Current Gold Standard Method Research Articles

3D photography is as accurate as digital planimetry tracing in determining burn wound area

BackgroundIn the paediatric population careful attention needs to be made concerning techniques utilised for wound assessment to minimise discomfort and stress to the child. AimTo investigate whether 3D photography is a valid measure of burn wound area in children compared to the current clinical gold standard method of digital planimetry using Visitrak™. MethodTwenty-five children presenting to the Stuart Pegg Paediatric Burn Centre for burn dressing change following acute burn injury were included in the study. Burn wound area measurement was undertaken using both digital planimetry (Visitrak™ system) and 3D camera analysis. Inter-rater reliability of the 3D camera software was determined by three investigators independently assessing the burn wound area. ResultsA comparison of wound area was assessed using intraclass correlation co-efficients (ICC) which demonstrated excellent agreement 0.994 (CI 0.986, 0.997). Inter-rater reliability measured using ICC 0.989 (95% CI 0.979, 0.995) demonstrated excellent inter-rater reliability. Time taken to map the wound was significantly quicker using the camera at bedside compared to Visitrak™ 14.68 (7.00)s versus 36.84 (23.51)s (p<0.001). In contrast, analysing wound area was significantly quicker using the Visitrak™ tablet compared to Dermapix® software for the 3D Images 31.36 (19.67)s versus 179.48 (56.86)s (p<0.001). ConclusionThis study demonstrates that images taken with the 3D LifeViz™ camera and assessed with Dermapix® software is a reliable method for wound area assessment in the acute paediatric burn setting.

Evaluation of toxicogenomics approaches for assessing the risk of nongenotoxic carcinogenicity in rat liver.

The current gold-standard method for cancer safety assessment of drugs is a rodent two-year bioassay, which is associated with significant costs and requires testing a high number of animals over lifetime. Due to the absence of a comprehensive set of short-term assays predicting carcinogenicity, new approaches are currently being evaluated. One promising approach is toxicogenomics, which by virtue of genome-wide molecular profiling after compound treatment can lead to an increased mechanistic understanding, and potentially allow for the prediction of a carcinogenic potential via mathematical modeling. The latter typically involves the extraction of informative genes from omics datasets, which can be used to construct generalizable models allowing for the early classification of compounds with unknown carcinogenic potential. Here we formally describe and compare two novel methodologies for the reproducible extraction of characteristic mRNA signatures, which were employed to capture specific gene expression changes observed for nongenotoxic carcinogens. While the first method integrates multiple gene rankings, generated by diverse algorithms applied to data from different subsamplings of the training compounds, the second approach employs a statistical ratio for the identification of informative genes. Both methods were evaluated on a dataset obtained from the toxicogenomics database TG-GATEs to predict the outcome of a two-year bioassay based on profiles from 14-day treatments. Additionally, we applied our methods to datasets from previous studies and showed that the derived prediction models are on average more accurate than those built from the original signatures. The selected genes were mostly related to p53 signaling and to specific changes in anabolic processes or energy metabolism, which are typically observed in tumor cells. Among the genes most frequently incorporated into prediction models were Phlda3, Cdkn1a, Akr7a3, Ccng1 and Abcb4.

Digital microfluidic assay for protein detection.

Global studies of the human proteome have revealed a plethora of putative protein biomarkers. However, their application for early disease detection remains at a standstill without suitable methods to realize their utility in the clinical setting. There thus continues to be tremendous interest in developing new technology for sensitive protein detection that is both low in cost and carries a small footprint to be able to be used at the point of care. The current gold standard method for protein biomarker detection is the ELISA, which measures protein abundance using bulky fluorescent scanners that lack portability. Here, we present a digital microfluidic platform for protein biomarker detection that is low in cost compared with standard optical detection methods, without any compromise in sensitivity. This platform furthermore makes use of simple electronics, enabling its translation into a portable handheld device, and has been developed in a manner that can easily be adapted to assay different types of proteomic biomarkers. We demonstrate its utility in quantifying not only protein abundance, but also activity. Interleukin-6 abundance could be assayed from concentrations as low as 50 pM (an order of magnitude lower than that detectable by a comparable laboratory designed ELISA) using less than 5 μL of sample, and Abelson tyrosine kinase activity was detectable in samples containing 100 pM of kinase.

