Scientists in Control: a progressive approach to preparing work ready graduates in QC, EQA and method evaluation

Introduction QC, EQA and method evaluation are integral to delivery of quality patient results. To ensure QUT graduates have a solid grounding in these key areas of practice, a theory-to-practice approach is used to progressively develop and consolidate these skills. Methods Using a BCG assay for serum albumin, each student undertakes an eight week project analysing two levels of QC alongside ‘patient’ samples. Results are assessed using both single rules and Multirules. Concomitantly with the QC analyses, an EQA project is undertaken; students analyse two EQA samples, twice in the semester. Results are submitted using cloud software and data for the full ‘peer group’ returned to students in spreadsheets and incomplete Youden plots. Youden plots are completed with target values and calculated ALP values and analysed for ‘lab’ and method performance. The method has a low-level positive bias, which leads to the need to investigate an alternative method. Building directly on the EQA of the first project and using the scenario of a lab that services renal patients, students undertake a method validation comparing BCP and BCG assays in another eight-week project. Precision and patient comparison studies allow students to assess whether the BCP method addresses the proportional bias of the BCG method and overall is a ‘better’ alternative method for analysing serum albumin, accounting for pragmatic factors, such as cost, as well as performance characteristics. Results Students develop understanding of the purpose and importance of QC and EQA in delivering quality results, the need to optimise testing to deliver quality results and importantly, a working knowledge of the analyses that go into ensuring this quality. In parallel to developing these key workplace competencies, students become confident, competent practitioners, able to pipette accurately and precisely and organise themselves in a busy, time pressured work environment.

A novel fully validated LC–MS/MS method for quantification of pyridoxal-5′-phosphate concentrations in samples of human whole blood

Quantification of pyridoxal-5´-phosphate (PLP) in biological samples is challenging due to the presence of endogenous PLP in matrices used for preparation of calibrators and quality control samples (QCs). Hence, we have developed an LC-MS/MS method for accurate and precise measurement of the concentrations of PLP in samples (20 µL) of human whole blood that addresses this issue by using a surrogate matrix and minimizing the matrix effect. We used a surrogate matrix comprising 2% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for making calibrators, QCs and the concentrations were adjusted to include the endogenous PLP concentrations in the surrogate matrix according to the method of standard addition. PLP was separated from the other components of the sample matrix using protein precipitation with trichloroacetic acid 10% w/v. After centrifugation, supernatant were injected directly into the LC-MS/MS system. Calibration curves were linear and recovery was > 92%. QCs were accurate, precise, stable for four freeze-thaw cycles, and following storage at room temperature for 17h or at -80 °C for 3 months. There was no significant matrix effect using 9 different individual human blood samples. Our novel LC-MS/MS method has satisfied all of the criteria specified in the 2012 EMEA guideline on bioanalytical method validation.

Modification and validation of a microwave-assisted digestion method for subsequent ICP-MS determination of selected heavy metals in sediment and fish samples in Agusan River, Philippines

This study investigated, validated, and applied the optimum conditions for a modified microwave assisted digestion method for subsequent ICP-MS determination of mercury, cadmium, and lead in two matrices relevant to water quality, that is, sediment and fish. Three different combinations of power, pressure, and time conditions for microwave-assisted digestion were tested, using two certified reference materials representing the two matrices, to determine the optimum set of conditions. Validation of the optimized method indicated better recovery of the studied metals compared to standard methods. The validated method was applied to sediment and fish samples collected from Agusan River and one of its tributaries, located in Eastern Mindanao, Philippines. The metal concentrations in sediment ranged from 2.85 to 341.06 mg/kg for Hg, 0.05 to 44.46 mg/kg for Cd and 2.20 to 1256.16 mg/kg for Pb. The results indicate that the concentrations of these metals in the sediments rapidly decrease with distance downstream from sites of contamination. In the selected fish species, the metals were detected but at levels that are considered safe for human consumption, with concentrations of 2.14 to 6.82 μg/kg for Hg, 0.035 to 0.068 μg/kg for Cd, and 0.019 to 0.529 μg/kg for Pb.

Validation of a new hand-held electronic data capture method for continuous monitoring of subjective appetite sensations

