Simultaneous assessment of loss of heterozygosity at multiple microsatellite loci using semi-automated fluorescence-based detection: subregional mapping of chromosome 4 in cervical carcinoma.

Detection of loss of heterozygosity (LOH) by comparison of normal and tumor genotypes using PCR-based microsatellite loci provides considerable advantages over traditional Southern blotting-based approaches. However, current methodologies are limited by several factors, including the numbers of loci that can be evaluated for LOH in a single experiment, the discrimination of true alleles versus "stutter bands," and the use of radionucleotides in detecting PCR products. Here we describe methods for high throughput simultaneous assessment of LOH at multiple loci in human tumors; these methods rely on the detection of amplified microsatellite loci by fluorescence-based DNA sequencing technology. Data generated by this approach are processed by several computer software programs that enable the automated linear quantitation and calculation of allelic ratios, allowing rapid ascertainment of LOH. As a test of this approach, genotypes at a series of loci on chromosome 4 were determined for 58 carcinomas of the uterine cervix. The results underscore the efficacy, sensitivity, and remarkable reproducibility of this approach to LOH detection and provide subchromosomal localization of two regions of chromosome 4 commonly altered in cervical tumors.

A rapid microtitre plate screening method for in vitro assessment of fibrinolysis: a preliminary report

A novel and precise assay that facilitates high-throughput screening of fibrinolytic agents was developed based on the automated assessment of the euglobulin clot lysis time in microtitre plates. Euglobulin fractions from fresh plasma samples were assessed over 28 days to determine the inter-assay and intra-assay precision. The intra-assay (coefficient of variation range, 0.7-2.6%) and inter-assay precision (coefficient of variation range, 6.8-12.1%) was found to be well within limits required by the Food and Drug Administration. On day 1 and day 28, the results of the microtitre plate euglobulin clot lysis time method were compared with tissue plasminogen activator activity, plasminogen activator inhibitor activity and results produced on fibrin plates. All comparisons were found to correlate significantly. The validity of this method for assaying fibrinolytic agents was assessed by comparing dose-response curves for streptokinase produced using fibrin plates and this method. The critical influence of ambient temperature on the inter-assay reproducibility of this method was established by testing samples over a range of temperatures between 20degreesC and 40degreesC. (C) 2004 Lippincott Williams Wilkins.

Reliability of a measure of prediagnosis physical activity for cancer survivors

Purpose: This study was conducted to examine the test-retest reliability of a measure of prediagnosis physical activity participation administered to colorecial cancer survivors recruited from a population-based state cancer registry. Methods: A total of 112 participants completed two telephone interviews. I month apart, reporting usual weekly physical activity in the year before their cancer diagnosis. Intraclass correlation coefficients (ICC) and standard en-or of measurement (SEM) were used to describe the test-retest reliability of the measure across the sample: the Bland-Altman approach was used to describe reliability at the individual level. The test-retest reliability for categorized total physical activity (active, insufficiently active, sedentary) was assessed using the kappa statistic. Results: When the complete sample was considered, the ICC ranged from 0.40 (95% Cl: 0.24, 0.55) for vigorous gardening to 0.77 (95% Cl: 0.68, 0.84) for moderate physical activity. The SEM, however, were large. indicating high measurement error. The Bland-Altman plots indicated that the reproducibility of data decreases as the aniount of physical activity reported each week increases The kappa coefficient for the categorized data was 0.62 (95% Cl: 0.48, 0.76). Conclusion: Overall. the results indicated low levels of repeatability for this measure of historical physical activity. Categorizing participants as active, insufficiently active, or sedentary provides a higher level of test-retest reliability.

Chromogenic in situ hybridisation testing for HER2 gene amplification in breast cancer produces highly reproducible results concordant with fluorescence in situ hybridisation and immunohistochemistry