Adjuncts to colonic cleansing before colonoscopy

Pre-procedural cleansing of the bowel can maximize the effectiveness and efficiency of colonoscopy. Yet, efficacy of the current gold standard colonic preparation method - high-volume oral administration of purgative agents 12-24 h prior to the procedure - is limited by several factors, such as patient compliance (due to poor palatability and inconvenience of the dosing regimen) and risks of complications (due to drug interactions or intolerance). Attempts to resolve these limitations have included providing adjunctive agents and methods to promote the colonic cleansing ability of the principal purgative agent, with the aim of lessening unpleasant side effects (such as bloating) and reducing the large ingested volume requirement. Several promising adjunctive agents are bisacodyl, magnesium citrate, senna, simethicone, metoclopramide, and prokinetics, and each are being investigated for their potential. This review provides an up to date summary of the reported investigations into the potencies and weaknesses of the key adjuncts currently being applied in clinic as supplements to the traditional bowel preparation agents. While the comparative analysis of these adjuncts showed that no single agent or method has yet achieved the goal of completely overcoming the limitations of the current gold standard preparation method, they at least provide endoscopists with an array of alternatives to help improve the suboptimal efficacy of the main cleansing solutions when used alone. To aid in this clinical endeavor, a subjective grade was assigned to each adjunct to indicate its practical value. In addition, the systematic review of the currently available agents and methods provides insight into the features of each that may be overcome or exploited to create novel drugs and strategies that may become adopted as effective bowel cleansing adjuncts or alternatives.

Allelic mapping bias in RNA-sequencing is not a major confounder in eQTL studies

RNA sequencing (RNA-seq) is the current gold-standard method to quantify gene expression for expression quantitative trait locus (eQTL) studies. However, a potential caveat in these studies is that RNA-seq reads carrying the non-reference allele of variant loci can have lower probability to map correctly to the reference genome, which could bias gene quantifications and cause false positive eQTL associations. In this study, we analyze the effect of this allelic mapping bias in eQTL discovery. We simulate RNA-seq read mapping over 9.5 M common SNPs and indels, with 15.6% of variants showing biased mapping rate for reference versus non-reference reads. However, removing potentially biased RNA-seq reads from an eQTL dataset of 185 individuals has a very small effect on gene and exon quantifications and eQTL discovery. We detect only a handful of likely false positive eQTLs, and overall eQTL SNPs show no significant enrichment for high mapping bias. Our results suggest that RNA-seq quantifications are generally robust against allelic mapping bias, and that this does not have a severe effect on eQTL discovery. Nevertheless, we provide our catalog of putatively biased loci to allow better controlling for mapping bias to obtain more accurate results in future RNA-seq studies.

Acoustic Pharyngometry Measurement of Minimal Cross-Sectional Airway Area Is a Significant Independent Predictor of Moderate-To-Severe Obstructive Sleep Apnea

The current gold-standard method of diagnosing obstructive sleep apnea (OSA) is polysomnography, which can be inefficient. We therefore sought to determine a method to triage patients at risk of OSA, without using subjective data, which are prone to mis-reporting. We hypothesized that acoustic pharyngometry in combination with age, gender, and neck circumference would predict the presence of moderate-to-severe OSA. Untreated subjects with suspected OSA were recruited from a local sleep clinic and underwent polysomnography. We also included a control group to verify differences. While seated in an upright position and breathing through the mouth, an acoustic pharyngometer was used to measure the minimal cross-sectional area (MCA) of the upper airway at end-exhalation. Sixty subjects were recruited (35 males, mean age 42 years, range 21-81 years; apnea-hypopnea index (AHI) 33 ± 30 events/h (mean ± standard deviation), Epworth Sleepiness Scale score 11 ± 6, body mass index 34 ± 8 kg/m(2)). In univariate logistic regression, MCA was a significant predictor of mild-no OSA (AHI < 15). A multivariate logistic regression model including MCA, age, gender, and neck circumference significantly predicted AHI < 15, explaining approximately one-third of the total variance (χ(2)(4) = 37, p < 0.01), with only MCA being a significant independent predictor (adjusted odds ratio 54, standard error 130; p < 0.01). These data suggest that independent of age, gender, and neck size, objective anatomical assessment can significantly differentiate those with mild versus moderate-to-severe OSA in a clinical setting, and may have utility as a component in stratifying risk of OSA.

AB1238 A comparison between automated and manual methods for detection antinuclear autoantibodies on human epithelial-2 cells

BackgroundA variety of antinuclear antibodies (ANAs) are found in serum samples obtained from patients with rheumatic autoimmune diseases1. Identification of the corresponding antigens and measurement of the antibody titer are...