Background When large scale trials are investigating the effects of interventions on appetite, it is paramount to efficiently monitor large amounts of human data. The original hand-held Electronic Appetite Ratings System (EARS) was designed to facilitate the administering and data management of visual analogue scales (VAS) of subjective appetite sensations. The purpose of this study was to validate a novel hand-held method (EARS II (HP® iPAQ)) against the standard Pen and Paper (P&P) method and the previously validated EARS. Methods Twelve participants (5 male, 7 female, aged 18-40) were involved in a fully repeated measures design. Participants were randomly assigned in a crossover design, to either high fat (>48% fat) or low fat (<28% fat) meal days, one week apart and completed ratings using the three data capture methods ordered according to Latin Square. The first set of appetite sensations was completed in a fasted state, immediately before a fixed breakfast. Thereafter, appetite sensations were completed every thirty minutes for 4h. An ad libitum lunch was provided immediately before completing a final set of appetite sensations. Results Repeated measures ANOVAs were conducted for ratings of hunger, fullness and desire to eat. There were no significant differences between P&P compared with either EARS or EARS II (p > 0.05). Correlation coefficients between P&P and EARS II, controlling for age and gender, were performed on Area Under the Curve ratings. R2 for Hunger (0.89), Fullness (0.96) and Desire to Eat (0.95) were statistically significant (p < 0.05). Conclusions EARS II was sensitive to the impact of a meal and recovery of appetite during the postprandial period and is therefore an effective device for monitoring appetite sensations. This study provides evidence and support for further validation of the novel EARS II method for monitoring appetite sensations during large scale studies. The added versatility means that future uses of the system provides the potential to monitor a range of other behavioural and physiological measures often important in clinical and free living trials.

Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes

The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson’s Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59–119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.

Development and validation of a ultra performance LC-ESI/MS method for analysis of metabolic phenotypes of healthy men in day and night urine samples

Ultra-performance LC coupled to quadrupole TOF/MS (UPLC-QTOF/MS) in positive and negative ESI was developed and validated to analyze metabolite profiles for urine from healthy men during the day and at night. Data analysis using principal components analysis (PCA) revealed differences between metabolic phenotypes of urine in healthy men during the day and at night. Positive ions with mass-to-charge ratio (m/z) 310.24 (5.35 min), 286.24 (4.74 min) and 310.24 (5.63 min) were elevated in the urine from healthy men at night compared to that during the day. Negative ions elevated in day urine samples of healthy men included m/z 167.02 (0.66 min), 263.12 (2.55 min) and 191.03 (0.73 min), whilst ions m/z 212.01 (4.77 min) were at a lower concentration in urine of healthy men during the day compared to that at night. The ions m/z 212.01 (4.77 min), 191.03 (0.73 min) and 310.24 (5.35 min) preliminarily correspond to indoxyl sulfate, citric acid and N-acetylneuraminic acid, providing further support for an involvement of phenotypic difference in urine of healthy men in day and night samples, which may be associated with notably different activities of gut microbiota, velocity of tricarboxylic acid cycle and activity of sialic acid biosynthesis in healthy men as regulated by circadian rhythm of the mammalian bioclock.

Colloidal drug probe : method development and validation for adhesion force measurement using atomic force microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between the drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from a dry powder inhaler (DPI) formulation. Initially model silica probes of approximately 4 μm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-Ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres preattached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

Colloidal drug probe : Method development and validation for adhesion force measurement using Atomic Force Microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between a model drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from dry powder inhaler (DPI) formulations. Model silica probes of approximately 4 lm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres pre-attached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/ hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

A new method for the in vivo identification of mechanical properties in arteries from cine MRI images: Theoretical framework and validation

Quantifying the stiffness properties of soft tissues is essential for the diagnosis of many cardiovascular diseases such as atherosclerosis. In these pathologies it is widely agreed that the arterial wall stiffness is an indicator of vulnerability. The present paper focuses on the carotid artery and proposes a new inversion methodology for deriving the stiffness properties of the wall from cine-MRI (magnetic resonance imaging) data. We address this problem by setting-up a cost function defined as the distance between the modeled pixel signals and the measured ones. Minimizing this cost function yields the unknown stiffness properties of both the arterial wall and the surrounding tissues. The sensitivity of the identified properties to various sources of uncertainty is studied. Validation of the method is performed on a rubber phantom. The elastic modulus identified using the developed methodology lies within a mean error of 9.6%. It is then applied to two young healthy subjects as a proof of practical feasibility, with identified values of 625 kPa and 587 kPa for one of the carotid of each subject.

Validation of a partial coherence interferometry method for estimating retinal shape

To validate a simple partial coherence interferometry (PCI) based retinal shape method, estimates of retinal shape were determined in 60 young adults using off-axis PCI, with three stages of modeling using variants of the Le Grand model eye, and magnetic resonance imaging (MRI). Stage 1 and 2 involved a basic model eye without and with surface ray deviation, respectively and Stage 3 used model with individual ocular biometry and ray deviation at surfaces. Considering the theoretical uncertainty of MRI (12-14%), the results of the study indicate good agreement between MRI and all three stages of PCI modeling with <4% and <7% differences in retinal shapes along horizontal and vertical meridians, respectively. Stage 2 and Stage 3 gave slightly different retinal co-ordinates than Stage 1 and we recommend the intermediate Stage 2 as providing a simple and valid method of determining retinal shape from PCI data.

