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Mechanical precision in two-dimensional electrophoresis can improve protein spot positional reproducibility.

Current methods for high resolution two-dimensional electrophoresis (2DE) of proteins are capable of separating over 5000 protein spots in one procedure. Running and analysing such 2DE gels requires skilled technical work. However, the variable reproducibility of spot positions means that, even under the best circumstances, one gel cannot be overlain directly on another for precise comparison. Therefore, new and improved technologies that enhance gel-to-gel reproducibility are required. To this end, we have designed and built a research instrument to test whether a precise mechanical device could improve the gel-to-gel reproducibility by reducing the amount of distortion and positional variation between the first and second dimension gels. Other causes of poor reproducibility, including sample type and preparation, gel matrices and running conditions were not varied in order to limit this study to the mechanical variations inherent in current 2DE systems. We found that the sample standard deviation of pooled data for measured protein spot-to-spot distances in the prototype device was 1.3 mm as compared to 4.3 mm in a conventional 2DE system. These improvements support the possibility that greater automation of the multistep 2DE process will enhance reproducibility. This approach seems justified in order to achieve significantly better matching between gels and between results from different laboratories.

Cerebrospinal Fluid Proteins↗

The reproducibility of the 50-g, 1-hour glucose screen for diabetes in pregnancy.

OBJECTIVE: To explore the day-to-day reproducibility of the 50-g, 1-hour glucose screening test performed on 2 consecutive days in the same women. METHODS: Eight women at 12-23.6 weeks' gestation (early subjects) and 80 women at 24-28 weeks (late subjects) without known diabetes mellitus were studied. The glucose screening test was performed in the morning on 2 consecutive days in the same women, under an identical or opposite sequence of fasting and fed conditions. The women were thus divided into four subgroups: fast-fast, fed-fed, fast-fed, and fed-fast. Duplicate serum glucose concentrations were measured by the glucose oxidase method. Paired Student t test was used to analyze day-1 versus day-2 glucose levels in each woman of each subgroup. RESULTS: The serum glucose concentrations were higher on day 1 than on day 2 in the subgroups fast-fast and fast-fed (P < .05) in both early and late patients, whereas the opposite was seen in the subgroups fed-fast (P < .05). No significant differences were observed in the subgroups of fed-fed. At three different glucose thresholds (130, 135, and 140 mg/dL), there was more than 90% daily reproducibility for normal results in both groups, and nearly 50 and 83% daily reproducibility for abnormal results in the early and late patients, respectively. CONCLUSIONS: Up to 28 weeks of pregnancy, the screening test had a high reproducibility for normal results. For abnormal results, daily reproducibility was better after than before 24 weeks' gestation, regardless of prior testing conditions. Depending on the pre-testing conditions, the use of a different serum glucose threshold seems warranted.

Adult↗

Reproducibility of regional brain metabolic responses to lorazepam.

UNLABELLED: Changes in regional brain glucose metabolism in response to benzodiazepine agonists have been used as indicators of benzodiazepine-GABA receptor function. The purpose of this study was to assess the reproducibility of these responses. METHODS: Sixteen healthy right-handed men underwent scanning with PET and [18F]fluorodeoxyglucose (FDG) twice: before placebo and before lorazepam (30 micrograms/kg). The same double FDG procedure was repeated 6-8 wk later on the men to assess test-retest reproducibility. RESULTS: The regional absolute brain metabolic values obtained during the second evaluation were significantly lower than those obtained from the first evaluation regardless of condition (p < or = 0.001). Lorazepam significantly and consistently decreased both whole-brain metabolism and the magnitude. The regional pattern of the changes were comparable for both studies (12.3% +/- 6.9% and 13.7% +/- 7.4%). Lorazepam effects were the largest in the thalamus (22.2% +/- 8.6% and 22.4% +/- 6.9%) and occipital cortex (19% +/- 8.9% and 21.8% +/- 8.9%). Relative metabolic measures were highly reproducible both for pharmacologic and replication condition. CONCLUSION: This study measured the test-retest reproducibility in regional brain metabolic responses, and although the global and regional metabolic values were significantly lower for the repeated evaluation, the response to lorazepam was highly reproducible.

