Repeatability Coefficients Of Variation Research Articles

Validation of an automated chromogenic assay of potency of factor VIII in commercial concentrates

The determination of factor VIII (FVIII) potency in FVIII concentrates can be performed using both manual and automated methods. This work aimed to validate the use of the chromogenic kit Coamatic® FVIII (Chromogenix) on the automated ACL® Elite PRO analyzer for evaluating the potency of FVIII in commercial preparations in pharmaceutical analytical laboratories. After setting the activation and reading times to 2 min and 3 min, respectively, the validation parameters, according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guideline Q2 (R1), were as follows: linearity, expressed by the adjusted model: log (Absorbance) = 1.848 + 0.777∙log (Concentration), with r² = 0.998; accuracy was verified (P-value = 0.6959); and the coefficient of variation for repeatability and intermediate precision was ≤6.5%. The Coamatic® FVIII kit method has been adapted to the ACL Elite PRO analyzer with improved performance compared with a manual microplate method.

Determination of avermectin and milbemycin residues in bovine muscle by liquid chromatography-tandem mass spectrometry and fluorescence detection using solvent extraction and low temperature cleanup

A simple and inexpensive sample preparation method based on solvent extraction, followed by low temperature cleanup, was demonstrated to be applicable for the determination of avermectin and milbemycin residues in bovine muscle by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography with fluorescence (LC-FL) detection. The analytical methodology was validated according to the Commission Decision 2002/657/EC, using LC-MS/MS for confirmatory and LC-FL for quantitative purposes. Mean recovery was between 88.9 and 100.7% in three distinct concentrations. The coefficient of variation for repeatability and within-laboratory reproducibility ranged from 0.78 to 5.1% and from 0.28 to 9.0%, respectively. Method precision led to satisfactory values of decision limits (CCα) and detection capabilities (CCβ). The proposed method has been applied in the Brazilian National Residue Control Plan since 2010 for the determination of avermectins and milbemycin residues in bovine muscle samples. A total of 760 samples were analyzed and none of them presented residues at concentrations above the permitted levels established by the more recently applied directives.

Performance of the Emit® II Plus 6-Acetylmorphine Assay on the Viva-E® Analyzer

We evaluated the performance of Emit(®) II Plus 6-Acetylmorphine Assay for human urine screening on the Viva-E(®) analyzer. Precision was evaluated using the cutoff and ±25% controls. Recovery and linearity were studied by spiking 6-acetylmorphine (6-AM) into human urine pools. Accuracy was evaluated using urine specimens and the results were compared to those from GC/MS. Cross-reactivity with structurally related drugs was assessed at different cross-reactant concentrations. Interferences were assessed in the presence of 7.5 and 12.5 ng/mL of 6-AM. The qualitative repeatability coefficients of variation (CV's) ranged from 0.3% to 0.4% and the within-lab CV's ranged from 2.0% to 2.2%. In analyte units (ng/mL), the repeatability CV's ranged from 1.3% to 2.2% and the within-lab CV's ranged from 2.6% to 4.3%. The limit of detection of the assay was found to be 2.1 ng/mL. Recovery was within 15% of expected value. Linearity was 2.1-20 ng/mL. Method comparison showed 99% agreement with GC/MS. The assay had minimal cross-reactivity with morphine, morphine-3-glucuronide, morphine-6-glucuronide and other opioids. No interference was observed with endogenous interferences and structurally unrelated drugs. The assay correctly classified CAP survey samples. The Emit(®) II Plus 6-Acetylmorphine Assay will be a suitable screening method for urine specimens in both qualitative and semi-quantitative analyses.