Statistical notes for clinical researchers: assessing normal distribution (2) using skewness and kurtosis

As discussed in the previous statistical notes, although many statistical methods have been proposed to test normality of data in various ways, there is no current gold standard method. The eyeball test may be useful for medium to large sized (e.g., n > 50) samples, however may not useful for small samples. The formal normality tests including Shapiro-Wilk test and Kolmogorov-Smirnov test may be used from small to medium sized samples (e.g., n < 300), but may be unreliable for large samples. Moreover we may be confused because ‘eyeball test’ and ‘formal normality test’ may show incompatible results for the same data. To resolve the problem, another method of assessing normality using skewness and kurtosis of the distribution may be used, which may be relatively correct in both small samples and large samples. 1) Skewness and kurtosis Skewness is a measure of the asymmetry and kurtosis is a measure of ’peakedness’ of a distribution. Most statistical packages give you values of skewness and kurtosis as well as their standard errors.

P196 Percutaneous Ultrasound-Guided Biopsy of Intrathoracic Parietal Masses – a Safe Alternative to CT-Guided Biopsy?

BackgroundThe current gold standard method for sampling intrathoracic lesions is via a CT-guided approach. However, most parietal-attached intrathoracic lesions can be visualised with ultrasound and sampled.AimsA prospective audit of consecutive...

A novel automated indirect immunofluorescence autoantibody evaluation

Autoantibodies (AAb), especially antinuclear (ANAs) and anticytoplasmatic antibodies (ACyA), are essential diagnosing markers for several autoimmune diseases. The current gold standard method for ANA detection is manual indirect immunofluorescence (IIF) on human epithelial-2 (HEp-2) cells. However, this technique is cost and time consuming, and characterized by considerable intra- and interlaboratory variability. Thus, an automated IIF-HEp-2 reader has been developed recently. In the current study, we compared the performance of the automated AAb IIF-HEp-2 interpretation to conventional detection methods. Autoantibody detection by IIF on HEp-2 cells was performed in a total of 260 sera of patients, including 34 with systemic lupus erythematosus, 111 with dermatomyositis or polymyositis, 74 with systemic sclerosis, 41 with rare AAb patterns, and 137 healthy individuals. Visual interpretation and routine immunoassays were compared with a novel automated IIF-HEp-2 system using Aklides pattern recognition algorithms. Positive AAbs were detected in 95-100% of rheumatic patients by automated interpretation, in 74-100% with manual reading, and in 64-100% by immunodot assay. Receiver operating characteristic curve analysis of fluorescent intensity revealed a high sensitivity and specificity for automated reading of AAb with an agreement ranging from 90% to 95% between manual and automated interpretation (kappa 0.554-0.69) for systemic sclerosis and myositis, respectively. This study demonstrates a good correlation between manual and automated interpretation of AAb including ANA and ACyA in patients with autoimmune diseases. Full automation of HEp-2 cell assay reading may minimize errors in ANA pattern interpretation and thus help in the standardization of ANA assessment.

Citations

Citations

Abstract 1234: Development of a quick sensitive real-time PCR assay for measuring telomere length

Abstract Telomeres are the ends of linear chromosomes with tandem hexameric nucleotide repeats. Its function is to protect the natural ends of chromosomes from being recognized as damaged DNA, hence contributing to chromosomal stability. Telomeric sequences shorten each time the DNA replicates; when the telomere shrinks to a certain threshold, the cell can no longer divide. Its metabolism slows down, the cell ages, and dies. This molecular clock mechanism is also associated with cancer progression and therapy. Turning off telomerase activity by shutting off telomerase activation in cancer cells will led to cellular senescence. The current gold standard method to measuring telomere length is by Southern blot; however, it is tedious and costly, and often takes up to two days to complete. Here, we describe a new, simplified automated real-time PCR assay to accurately detect the abundance of these 5’-TTAGGG-3’nucleotide repeats and determine frequency and the length of Telomere in 47 min. The assay demonstrated to be highly reproducible and accurate for human peripheral cells. The result has good correlation of R2=0.9939, slope=3.41, sensitivity to 1ng of gDNA and has an amplification efficiency of 100%. We evaluated this assay with 299 healthy human subjects to date ranging from 0 (core blood) to 99-year of age. The results are positive with an inverse correlation between telomere length and age, as telomeres were shorter with aging. The telomere length was measured in 13 samples using the gold standard Southern blot method and the new q-PCR method (R2 = 0.8623), significantly higher than the originally described (Cawthon NAS 2002). We believe this fast, sensitive methodology is a powerful tool and provides a new avenue for investigators to address the full extent and significance of telomere shortening in the tumorigenic process. This new assay described here will allow for more widespread use of this technique among cancer researchers and for study of age related telomere diseases and conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1234. doi:10.1158/1538-7445.AM2011-1234