Adult↗

Radiographic assessment of knee osteoarthritis: reproducibility and sensitivity to change.

OBJECTIVE: To determine the cross sectional and longitudinal reproducibility of various measures used for assessing radiographic knee osteoarthritis (OA) and to compare the sensitivity to change over a one year period of these measures. METHODS: We studied 55 patients referred to hospital with knee OA (clinical and radiographic ACR criteria). Anteroposterior radiographs at baseline and after 12 months were read by methods both qualitative (Kellgren and Lawrence grading scale, joint space narrowing scale) and quantitative (joint space width measurement at 3 different points). All qualitative methods used standardized atlases. The intraclass correlation coefficient and the graphical method of Bland and Altman were used to assess cross sectional and longitudinal reproducibility. Reproducibility was tested using 2 readers (interreader) and 2 readings for one of the readers (intrareader). Sensitivity to change was assessed using standardized response mean (SRM). RESULTS: All methods tested were shown to be reproducible both for cross sectional and longitudinal data. Intrareader was higher than interreader reproducibility for most radiographic features. Significant changes were observed after one year for methods measuring joint space narrowing. The sensitivity to change of the methods assessing joint space narrowing (joint space narrowing scale and joint space width measurement) was higher, with SRM varying from 0.37 to 0.57, than for the Kellgren and Lawrence grading system (SRM 0.19 and 0.23). CONCLUSION: These data suggest that methods measuring narrowing should be preferred as outcome measures in clinical trials or longitudinal epidemiologic studies; and show that in a particular subset of patients with very active disease, significant radiographic changes in knee OA can be detected after a one year period.

Aged↗

Reproducibility of proximal probe pH parameters in 24-hour ambulatory esophageal pH monitoring.

OBJECTIVES: To assess the reproducibility and reliability of the proximal pH probe in detecting acid reflux into the proximal esophagus. METHODS: Using dual probe ambulatory esophageal pH monitoring, we studied 32 subjects (11 healthy control subjects, 10 patients with distal esophageal acid reflux, and 11 patients with both distal and proximal esophageal acid exposure) on two separate days within a 20-day period. The distal pH probe was placed 5 cm above the manometrically determined lower esophageal sphincter, and the proximal probe was positioned immediately distal to the upper esophageal sphincter. Patients were categorized on the basis of the esophageal pH data obtained during the first study. Reflux parameters assessed were the percentages of time in which pH was <4 in the total, upright, and supine positions. To be considered reproducible, all three of the above parameters had to remain in the same category as the first day's results. RESULTS: Intrasubject reproducibility of the proximal probe was 91-100% in healthy subjects, 70-90% in patients with distal esophageal acid reflux, and 45-73% in patients with proximal esophageal acid reflux. The proximal probe reproducibility for the overall diagnosis of gastroesophageal reflux disease was 91% in healthy subjects, 70% in patients with distal esophageal acid reflux, and only 55% in those with proximal esophageal acid reflux. Statistical analysis demonstrated only a fair index of concordance (kappa = 0.40) for the proximal probe. CONCLUSIONS: The proximal pH probe has excellent specificity (91%) but poorer sensitivity and reproducibility (55%) for identifying abnormal amounts of proximal esophageal acid reflux. Therefore, a negative test result does not exclude proximal reflux with microaspiration as a cause of atypical reflux symptoms.

Adult↗

[Reproducibility of malignancy grading in prostatic cancers using the Gleason-Böckling system].

Histologic slides from 50 cases of prostatic adenocarcinoma were evaluated by 5 pathologists, in order to test the reproducibility of grading in two systems. Twenty-five needle core biopsies and 25 surgical (adenomectomy) specimens were graded in two sessions, according to the histomorphologic criteria of Gleason and Böcking. The results were analyzed by the kappa statistics. In surgical specimens, there were no significant differences in the interobserver reproducibility of microscopically assessed categories. In needle biopsies, however, Gleason's primary pattern (62%, kappa = 0.42), and Böcking's histological pattern (63%, kappa = 0.37) showed the highest level of agreement. Among the computed (derivated) classification terms, those consisting of only 3 groups (Gleason grouping, kappa = 0.39; Böcking grade, kappa = 0.39) proved to be better reproducible than the corresponding score values (p < 0.05). When compressing both systems into two grades (high and "non-high"), reproducibility was improved (kappa = 0.52). For a substantial improvement of grading results, more accurate grade definitions, continuing training and regular consultation of pathologists are necessary. Based on the results obtained by intraobserver analysis we conclude that kappa statistics is of limited value when analyzing the role of individual experience at grading reproducibility.

