions on cardiac autonomic tone.

Adult↗

Interlaboratory studies to determine optimal analytical methods for measuring the oxidation index of UHMWPE.

Fourier transform infrared spectroscopy has emerged as the technique of choice for the quantification of oxidation in ultra-high molecular weight polyethylene used in orthopedic implants. We initiated interlaboratory studies to determine the method of normalization, hence quantification, that provided the highest level of reproducibility across multiple institutions. The goal of this research was to identify optimal normalization methods that minimize the experimental uncertainties associated with interlaboratory reproducibility and intralaboratory repeatability of oxidation index measurements. Test samples were prepared from GUR 4150 HP, gamma irradiated in air, and had a shelf age of two years. Samples were analyzed according to ten oxidation index test methods during two interlaboratory studies, which were conducted in accordance with ASTM E691. Variations in reproducibility and repeatability were evaluated using analysis of variance (ANOVA). The basis of the test methods (peak area-based vs. peak height-based), as well as the normalization method, were both found to be associated with significant differences in reproducibility (p = 0.0006 andp < 0.0001, respectively). Normalization techniques based on the 1370 and 2022cm(-1) peaks areas were found to be the most reproducible methods, and were associated with mean interlaboratory uncertainties of 16.5% and 24.2%, respectively. Repeatability of the test methods was not sensitive to the normalization technique; the mean intralaboratory repeatability for all of oxidation index measurements was found to be 10.2%. The results of this interlaboratory research will be a useful basis for the development of a new oxidation index standard for the orthopedics community.

Biocompatible Materials↗

Ion-pair column chromatographic determination of trimethobenzamide hydrochloride in capsule and injection dosage forms: collaborative study.

An ion-pair column chromatographic method was developed for the determination of trimethobenzamide hydrochloride in capsules and injection dosage forms. Detection is by UV spectrophotometry at 261 nm. Recoveries by the Associate Referee ranged from 98.3 to 101.0% for the drug substance. Results by 5 collaborators for capsules averaged 99.1% of labeled or theoretical with coefficients of variation (CVs) of 1.81% (reproducibility) and 1.17% (repeatability); results for injections averaged 100.4% of labeled or theoretical with CVs of 1.91% (reproducibility) and 0.69% (repeatability). The method has been adopted official first action.

Benzamides↗

Report of a collaborative trial to investigate the performance of the R5 enzyme linked immunoassay to determine gliadin in gluten-free food.

OBJECTIVE: Analytical methods for measurements of gluten in the low level range of 20-200 ppm of gluten, as required for gluten-free food, have never been endorsed by the Codex Alimentarius. With the aim of investigating standardized and reliable methods for the detection of gliadin in food with detection limits lower than 200 ppm, as proposed by the Codex Alimentarius, the Working Group on Prolamin Analysis and Toxicity (WGPAT) coordinated a large collaborative study to validate a monoclonal antibody based ELISA, which uses an antibody (R5) against rye secalins. METHODS: Twelve food samples in which gliadin was present in the range of 0-168 ppm were analysed with two different commercially available R5 ELISA tests and a special extraction solvent, based on a reducing and a dissociating agent designed to extract heat denatured proteins. Twenty laboratories participated in this study. RESULTS: Recovery values ranged from 65 to 110% in general. The repeatability (RSDr) and reproducibility (RSDR) figures ranged between 13 and 25, resp. 23 and 47 for one test, and between 11 and 22, resp. 25 and 33 for the other. CONCLUSION: Both assays are comparable and robust. The repeatability and reproducibility data are in a range that is acceptable for ELISAs. Kits from both suppliers fulfilled performance criteria of regular ELISA methods, and it is shown that both ELISA kits guarantee a sensitivity of 1.5 ppm gliadin for gluten-free food.

Antibodies, Monoclonal↗

Extension of dry ash atomic absorption and spectrophotometric methods to determination of minerals and phosphorus in soy-based, whey-based, and enteral formulae (modification of AOAC Official Methods 985.35 and 986.24): collaborative study.