Aims: The objective of this study was to evaluate the accuracy, ease of use and reproducibility of chromogenic in situ hybridisation (CISH) for HER2 testing by studying its inter-laboratory concordance in five Australian pathology laboratories. Methods: The HER2 status of 49 breast cancers was determined by CISH twice in two different laboratories. Each sample had previously been tested by immunohistochemistry (IHC; 2+ and 3+ cases selected) and fluorescence in situ hybridisation ( FISH). Participating laboratories were blinded to these test results. Oestrogen receptor ( ER) status was also evaluated for each cancer. Results: High correlation was observed between FISH and CISH results. No cases showing high gene amplification by FISH were scored as non-amplified by CISH ( kappa coefficient=1). High correlation was observed between IHC and CISH, all IHC 3+ samples showing amplification by CISH. Inter-laboratory CISH concordance was also good ( kappa coefficient=0.67). Fifty-six per cent of HER2-amplified samples tested ER positive, while 42% of ER-positive cases showed HER2 gene amplification, confirming that HER2 testing should not be confined to ER-negative breast cancers. Conclusions: These findings demonstrate that CISH is a robust test to assess HER2 status in breast cancer and therefore is an important addition to the HER2 testing algorithm.

Clinical evaluation of the MPS 9000 Macular Pigment Screener

Background/aims The MPS 9000 uses a psychophysical technique known as heterochromatic flicker photometry to measure macular pigment optical density (MPOD). Our aim was to determine the measurement variability (noise) of the MPS 9000. Methods Forty normally sighted participants who ranged in age from 18 to 50 years (25.4±8.2 years) were recruited from staff and students of Aston University (Birmingham, UK). Data were collected by two operators in two sessions separated by 1 week in order to assess test repeatability and reproducibility. Results The overall mean MPOD for the cohort was 0.35±0.14. There was no significant negative correlation between MPS 9000 MPOD readings and age (r=-0.192, p=0.236). Coefficients were 0.33 and 0.28 for repeatability, and 0.25 and 0.26 for reproducibility. There was no significant correlation between mean and difference MPOD values for any of the four pairs of results. Conclusions When MPOD is being monitored over time then any change less than 0.33 units should not be considered clinically significant as it is very likely to be due to measurement noise. The size of the coefficient appears to be positively correlated with MPOD.

Clinical evaluation of the Macuscope macular pigment densitometer

Background/aims. The MacuScope uses a psychophysical technique called heterochromic flicker photometry to measure macular pigment optical density (MPOD). Our aim was to determine the measurement variability (noise) of the MacuScope. Methods. Thirty-eight normally sighted participants who ranged in age from 19 to 46 years (25.7±7.6 years) were recruited from staff and students of Aston University. Data were collected by two operators, HB and JA, in two sessions separated by 1 week in order to assess test repeatability and reproducibility. Results. The overall mean MPOD for the cohort was 0.47±0.14. There was a significant negative correlation between MacuScope MPOD readings and age (r=-0.368, p=0.023). Coefficients were 0.45 and 0.58 for repeatability, and 0.49 and 0.36 for reproducibility. For each pair of results, there was a significant positive correlation between mean and difference MPOD values. Conclusions. If MPOD is being monitored over time then any change less than 0.58 units should not be considered clinically significant as it is very likely to be due to instrument noise. The size of the coefficient appears to be positively correlated with MPOD.

The development of a testing procedure for dry friction brakes

A description of the background to testing friction materials for automotive brakes explains the need for a rapid, inexpensive means of assessing their behaviour in a way which is both accurate and meaningful. Various methods of controlling inertia dynamometers to simulate road vehicles are rejected in favour of programming by means of a commercially available XY plotter. Investigation of brake service conditions is used to set up test schedules, and a dynamometer programming unit built to enable service conditions on vehicles to be simulated on a full scale dynamometer. A technique is developed by which accelerated testing can be achieved without operating under overload conditions, saving time and cost without sacrificing validity. The development of programming by XY plotter is described, with a method of operating one XY plotter to programme the machine, monitor its own behaviour, and plot its own results in logical sequence. Commissioning trials are described and the generation of reproducible results in frictional behaviour and material durability is discussed. Teclmiques are developed to cross check the operation of the machine in retrospect, and retrospectively correct results in the event of malfunctions. Sensitivity errors in the measuring circuits are displayed between calibrations, whilst leaving the recorded results almost unaffected by error. Typical results of brake lining tests are used to demonstrate the range of performance parameters which can be studied by use of the machine. Successful test investigations completed on the machine are reported, including comments on behaviour of cast iron drums and discs. The machine shows that materials can repeat their complex friction/ temperature/speed/pressure relationships at a reproducibility of the order of +-0.003u and +~ 0.0002 in. thickness loss during wear tests. Discussion of practical and academic implications completes the report with recommendations for further work in both fields.