Adenocarcinoma↗

[Reproducibility of measurements of blood gas exchange during exercise in mild cardiac failure: need for a preliminary test?].

The reproducibility of blood gas exchange measurements on exercise in chronic cardiac failure has already been established in patients familiar with this technique. The aim of this prospective study was to evaluate the reproducibility of cardiopulmonary parameters on exercise in a population of patients who had never undergone this type of investigation. Twenty patients with chronic cardiac failure in classes I to III of the NYHA classification, with a mean age of 55 +/- 11.5 years and a mean LV ejection fraction of 31.2 +/- 9%, underwent two cardiopulmonary exercise tests (CPX Medgraphic) performed on a bicycle ergometer. Patients underwent maximal exercise stress testing attaining 89% of the theoretical maximal heart rate and 1.14 of the respiratory quotient during the first test. There was no significant change in peak VO2 (22.5 ml/min/kg vs 22.6 ml/min/kg) or in ventilatory anaerobic threshold (12.8 ml/min/kg vs 12.7 ml/min/kg) between the two tests. The ventilatory anaerobic threshold could not be measured in one patient and seemed less reproducible than peak VO2 with a standard deviation of relative differences (T2-T1/T1) of 10.4 versus 7.8%. There was a significant increase in the duration of exercise (7.4 +/- 9.2%; p < 0.002) and a ventilatory flow (4.5 +/-, p = 0.03). This study shows that peak VO2 is a reproducible measurement in mild to moderate chronic cardiac failure, even in the absence of a preliminary test to familiarize the patient with the equipment. The reproducibility of the ventricular anaerobic threshold is less satisfactory than that of peak VO2. The increase in the duration of exercise is more dependent on motivation and should not be taken into account alone in the functional evaluation of chronic cardiac failure.

Adult↗

Test/retest reproducibility of iodine-123-betaCIT SPECT brain measurement of dopamine transporters in Parkinson's patients.

UNLABELLED: Iodine-123-beta-CIT has been used as a probe of dopamine transporters in Parkinson's disease patients using SPECT. We studied the test/retest reproducibility of SPECT measures in Parkinson's disease patients and healthy controls obtained after injection of [123I])beta-CIT in part to assess the utility of this tracer for longitudinal evaluation of striatal dopamine transporters as a marker of disease progression. METHODS: Seven Parkinson's disease patients and seven healthy control subjects participated in two [123I]beta-CIT SPECT scans separated by 7-21 days. Subjects were imaged at 24 hr post injection of 360 MBq (9.7 mCi) of [123I]beta-CIT. Two outcome measures were evaluated; 1) the ratio of specific striatal (activity associated with DA transporter binding) to nondisplaceable uptake, also designated V3," and 2) the total specific striatal uptake (%SSU) expressed as a percentage of injected radiotracer dose. For both measures, test/retest variability was calculated as the absolute difference of test minus retest divided by the mean of test/retest and expressed as a percent. In addition, the reproducibility of left and right striatal asymmetry and putamen:caudate ratios were determined. RESULTS: The two outcome measures demonstrated excellent test/retest reproducibility for both the Parkinson's disease and healthy subject groups with variability of striatal V3" = 16.8 +/- 13.3% and percent striatal uptake = 6.8 +/- 3.4% for Parkinson's disease patients and V3" = 12.8 +/- 8.9% and %SSU = 7.0 +/- 3.9% for control subjects. There were no statistically significant differences in test/retest variability between control subjects and Parkinson's disease patients for either outcome measure. The reproducibility of left/right asymmetry indices and putamen-to-caudate ratios showed no patient versus control subject differences. The asymmetry index had greater test/retest variability than the other outcome measures. CONCLUSION: These data suggest that SPECT imaging performed at 24 hr postinjection of [123I]beta-CIT permits calculation of reliable and reproducible measures of dopamine transporters in both Parkinson's disease patients and control subjects and supports the feasibility of using [123I]beta-CIT in the evaluation of disease progression in Parkinson's disease.