Determination of patulin in apple juice by HPLC using a simple and fast sample preparation method

The goal of this work was to develop a simple and rapid preparation method for patulin analysis in apple juice without previous clean-up. This method combined sonication and liquid extraction techniques and was used for determination of patulin in 37 commercial apple juices available on the market in the South of Brazil. The method performance characteristics were determined using a sample obtained in a local market fortified at five concentration levels of patulin and done in triplicates. The coefficient of variation for repeatability at the fortification level of 20.70μg.L-1 was 3.53 % and the recovery 94.63 %, respectively. The correlation coefficient was 0.9996 and agrees with the requirements for a linear analytical method value. The detection limit was 0.21μg.L-1 and the quantification limit 0.70 μg.L-1. Only three of the analyzed samples were upper the allowed level of 50.00 μg.L-1 recommended for the World Health Organization.

Quantification of Saponins in Extractive Solution of Mate Leaves (Ilex paraguariensisA. St. Hil.)

Mate (Ilex paraguariensis A. St. Hil.) is a raw material used to prepare popular beverages in South America, whose healthy effects have been pointed out. The leaves and stems of mate contain a complex assemblage of saponins, mainly ursolic acid and oleanolic acid glycosides, and their quantification presents difficulties such as weak cromophorous sites and interference of other compounds. On the other hand, the sugar moieties are a chief character that distinguishes them. We developed and validated a high-performance liquid chromatography method using a refractometer detector for quantifying the major saponins in extractive solution of mate leaves. The validation indicates the suitability of the method, presenting linearity in the concentration range between 102.5 to 854.0 microg/mL to matesaponin 1 and between 103.0 to 515.0 microg/mL to matesaponin 3. The repeatability coefficient of variation was 4.70% and 6.61% for matesaponin 1 and matesaponin 3, respectively. The inter-day coefficient of variation was 6.77% and 7.79% for matesaponin 1 and matesaponin 3, respectively. The analysis of plants from Brazilian States Mato Grosso do Sul and Rio Grande do Sul showed the predominance of matesaponins 1, 2, and 3 in the corresponding extractive solution. The sum of these three saponins ranged between 3 and 10 mg/g for mature leaves in the majority of sampled plants.

Determination of patulin in apple juice by HPLC using a simple and fast sample preparation method

The goal of this work was to develop a simple and rapid preparation method for patulin analysis in apple juice without previous clean-up. This method combined sonication and liquid extraction techniques and was used for determination of patulin in 37 commercial apple juices available on the market in the South of Brazil. The method performance characteristics were determined using a sample obtained in a local market fortified at five concentration levels of patulin and done in triplicates. The coefficient of variation for repeatability at the fortification level of 20.70µg.L-1 was 3.53 % and the recovery 94.63 %, respectively. The correlation coefficient was 0.9996 and agrees with the requirements for a linear analytical method value. The detection limit was 0.21µg.L-1 and the quantification limit 0.70 µg.L-1. Only three of the analyzed samples were upper the allowed level of 50.00 µg.L-1 recommended for the World Health Organization.

Interlaboratory Trial on the Analysis of Alkylphenols, Alkylphenol Ethoxylates, and Bisphenol A in Water Samples According to ISO/CD 18857-2

ISO/CD 18857-2 (International Organization for Standardization, Geneva) describes a new international standard method for the determination of octylphenol, nonylphenol, their mono- and diethyoxylates, and bisphenol A in nonfiltered samples of drinking, ground, surface, and wastewater. The method is based on the extraction of the analytes from an acidified water sample by solid phase extraction, solvent elution, derivatization, and determination by gas chromatography with mass spectrometric detection. For validation of this method, 14 laboratories from 4 different countries in Europe and Canada participated in an interlaboratory trial to determine the performance characteristics of the method, which are intended for publication in the corresponding standard. The interlaboratory trial was evaluated according to ISO 5725-2 and included two duplicate nonfiltered water samples: surface water containing the target compounds in an analyte concentration range from 0.05 to 0.4 microg/L and wastewater containing the target compounds in a concentration ranged from 0.1 to 5 microg/L. The repeatability variation coefficients (within-laboratory precision) varied for all samples and compounds between 1.9 and 7.8%, showing a sufficiently high repeatability of the method. The reproducibility variation coefficients (between-laboratory precision) were found to vary within a satisfactory range of 10.0-29.5% for surface water and 10.8-22.5% for wastewater. The recoveries as a measure of accuracy varied from 98.0 to 144.1% for surface water and from 95.4 to 108.6% for wastewater. The determined concentrations of the samples compared well to the "true" values, thus showing very satisfactory accuracy of the method. In the chromatogram of the surface water sample, a high unresolved background made up of coextractable matrix compounds was apparent. It is conceivable that compounds from this background may be responsible for enhanced recoveries of 144.1% for 4-nonylphenol (mixture of isomers) and of 123.4% for 4-nonylphenol monoethoxylate (mixture of isomers) in the surface water samples. The isotope-marked standard compounds developed in this context proved to be reliable internal standards that allow a precise and accurate quantitation of all compounds specified in ISO/CD 18857-2. The results of the interlaboratory trial confirmed that the analytical method is robust and reliable and can be used as a standard method to analyze the target compounds in water samples.