Eight laboratories participated in a collaborative study of AOAC Official Method 985.35, Minerals in Ready-to-Feed Milk-Based infant Formula and Pet Foods, Atomic Absorption Spectrophotometric Method; and 7 laboratories participated in a study of AOAC Official Method 986.24, Phosphorus in Milk-Based infant Formula, Spectrophotometric Method, to extend these methods to infant formulae (other than milk-based) and enteral products. Three ready-to-feed soy-based formulae and 2 soy-based powder formulae were chosen to represent the plant matrix. A whey-based formula and a casein-based enteral formula were also included in the study. Soy formulae containing nearly identical concentrations of particular elements were matched, and an application of the Youden "closely matched pair" approach was used to estimate repeatability parameters. Average reproducibility values were as follows: calcium, 9.3%; copper, 9.7%; Iron, 5.5%; potassium, 4.0%; magnesium, 5.2%; manganese, 10.6%; sodium, 4.7%; phosphorus, 10.5%; and zinc, 7.3%. At similar analyte concentrations, the between-laboratory variabilities compared well with those reported for the official methods. Most repeatability and reproducibility parameters compared well with the original collaborative study. AOAC Official Methods 985.35 and 966.24 have been modified to extend their applicability to infant formulae (other than milk-based) and enteral products.

Animal Feed↗

Determination of the total nitrogen content of hard, semihard, and processed cheese by the Kjeldahl method: collaborative study.

The objective of this collaborative study was to determine interlaboratory performance statistics for a modified and optimized version of AOAC Method 920.123 for the determination of the total nitrogen content of hard, semihard, and processed cheese by Kjeldahl analysis. Details included addressing the issues of material homogeneity, test portion size (1 g), quantitative transfer (weighing on to filter paper), ensuring system suitability (nitrogen recoveries), and using AOAC Method 991.20 as the basis for nitrogen analysis. Fifteen laboratories tested 18 pairs of blind duplicate cheese materials with a crude protein content between 18 and 36%. Materials represented hard, semihard, and processed commercial cheeses with a wide range of composition. Statistical performance parameters expressed as crude protein (nitrogen x 6.38), g/100 g, with invalid and outlier data removed were mean = 26.461, repeatability standard deviation (Sr) 0.111, reproducibility standard deviation (S(R)) = 0.153, repeatability relative standard deviation (RSDr) = 0.42%, reproducibility relative standard deviation (RSDR) = 0.58%, repeatability (r) = 0.312, and reproducibility (R) = 0.428. The interlaboratory study results were acceptable and comparable to those for the milk Kjeldahl nitrogen method on a relative nitrogen basis. The Study Directors recommend that this modified method for the determination of total nitrogen in hard, semihard, and processed cheese by Kjeldahl analysis be adopted First Action as an improved method to replace Method 920.123.

Cheese↗

In vivo epidermal thickness measurement: ultrasound vs. confocal imaging.

BACKGROUND/PURPOSE: In this study, in vivo skin imaging methods, ultrasound (US) and confocal microscopy (CM) were compared with regards to their accuracy in measuring the epidermal thickness. In addition an attempt was made to clarify the biological significance of the second echo-rich line observed on US skin images, i.e. whether it represents the dermal-epidermal junction or the papillar-reticular dermis limit. METHODS: US images were obtained with an in-house device (22 MHz probe) and the CM images with the VivaScope 1000 (Lucid Inc., Rochester, NY, USA). Skin from the dorsal forearm, the back of hand and the palm skin of 11 subjects (25-40 years) were examined. Repeatability of the procedure and reproducibility of the results were evaluated on repeated measurements taken at 1-month interval. RESULTS: Both techniques are correlated. When a CM measurement is performed from the stratum corneum (SC) surface to the bottom of the papillae, results obtained with US and CM are very similar. Thus, the second echo-rich line on US skin imaging is likely to reflect a virtual line joining the bottom of the papillae. CM is limited to the measurement of a relative thin epidermis, due to the signal-to-noise ratio, which decreases with depth. US technique offers a better repeatability and reproducibility, particularly for SC measurement. This is mainly due to the small size of the investigated field of view in CM. CONCLUSIONS: This study confirms the accuracy of US and the feasibility of CM imaging techniques for in vivo epidermal thickness measurement. Echography probably measures a maximal epidermal thickness since it encompasses the bottom of the papillae.

Adult↗