Adult↗

Reproducibility of color Doppler imaging in orbital vessels.

The purpose of this study was to evaluate the short-term reproducibility, the long-term reproducibility, and the reproducibility of the measurements made between two different examiners of blood-flow velocity in extraocular vessels using color Doppler imaging (CDI). In a group of 10 healthy volunteers, measurements of the peak-systolic velocity (PSV), end-diastolic velocity (EDV), and resistivity index (RI) in the ophthalmic artery and the central retinal artery as well as one lateral and one medial short posterior ciliary artery were performed. The best intraindividual reproducibility, expressed as the relative error, was found for the PSV and RI measured in the ophthalmic artery (OA; PSV 5.9%, RI 3.1%) and the central retinal artery (CRA; PSV 7.7%, RI 4.7%). The PSV and RI determined in the posterior ciliary arteries were less reproducible but as much so as the EDV measured in the OA (11.8%) and CRA (19.9%). No systematic trend could be found between the first and second measurements. However, the long-term fluctuation was considerable high. Measurements of PSVs showed good concurrence between the two observers in all four vessels measured. The EDVs and RIs differed statistically significantly between the two observers. This difference was most pronounced in the posterior ciliary arteries.

Adult↗

Peripapillary nerve fiber layer thickness measurement reproducibility using scanning laser polarimetry.

PURPOSE: The authors assess the reproducibility of retinal nerve fiber layer (RNFL) thickness measurements using scanning laser polarimetry. METHODS: The right eyes of five healthy individuals were scanned by two trained operators (S.T.H., H.I.) using the Nerve Fiber Analyzer II (Laser Diagnostic Technologies, Inc., San Diego, CA, U.S.A.). Each participant was scanned six consecutive times per session for five sessions carried out over separate days within a two-week period. Intra- and interoperator measurement reproducibility was assessed. RESULTS: Intraoperator reproducibility was high. Mean coefficients of variation for total RNFL thickness measurements were 4.48 +/- 1.76% and 4.92 +/- 2.32% for operators 1 and 2, respectively. Interoperator reproducibility was high (p = 0.20-0.93) if a single ellipse was applied to the images of both examiners. However, measurement reproducibility worsened if each operator created his own measurement ellipse (p < or = 0.05 for 3 out of 5 participants). CONCLUSION: Interoperator error in image analysis can be minimized by using a single ellipse for the baseline and all subsequent scans even if subsequent scans are acquired by different operators.

Adult↗

Reproducibility of lung volume measurements.

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

Adult↗

Reproducibility of technetium-99m-MAG3 clearance using the Bubeck method.

UNLABELLED: The objective of this study was to estimate the reproducibility of 99mTc-mercaptoacetyltriglycine (99mTc-MAG3) clearance calculated using a single-sample method. METHODS: One hundred forty-seven patients with urological or ear, nose and throat cancer were analyzed in a retrospective study. Each patient had at least two clearance studies with 99mTc-MAG3 before chemotherapy treatments to monitor renal function. Up to five clearance studies per patient were considered. The reproducibility was estimated by comparing two consecutive investigations. Pairs of investigations with a change in split renal function of more than 5% or an interval of more than 50 days were excluded. Clearance was determined using the Bubeck method. For each pair of consecutive clearance data, the difference between the first and the second measurements was expressed as a percentage of the mean value of the two measurements. The mean of these normalized differences represents the systematic deviation, and the s.d. represents the reproducibility of the compared clearances. RESULTS: After the selection, 242 pairs of consecutive clearance data remained for comparison. Significantly different clearances were observed only between investigations 0 and 1 and between 4 and 5. The systematic deviation of these comparisons totaled -3.8% and -5.7%, respectively. In the other comparisons, no significant deviation induced by the chemotherapy was found. The reproducibility calculated for all comparisons totaled 11.7%. CONCLUSION: The error of reproducibility of 99mTc-MAG3 clearance using the Bubeck method was < or =11.7%. This was an acceptable value, taking into account the greater fluctuation of tubular function compared with the glomerular filtration rate.