Caffeine biomonitoring: using LC‐MS/MS to estimate caffeine exposure in the U.S. population

Caffeine is a psycho‐stimulatory plant alkaloid commonly encountered in the diet. Food frequency reporting is most often used for the epidemiological assessment of caffeine consumption. Biomarker measurement has been proposed as a means of assessing caffeine exposure but is not widely used at present. Our laboratory has developed an isotope‐dilution LC‐MS/MS analysis method that will be used to develop reference ranges for urinary caffeine and its metabolites for the 2009‐2010 National Health and Nutritional Examination Survey (NHANES). These ranges will provide biomarker estimates of caffeine consumption and may also be used to estimate the prevalence of several detoxification enzyme activity classifications (i.e., phenotypes) in the U.S. population. The method is capable of measuring caffeine and all its anticipated metabolites in urine at sub‐micromolar detection limits. Metabolites proposed for assessing caffeine exposure or enzyme activity phenotyping could be quantified in >99% of samples in a random subset. Between‐run coefficients of variation (CVs) were generally <10% for these compounds at concentrations at or above the subset 10th percentile, and within‐run repeatability CVs were <3%. In this paper we present and discuss the analytical performance of our method in the perspective of population biomonitoring studies such as NHANES.

Optical Scanner for Immunoassays With Up-Converting Phosphorescent Labels

A 2-D optical scanner was developed for the imaging and quantification of up-converting phosphor (UCP) labels in immunoassays. With resolution better than 500 microm, a scan rate of 0.4 mm/s, and a 1-2% coefficient of variation for repeatability, this scanner achieved a detection limit of fewer than 100 UCP particles in an 8.8. x 10(4) microm(2) area and a dynamic range that covered more than three orders of magnitude. Utilizing this scanner, a microfluidic chip immunoassay for the cytokine interferon-gamma (IFN-gamma) was developed: concentrations as low as 3 pM (50 pg/mL) were detected from 100 microL samples with a total assay time of under an hour, including the 8 min readout. For this UCP-based assay, 2-D images of the capture antibody lines were scanned, image processing techniques were employed to extract the UCP emission signals, a response curve that spanned 3-600 pM IFN-gamma was generated, and a five-parameter logistic mathematical model was fitted to the data for determination of unknown IFN-gamma concentrations. Relative to common single-point or 1-D scanning optical measurements, our results suggest that a simple 2-D imaging system can speed assay development, reduce errors, and improve accuracy by characterizing the spatial distribution and uniformity of surface-captured optical labels as a function of assay conditions and device parameters.