Epidermal Growth Factor Receptor (EGFR) Is Overexpressed in High-Grade Dysplasia and Adenocarcinoma of the Esophagus and May Represent a Biomarker of Histological Progression in Barrett's Esophagus (BE)

The assessment of cancer risk in patients with Barrett's esophagus (BE) is currently fraught with difficulty. The current gold standard method of assessing cancer risk is histological assessment, with the appearance of high-grade dysplasia (HGD) as the key event monitored. Sampling error during endoscopy limits the usefulness of this approach, and there has been much recent interest in supplementing histological assessment with molecular markers, which may aid in patient stratification. No molecular marker has been yet validated to accurately correlate with esophageal histological progression. Here, we assessed the suitability of several membranous proteins as biomarkers by correlating their abundance with histological progression. In all, 107 patient samples, from 100 patients, were arranged on a tissue microarray (TMA) and represented the various stages of histological progression in BE. This TMA was probed with antibodies for eight receptor proteins (mostly membranous). Epidermal growth factor receptor (EGFR) staining was found to be the most promising biomarker identified with clear increases in staining accompanying histological progression. Further, immunohistochemistry was performed using the full-tissue sections from BE, HGD, and adenocarcinoma tissues, which confirmed the stepwise increase in EGFR abundance. Using a robust H-score analysis, EGFR abundance was shown to increase 13-fold in the adenocarcinoma tissues compared to the BE tissues. EGFR was "overexpressed" in 35% of HGD specimens and 80% of adenocarcinoma specimens when using the H-score of the BE patients (plus 3 s.d.) as the threshold to define overexpression. EGFR staining was also noted to be higher in BE tissues adjacent to HGD/adenocarcinoma. Western blotting, although showing more EGFR protein in the adenocarcinomas compared to the BE tissue, was highly variable. EGFR overexpression was accompanied by aneuploidy (gain) of chromosome 7, plus amplification of the EGFR locus. Finally, the bile acid deoxycholic acid (DCA) (at neutral and acidic pH) and acid alone was capable of upregulating EGFR mRNA in vitro, and in the case of neutral pH DCA, this was NF-κB dependent. EGFR is overexpressed during the histological progression in BE tissues and hence may be useful as a biomarker of histological progression. Furthermore, as EGFR is a membranous protein expressed on the luminal surface of the esophageal mucosa, it may also be a useful target for biopsy guidance during endoscopy.

P26 Home serial spirometry as an adjunct in the diagnosis of vocal cord dysfunction

IntroductionThe diagnosis of vocal cord dysfunction (VCD) is often difficult. Visualisation of the vocal cords using laryngoscopy is the current gold standard method of diagnosis, but may not be diagnostic...

Malignant hyperthermia in India

Malignant hyperthermia in India

Automated anatomical demarcation using an active shape model for videofluoroscopic analysis in swallowing

The current gold standard method in the clinical assessment of swallowing is the visual inspection of videofluoroscopic frames. Specific clinical measurements are estimated based on various anatomical and bolus positional information with respect to time (or frame number). However, due to the subjective nature of visual inspection clinicians face intra- and inter-observer repeatability issues and bias when making these estimations. The correct demarcations of reference lines highlighting the positions of important anatomical landmarks would serve as a visual aid and could also be used in conjunction with bolus detection methods to objectively determine these desirable measurements. In this paper, we introduce and test the reliability of applying a 16-point Active Shape Model as a deformable template to demarcate the boundaries of salient anatomical boundaries with minimal user input. A robust end and corner point detection algorithm is also used to provide image information for the suggested movement of the template during the fitting stage. Results show the model deformation constraints calculated from a training set of images are clinically coherent. The Euclidean distances between the fitted model points against their corresponding target points were measured. Test images were taken from two different data sets from frames acquired using two different videofluoroscopy units. Overall, fitting was found to be more reliable on the vertebrae and inferior points of the larynx compared to the superior laryngeal points and hyoid bone, with the model always fitting the C7 vertebra with discrepancies no higher than a distance of 23 pixels (3.2% of the image width, approximately 7.6 mm).