Validity and repeatability of the Aladdin ocular biometer

Aim To assess the accuracy and reproducibility of biometry undertaken with the Aladdin (Topcon, Tokyo, Japan) in comparison with the current gold standard device, the IOLMaster 500 (Zeiss, Jena, Germany). Setting University Eye Clinic, Birmingham, UK and Refractive Surgery Centre, Kiel, Germany. Methods The right eye of 75 patients with cataracts and 22 healthy participants were assessed using the two devices. Measurements of axial length (AL), anterior chamber depth (ACD) and keratometry (K) were undertaken with the Aladdin and IOLMaster 500 in random order by an experienced practitioner. A second practitioner then obtained measurements for each participant using the Aladdin biometer in order to assess interobserver variability. Results No statistically significant differences ( p≥0.05) between the two biometers were found for average difference (AL)±95% CI=0.01±0.06 mm), ACD (0.00 ±0.11 mm) or mean K values (0.08±0.51 D). Furthermore, interobserver variability was very good for each parameter (weighted κ≥0.85). One patient's IOL powers could not be calculated with either biometer measurements, whereas a further three could not be analysed by the IOLMaster 500. The IOL power calculated from the valid measurements was not statistically significantly different between the biometers (p=0.842), with 91% of predictions within±0.25 D. Conclusions The Aladdin is a quick, easy-to-use biometer that produces valid and reproducible results that are comparable with those obtained with the IOLMaster 500.

Comparison of subjective and objective methods to determine the retinal arterio-venous ratio using fundus photography

Purpose: To assess the inter and intra observer variability of subjective grading of the retinal arterio-venous ratio (AVR) using a visual grading and to compare the subjectively derived grades to an objective method using a semi-automated computer program. Methods: Following intraocular pressure and blood pressure measurements all subjects underwent dilated fundus photography. 86 monochromatic retinal images with the optic nerve head centred (52 healthy volunteers) were obtained using a Zeiss FF450+ fundus camera. Arterio-venous ratios (AVR), central retinal artery equivalent (CRAE) and central retinal vein equivalent (CRVE) were calculated on three separate occasions by one single observer semi-automatically using the software VesselMap (ImedosSystems, Jena, Germany). Following the automated grading, three examiners graded the AVR visually on three separate occasions in order to assess their agreement. Results: Reproducibility of the semi-automatic parameters was excellent (ICCs: 0.97 (CRAE); 0.985 (CRVE) and 0.952 (AVR)). However, visual grading of AVR showed inter grader differences as well as discrepancies between subjectively derived and objectively calculated AVR (all p < 0.000001). Conclusion: Grader education and experience leads to inter-grader differences but more importantly, subjective grading is not capable to pick up subtle differences across healthy individuals and does not represent true AVR when compared with an objective assessment method. Technology advancements mean we no longer rely on opthalmoscopic evaluation but can capture and store fundus images with retinal cameras, enabling us to measure vessel calibre more accurately compared to visual estimation; hence it should be integrated in optometric practise for improved accuracy and reliability of clinical assessments of retinal vessel calibres. © 2014 Spanish General Council of Optometry.

A comparison of two distributed large-volume measurement systems:the mobile spatial co-ordinate measuring system and the indoor global positioning system

Advances in the area of industrial metrology have generated new technologies that are capable of measuring components with complex geometry and large dimensions. However, no standard or best-practice guides are available for the majority of such systems. Therefore, these new systems require appropriate testing and verification in order for the users to understand their full potential prior to their deployment in a real manufacturing environment. This is a crucial stage, especially when more than one system can be used for a specific measurement task. In this paper, two relatively new large-volume measurement systems, the mobile spatial co-ordinate measuring system (MScMS) and the indoor global positioning system (iGPS), are reviewed. These two systems utilize different technologies: the MScMS is based on ultrasound and radiofrequency signal transmission and the iGPS uses laser technology. Both systems have components with small dimensions that are distributed around the measuring area to form a network of sensors allowing rapid dimensional measurements to be performed in relation to large-size objects, with typical dimensions of several decametres. The portability, reconfigurability, and ease of installation make these systems attractive for many industries that manufacture large-scale products. In this paper, the major technical aspects of the two systems are briefly described and compared. Initial results of the tests performed to establish the repeatability and reproducibility of these systems are also presented. © IMechE 2009.