Virtual design process : design validation tool schematic approach in car carrier

An important aspect of designing any product is validation. Virtual design process (VDP) is an alternative to hardware prototyping in which analysis of designs can be done without manufacturing physical samples. In recent years, VDP have been generated either for animation or filming applications. This paper proposes a virtual reality design process model on one of the applications when used as a validation tool. This technique is used to generate a complete design guideline and validation tool of product design. To support the design process of a product, a virtual environment and VDP method were developed that supports validation and an initial design cycle performed by a designer. The product model car carrier is used as illustration for which virtual design was generated. The loading and unloading sequence of the model for the prototype was generated using automated reasoning techniques and was completed by interactively animating the product in the virtual environment before complete design was built. By using the VDP process critical issues like loading, unloading, Australian Design rules (ADR) and clearance analysis were done. The process would save time, money in physical sampling and to large extent in complete math generation. Since only schematic models are required, it saves time in math modelling and handling of bigger size assemblies due to complexity of the models. This extension of VDP process for design evaluation is unique and was developed, implemented successfully. In this paper a Toll logistics and J Smith and Sons car carrier which is developed under author’s responsibility has been used to illustrate our approach of generating design validation via VDP.

Development of diagnostic and prognostic algorithms for SF6 puffer circuit breakers from transient waveforms : a validation proposal

- This paper presents a validation proposal for development of diagnostic and prognostic algorithms for SF6 puffer circuit-breakers reproduced from actual site waveforms. The re-ignition/restriking rates are duplicated in given circuits and the cumulative energy dissipated in interrupters by the restriking currents. The targeted objective is to provide a simulated database for diagnosis of re-ignition/restrikes relating to the phase to earth voltage and the number of re-ignition/restrikes as well as estimating the remaining life of SF6 circuit-breakers. The model-based diagnosis of a tool will be useful in monitoring re-ignition/restrikes as well as predicting a nozzle’s lifetime. This will help ATP users with practical study cases and component data compilation for shunt reactor switching and capacitor switching. This method can be easily applied with different data for the different dielectric curves of circuit breakers and networks. This paper presents modelling details and some of the available cases, required project support, the validation proposal, the specific plan for implementation and the propsed main contributions.

Cues to starting CPAP in obstructive sleep apnoea : development and validation of the cues to CPAP Use Questionnaire (CCUQ)

Background: The reasons that a patient has to start treatment, their “Cues to Action”, are important for determining subsequent health behaviours. Cues to action are an explicit component of the Health Belief Model of CPAP acceptance adherence. At present there is no scale available to measure this construct for individuals with Obstructive Sleep Apnoea (OSA). This paper aims to develop, validate and describe responding patterns within an OSA patient sample to the Cues to CPAP Use Questionnaire (CCUQ).----- Method: Participants were 63 adult patients diagnosed with OSA who had never tried CPAP when initially recruited. The CCUQ was completed at one month after being prescribed CPAP.----- Results: Exploratory factor analysis (EFA) showed a three factor structure of the 9-item CCUQ, with “Health Cues”, “Partner Cues” and “Health Professional Cues” subscales accounting for 59.91% of the total variance. The CCUQ demonstrated modest internal consistency and split-half reliability. The questionnaire is brief and user-friendly, with readability at a 7th grade level. The most frequently endorsed cues for starting CPAP were Health Professional Cues (prompting by the sleep physician) and Health Cues such as tiredness and concern about health outcomes.----- Conclusions: This study validates a measure of an important motivational component of the Health Belief Model. Health Professional Cues and internal Health Cues were reported to be the most important prompts to commence CPAP by this patient sample.

Motivation, development and validation of a new spectral greenness index : a spectral dimension related to foliage projective cover

A method is presented for the development of a regional Landsat-5 Thematic Mapper (TM) and Landsat-7 Enhanced Thematic Mapper plus (ETM+) spectral greenness index, coherent with a six-dimensional index set, based on a single ETM+ spectral image of a reference landscape. The first three indices of the set are determined by a polar transformation of the first three principal components of the reference image and relate to scene brightness, percent foliage projective cover (FPC) and water related features. The remaining three principal components, of diminishing significance with respect to the reference image, complete the set. The reference landscape, a 2200 km2 area containing a mix of cattle pasture, native woodland and forest, is located near Injune in South East Queensland, Australia. The indices developed from the reference image were tested using TM spectral images from 19 regionally dispersed areas in Queensland, representative of dissimilar landscapes containing woody vegetation ranging from tall closed forest to low open woodland. Examples of image transformations and two-dimensional feature space plots are used to demonstrate image interpretations related to the first three indices. Coherent, sensible, interpretations of landscape features in images composed of the first three indices can be made in terms of brightness (red), foliage cover (green) and water (blue). A limited comparison is made with similar existing indices. The proposed greenness index was found to be very strongly related to FPC and insensitive to smoke. A novel Bayesian, bounded space, modelling method, was used to validate the greenness index as a good predictor of FPC. Airborne LiDAR (Light Detection and Ranging) estimates of FPC along transects of the 19 sites provided the training and validation data. Other spectral indices from the set were found to be useful as model covariates that could improve FPC predictions. They act to adjust the greenness/FPC relationship to suit different spectral backgrounds. The inclusion of an external meteorological covariate showed that further improvements to regional-scale predictions of FPC could be gained over those based on spectral indices alone.