Removing nasal valve obstruction in peak nasal inspiratory flow measurement

Peak nasal inspiratory flow (PNIF) measurements are used to evaluate nasal obstruction and as a surrogate for disease activity in allergic rhinitis and other nasal complaints. This measurement can give erroneous results when forced inspiration leads to nasal valve collapse. To determine the effects of 2 different nasal stents (Sinuscone and Nozovent) on valve collapse and repeatability of PNIF measurements. Repeated measurements of PNIF were obtained in 74 individuals with and without 2 different nasal stents: Sinuscone and Nozovent. The mean (95% confidence interval) improvement in PNIF was 1.7 L/min (-2.4 to 5.8 L/min; P = .42) with Nozovents and 25.4 L/min (11.4 to 39.4 L/min; P = .001) with Sinuscones. The PNIF coefficient of variation for repeatability was 11.6% without stents, 16.0% using Nozovents, and 10.4% using Sinuscones. Sinuscones, but not Nozovents, significantly improved PNIF. Repeatability of PNIF measurements was worse with Nozovents and only marginally improved with Sinuscones.

Accuracy, repeatability, and reproducibility of Artemis very high-frequency digital ultrasound arc-scan lateral dimension measurements

To determine the accuracy, repeatability, and reproducibility of measurement of lateral dimensions using the Artemis (Ultralink LLC) very high-frequency (VHF) digital ultrasound (US) arc scanner. London Vision Clinic, London, United Kingdom. A test object was measured first with a micrometer and then with the Artemis arc scanner. Five sets of 10 consecutive B-scans of the test object were performed with the scanner. The test object was removed from the system between each scan set. One expert observer and one newly trained observer separately measured the lateral dimension of the test object. Two-factor analysis of variance was performed. The accuracy was calculated as the average bias of the scan set averages. The repeatability and reproducibility coefficients were calculated. The coefficient of variation (CV) was calculated for repeatability and reproducibility. The test object was measured to be 10.80 mm wide. The mean lateral dimension bias was 0.00 mm. The repeatability coefficient was 0.114 mm. The reproducibility coefficient was 0.026 mm. The repeatability CV was 0.38%, and the reproducibility CV was 0.09%. There was no statistically significant variation between observers (P = .0965). There was a statistically significant variation between scan sets (P = .0036) attributed to minor vertical changes in the alignment of the test object between consecutive scan sets. The Artemis VHF digital US arc scanner obtained accurate, repeatable, and reproducible measurements of lateral dimensions of the size commonly found in the anterior segment.

Effects of intranasal corticosteroid on nasal adenosine monophosphate challenge in persistent allergic rhinitis*

Response to a single dose nasal adenosine monophosphate challenge has been used as a surrogate inflammatory marker for allergic rhinitis. Attenuation of response following intranasal corticosteroid would further validate the challenge. To assess the effect of 4 weeks of 200 mcg once daily mometasone furoate nasal spray on a simplified (single 160 mg dose) nasal adenosine monophosphate challenge. Twenty participants with persistent allergic rhinitis completed a double blind placebo-controlled crossover study. Outcome measures were the peak nasal inspiratory flow and total nasal symptoms score responses to nasal adenosine monophosphate challenge, as well as domiciliary peak nasal inspiratory flow and patient symptom diary cards. Mometasone significantly (P < 0.05) attenuated response time profiles vs. placebo for peak nasal inspiratory flow but not total nasal symptom scores. For the maximum percentage fall this amounted to a mean difference of 9.6% (95% confidence interval 1.3-17.9%). The coefficient of variation for repeatability was 48.7%. Improvements were seen in prechallenge and domiciliary measurements of peak nasal inspiratory flow (both P < 0.05) and total nasal symptom scores (both P < 0.01). Mometasone attenuates the peak nasal inspiratory flow response to a single 160 mg nasal adenosine monophosphate challenge. Such challenges have been shown to be sensitive to the effects of antihistamines, antileukotrienes and now nasal steroids. This further supports their application as surrogate inflammatory markers for therapeutic trials in allergic rhinitis, potentially as 20 min challenges which can be conducted in a non-hospital setting.