An Application of ISO 5725 in Brewing Industry

2002 Mathematics Subject Classification: 62P30, 62P10.

Absolute abundances of dinoflagellate cysts in surface sediment samples from four sites: Lycopodium marker-grain method

Absolute abundances (concentrations) of dinoflagellate cysts are often determined through the addition of Lycopodium clavatum marker-grains as a spike to a sample before palynological processing. An inter-laboratory calibration exercise was set up in order to test the comparability of results obtained in different laboratories, each using its own preparation method. Each of the 23 laboratories received the same amount of homogenized splits of four Quaternary sediment samples. The samples originate from different localities and consisted of a variety of lithologies. Dinoflagellate cysts were extracted and counted, and relative and absolute abundances were calculated. The relative abundances proved to be fairly reproducible, notwithstanding a need for taxonomic calibration. By contrast, excessive loss of Lycopodium spores during sample preparation resulted in non-reproducibility of absolute abundances. Use of oxidation, KOH, warm acids, acetolysis, mesh sizes larger than 15 µm and long ultrasonication (> 1 min) must be avoided to determine reproducible absolute abundances. The results of this work therefore indicate that the dinoflagellate cyst worker should make a choice between using the proposed standard method which circumvents critical steps, adding Lycopodium tablets at the end of the preparation and using an alternative method.

Introduction of novel video-based tasks to clinical fMRI: Comparing traditional fMRI tasks with novel video-based tasks for mapping brain language areas

Recently, blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) has become a routine clinical procedure for localization of language and motor brain regions and has been replacing more invasive preoperative procedures. However, the fMRI results from these tasks are not always reproducible even from the same patient. Evaluating the reproducibility of language and speech mapping is especially complicated due to the complex brain circuitry that may become activated during the functional task. Non-language areas such as sensory, attention, decision-making, and motor brain regions may also be activated in addition to the specific language regions during a traditional sentence-completion task. In this study, I test a new approach, which utilizes 4-minute video-based tasks, to map language and speech brain regions for patients undergoing brain surgery. Results from 35 subjects have shown that the video-based task activates Wernicke’s area, as well as Broca’s area in most subjects. The computed laterality indices, which indicate the dominant hemisphere from that functional task, have indicated left dominance from the video-based tasks. This study has shown that the video-based task may be an alternative method for localization of language and speech brain regions for patients who are unable to complete the sentence-completion task.

Second worldwide proficiency study on variable number of tandem repeats typing of Mycobacterium tuberculosis complex

Global Network for the Molecular Surveillance of Tuberculosis 2010: A. Miranda (Tuberculosis Laboratory of the National Institute of Health, Porto, Portugal)

Impact of Routine Fractional Flow Reserve Evaluation During Coronary Angiography on Management Strategy and Clinical Outcome: One-Year Results of the POST-IT.

Penetration of fractional flow reserve (FFR) in clinical practice varies extensively, and the applicability of results from randomized trials is understudied. We describe the extent to which the information gained from routine FFR affects patient management strategy and clinical outcome. METHODS AND RESULTS: Nonselected patients undergoing coronary angiography, in which at least 1 lesion was interrogated by FFR, were prospectively enrolled in a multicenter registry. FFR-driven change in management strategy (medical therapy, revascularization, or additional stress imaging) was assessed per-lesion and per-patient, and the agreement between final and initial strategies was recorded. Cardiovascular death, myocardial infarction, or unplanned revascularization (MACE) at 1 year was recorded. A total of 1293 lesions were evaluated in 918 patients (mean FFR, 0.81±0.1). Management plan changed in 406 patients (44.2%) and 584 lesions (45.2%). One-year MACE was 6.9%; patients in whom all lesions were deferred had a lower MACE rate (5.3%) than those with at least 1 lesion revascularized (7.3%) or left untreated despite FFR≤0.80 (13.6%; log-rank P=0.014). At the lesion level, deferral of those with an FFR≤0.80 was associated with a 3.1-fold increase in the hazard of cardiovascular death/myocardial infarction/target lesion revascularization (P=0.012). Independent predictors of target lesion revascularization in the deferred lesions were proximal location of the lesion, B2/C type and FFR. CONCLUSIONS: Routine FFR assessment of coronary lesions safely changes management strategy in almost half of the cases. Also, it accurately identifies patients and lesions with a low likelihood of events, in which revascularization can be safely deferred, as opposed to those at high risk when ischemic lesions are left untreated, thus confirming results from randomized trials.