Humans↗

Quantitative discomanometry: technique and reproducibility in vitro.

Quantitative discomanometry is a study of intradiscal pressure and volume measurements during the injection of fluid into an endplate-disc-endplate complex. The purpose of this article was to describe the technique of quantitative discomanometry, determine the reproducibility of the injection technique in a cadaveric thoracolumbar spine, and standardize the technique for future clinical investigations. Nineteen fresh human cadaveric thoracolumbar discs were injected using quantitative discomanometry to determine: (a) the time necessary for a disc to return to a baseline pressure-volume curve, (b) the reproducibility of the technique in vitro, (c) effects of the injection approach and position of the needle in the disc, and (d) effects of the type and length of tubing as well as gauge of spinal needle. A pressure-volume curve was obtained for each disc injection. Reproducibility was measured by nine parameters obtained from each pressure-volume curve: intrinsic pressure, leakage pressure, initial slope, slope between 0-0.1 ml, slope between 1-4 ml, pressure at 2 ml, pressure at 4 ml, maximum pressure, and volume at maximum pressure. The results demonstrated that (a) the injector apparatus was reproducible, (b) the time necessary for a disc to return to a baseline pressure-volume curve was 24 h, (c) the technique using fresh human cadaveric thoracolumbar discs was reproducible, (d) the anterior and posterolateral approaches had similar results if the needle was placed into the center of the nucleus pulposus using radiographic control, and (e) the type and length of tubing, and gauge of needle did not affect the results.

Aged↗

Accuracy and reproducibility of instrumented knee-drawer tests.

Instrumented devices for knee-drawer tests have become popular in orthopaedics relatively recently. The objective of the present study was to document the effects of several parameters on the accuracy and reproducibility of anterior-posterior (AP) drawer measurements. An instrumented knee-drawer tester for AP laxity evaluations was constructed, based on the differential displacement method, measuring shifts of the tuberosity relative to the patella. The accuracy of the AP-shift was determined with the parallel use of a highly accurate roentgen stereo photogrammetric (RSP) measurement system on two postmortem leg specimens. The effects of relative motion between patella and femur were negligible. In addition to AP shifts, significant knee flexion and tibial rotations occurred, although the foot and the thigh were fixed as well as possible. The differential displacement method was effective in circumventing this problem. The accuracy of the AP shift was greater than 10%. The reproducibility of the AP drawer parameters (shifts and compliances) was determined in normal subjects and patients. Tests were made to evaluate the effects of different observers, time sequences, and different days. In addition, effects of muscle relaxation were studied. Overall, the shift parameters at different forces were found to be reproducible to between 5 and 15%. The slopes (compliances) of the laxity curves, at different forces, were found to be reproducible between 20 and 40%. The reproducibility was principally affected by deviations in the subject positioning procedure.

Biomechanical Phenomena↗

Reproducibility of relaxation and spin-density parameters in phantoms and the human brain measured by MR imaging at 1.5 T.

The reproducibility of T1, T2, and proton density, measured in phantoms and the human brain was evaluated by proton imaging techniques. The sequence used to derive T1 and density values was a multiple-saturation recovery which consists of four pairs of 90 degrees pulses, followed by a 180 degrees phase reversal pulse, generating four T1-weighted images. T2 was derived from a multiple-echo sequence, generating four T2-weighted images. The data were analyzed by fitting the pixel intensities to the respective equations by means of nonlinear multiparameter least-squares analysis. Short-term reproducibility between four consecutive scans was evaluated to be 1-4% depending on location with a phantom covering the entire span of physiologic T1 and T2 values. A second phantom containing a series of identical samples served to study the dependence of the apparent T1 and T2 on position, both radially and axially, with respect to magnet isocenter. Reproducibility across the field of view was found to be better than 7% (T1 and T2). This phantom was further used to evaluate effects of long-term reproducibility, which at each location varied from 5-14% (T1) and 2-10% (T2). Finally, interinstrument reproducibility, tested by means of the same protocol on three different instruments, all operating at the same magnetic field and using largely identical hardware for each location, was found to be 1-14% (T1) and 2-10% (T2). The positional dependence of the apparent relaxation times appears to be systematic and may be due to variations in the effective field, caused by magnet and rf inhomogeneity. Finally, brain tissue relaxation and spin-density data were determined using the same protocol in 37 scans performed on 27 normal volunteers. The tissues analyzed were putamen, thalamus, caudate nucleus, centrum semiovale, internal capsule, and corpus callosum. Excellent accordance was further obtained between left and right hemispheres.