Lung volume and pharyngeal stability in sleeping humans: the knee bone is connected to the thigh bone

in 1988 the Journal of Applied Physiology published a seminal article by William Van de Graaff ([13][1]) demonstrating that inflation of the thorax in anesthetized dogs decreased upper airway resistance independently of upper airway muscle activity. The mechanism underlying this effect was shown to

Numerical Validation of a New Method to Assess Aortic Pulse Wave Velocity from a Single Recording of a Brachial Artery Waveform with an Occluding Cuff

Recently a new method has been proposed as a tool to measure arterial pulse wave velocity (PWV), a measure of the stiffness of the large arteries and an emerging parameter used as indicator of clinical cardiovascular risk. The method is based on measurement of brachial blood pressure during supra-systolic pressure inflation of a simple brachial cuff [the device is known as the Arteriograph (Tensiomed, Budapest, Hungary)]. This occlusion yields pronounced first and secondary peaks in the pressure waveform, the latter ascribed to a reflection from the aortic bifurcation, and PWV is calculated as the ratio of twice the jugulum-symphysis distance and the time difference between the two peaks. To test the validity of this working principle, we used a numerical model of the arterial tree to simulate pressures and flows in the normal configuration, and in a configuration with an occluded brachial artery. A pronounced secondary peak was indeed found in the brachial pressure signal of the occluded model, but its timing was only related to brachial stiffness and not to aortic stiffness. We also compared PWV's calculated with three different methods: PWVATG (approximately Arteriograph principle), PWVcar-fem (approximately carotid-femoral PWV, the current clinical gold standard method), and PWVtheor (approximately Bramwell-Hill equation). Both PWVATG (R2=0.94) and PWVcar-fem (R2=0.95) correlated well with PWVtheor, but their numerical values were lower (by 2.17+/-0.42 and 1.08+/-0.70 m/s for PWVATG and PWVcar-fem, respectively). In conclusion, our simulations question the working principle of the Arteriograph. Our data indicate that the method picks up wave reflection phenomena confined to the brachial artery, and derived values of PWV rather reflect the stiffness of the brachial arteries.

Application of the Ibis-T5000 Pan-Orthopoxvirus Assay to Quantitatively Detect Monkeypox Viral Loads in Clinical Specimens from Macaques Experimentally Infected with Aerosolized Monkeypox Virus

Monkeypox virus (MPXV), a member of the family Poxviridae and genus Orthopoxvirus, causes a smallpox-like disease in humans. A previously described pan-Orthopoxvirus assay, based on a broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), was evaluated for its ability to detect MPXV from spiked human and aerosol-infected cynomolgous macaque (Macaca fascicularis) samples. Detection of MPXV DNA from macaque tissue, blood, and spiked human blood by the PCR/ESI-MS pan-Orthopoxvirus assay was comparable, albeit at slightly higher levels, to the current gold standard method of real-time PCR with the pan-Orthopoxvirus assay and had a limit of detection of 200 plaque-forming units. Furthermore, the platform was able to distinguish MPXV and vaccinia viruses that were spiked into macaque blood samples at various concentrations. This platform provides a new tool for the diagnosis and monitoring of orthopoxviral loads during vaccine or antiviral studies, but also could provide rapid identification during natural outbreaks or bioterrorism attacks.

A modified low-cost colorimetric method for paracetamol (acetaminophen) measurement in plasma

Background. Despite a significant increase in the number of patients with paracetamol poisoning in the developing world, plasma paracetamol assays are not widely available. The purpose of this study was to assess a low-cost modified colorimetric paracetamol assay that has the potential to be performed in small laboratories with restricted resources. Methods. The paracetamol assay used in this study was based on the Glynn and Kendal colorimetric method with a few modifications to decrease the production of nitrous gas and thereby reduce infrastructure costs. Preliminary validation studies were performed using spiked aqueous samples with known concentrations of paracetamol. Subsequently, the results from the colorimetric method for 114 stored clinical samples from patients with paracetamol poisoning were compared with those from the current gold-standard high-performance liquid chromatography method. A prospective survey, assessing the clinical use of the paracetamol assay, was performed on all patients with paracetamol poisoning attending the Peradeniya General Hospital, Sri Lanka, over a 10-month period. Results. The recovery study showed an excellent correlation (r2 > 0.998) for paracetamol concentrations from 25 to 400 mg/L. The final yellow color was stable for at least 10 min at room temperature. There was also excellent correlation with the high-performance liquid chromatography method (r2 = 0.9758). In the clinical cohort study, use of the antidote N-acetylcysteine was avoided in over a third of patients who had the plasma paracetamol concentration measured. The cost of consumables used per assay was $0.50 (US). Conclusions. This colorimetric paracetamol assay is reliable and accurate and can be performed rapidly, easily, and economically. Use of this assay in resource-poor clinical settings has the potential to have a significant clinical and economic impact on the management of paracetamol poisoning.