Brain↗

Ambulatory 24-hour esophageal pH monitoring. Reproducibility and variability of pH parameters.

If 24-hour esophageal pH monitoring is to be a useful diagnostic tool, it must reliably discriminate gastroesophageal reflux patients despite daily variations in distal esophageal acid exposure. To address this issue, we studied 53 subjects (14 healthy normals, 14 esophagitis patients, and 25 patients with atypical symptoms) with two ambulatory pH tests performed within 10 days of each other. Intrasubject reproducibility of 12 pH parameters to discriminate the presence of abnormal acid reflux was determined. As a group, the parameters of percent time with pH less than 4 (total, upright, recumbent) were most reproducible (80%). Therefore, a subject was defined as having gastroesophageal reflux disease if at least one of these three values were abnormal. Intrasubject reproducibility for the diagnosis of reflux disease was 89% for the entire sample. Among subsets, the reproducibility was 93% for the normals and esophagitis patients and 84% for the atypical symptom patients. Total percent time with pH less than 4 was the single most discriminate pH parameter (85%) and nearly equaled that of the three combined parameters (89%). The intrasubject variability of this parameter was determined by the mean +/- 2 SD of the relative differences between the two test results for all 53 subjects. Total percent time with pH less than 4 may vary between tests by a factor of 3.2-fold or less (218% higher to 69% lower). We conclude: (1) ambulatory 24-hr esophageal monitoring is a reproducible test for the diagnosis of gastroesophageal reflux disease; and (2) the large intrastudy variability in 24-hr total acid exposure may limit this test's usefulness as a measurement of therapeutic improvement.

Adult↗

Reproducibility of incremental maximal cycle ergometer tests in patients with mild to moderate obstructive lung diseases.

This study was designed to examine the reproducibility and the learning effects of incremental maximal cycle ergometer testing in patients with obstructive lung diseases who are untrained and have never done such a test before. Eleven patients were studied on 2 consecutive days with an interval of 24 h. The reproducibility (expressed in relative duplicate error) of most of the parameters tested was good: workload (watt) 4.5%, maximal oxygen consumption (VO2max 3.5%, gas exchange ratio (R) 3.4%, and heart rate 3.7%. Moderate reproducibility was found in maximal ventilation (VEmax) 6.6%, carbon dioxide production (VCO2max 6.0%, and respiration frequency (RF) 8.3%. The reproducibility of the time between the start of the test and the minimum value of VE/VO2 and VE/VCO2 was low. We concluded that in these patients the results of incremental cycle ergometer testing showed good reproducibility in a test-retest procedure. There were no learning effects.

Adult↗

Reproducibility of exercise-induced ventricular arrhythmia after myocardial infarction.

To evaluate the reproducibility of exercise-induced ventricular arrhythmia, 155 men with a mean age of 53 +/- 8 years underwent serial exercise testing 3 to 52 weeks after myocardial infarction. The reproducibility of categorical test responses, that is, the presence or absence of ventricular arrhythmia, was evaluated with the kappa coefficient, which considers negative as well as possible test responses and expresses reproducibility above the chance level. Reproducibility was highest at an intertest interval of 1 to 5 days and was not enhanced by further categorizing premature ventricular complexes as simple or complex based on their frequency or configuration. Continuous response measures such as frequency of premature ventricular complexes yielded higher reproducibility than categorical responses. Continuous response measures appear preferable to categorical responses for evaluating the clinical significance and response to antiarrhythmic therapy of ventricular arrhythmias.

Arrhythmias, Cardiac↗