Marked Interindividual Variability in the Response to Selective Inhibitors of Cyclooxygenase-2

Variability in response to drugs may influence both efficacy and safety. Cyclooxygenase (COX)-2 inhibitors pose a cardiovascular risk by potentially increasing the likelihood of thrombosis, hypertension, and atherogenesis. Differences between individuals in the response to COX-2 inhibitors would be expected to influence their susceptibility to cardiovascular complications. We examined the variability in degree and selectivity of COX-2 inhibition in humans in response to celecoxib and rofecoxib. Fifty healthy volunteers received placebo, rofecoxib (25 mg), and celecoxib (200 mg), randomized by order. COX-1 and COX-2 inhibition was determined using ex vivo and in vivo indices of enzymatic activity. A subset of 5 individuals underwent 5 replicate studies to estimate variability in drug response both within and between subjects. Despite the higher COX-2 selectivity of rofecoxib in vitro, the average selectivity attained by 25 mg rofecoxib and 200 mg celecoxib in vivo were not different. However, there was considerable variability at an individual level in the degree of COX-2 inhibition and selectivity attained by both drugs. Approximately one third of the variability was attributable to differences between individuals, suggesting the contribution of genetic sources of variance, such as candidate polymorphisms detected in COX-1 and CYP2C9. The actual degree of selectivity for inhibition of COX-2 achieved by the coxibs relates both to chemical properties of the drug and to factors within an individual that modulate drug response. These sources of variability might be exploited to identify patients uniquely susceptible to benefit or at developing risk of cardiovascular complications.

Statistics of Assay Validation in High Throughput Cell Imaging of Nuclear Factor κB Nuclear Translocation

This report describes statistical validation methods implemented on assay data for inhibition of subcellular redistribution of nuclear factor kappaB (NF kappaB) in HeLa cells. We quantified cellular inhibition of cytoplasmic-nuclear translocation of NF kappaB in response to a range of concentrations of interleukin-1 (IL-1) receptor antagonist in the presence of IL-1alpha using eight replicate rows in each four 96-well plates scanned five times on each of 2 days. Translocation was measured as the fractional localized intensity of the nucleus (FLIN), an implementation of our more general fractional localized intensity of the compartments (FLIC), which analyzes whole compartments in the context of the entire cell. The NF kappaB antagonist assay (inhibition of IL-1- induced NF kappaB translocation) data were collected on a Q3DM (San Diego, CA) EIDAQtrade mark 100 high throughput microscopy system. [In 2003, Q3DM was purchased by Beckman Coulter Inc. (Fullerton, CA), which released the IC 100 successor to the EIDAQ 100.] The generalized FLIC method is described along with two-point (minimum-maximum) and multiple point titration statistical methods. As a ratio of compartment intensities that tend to change proportionally, FLIN was resistant to photobleaching errors. Two-point minimum-maximum statistical analyses yielded the following: a Z' of 0.174 with the data as n = 320 independent well samples; Z' by row data in a range of 0.393-0.933, with a mean of 0.766; by-plate Z' data of 0.310, 0.443, 0.545, and 0.794; and by-plate means of columns Z' data of 0.879, 0.927, 0.945, and 0.963. The mean 50% inhibitory concentration (IC50) for IL-1 receptor antagonist over all experiments was 213 ng/ml. The combined IC50 coefficients of variation (CVs) were 0.74%, 0.85%, 2.09%, and 2.52% for the four plates. Repeatability IC50 CVs were as follows: day to day 3.0%, row to row 8.0%, plate to plate 2.8%, and day to day 0.6%. The number of cells required for statistically resolvable differences in dose concentrations, plotted in a family of FLIN sigma/deltamicro (SD/range) curves and tabulated, demonstrated cell-by-cell assay precision with our combined sigma/deltamicro = 0.32 that required approximately 10-fold fewer cells than in a previously reported NF kappaB assay with sigma/deltamicro = 1.52. To better understand the relationship between cell-by-cell measurements and IC50 precision, 500 Monte Carlo simulations with varying cell-measurement SDs were used to explore three-, five-, seven-, and 11-point model titrations. The reductions in deltaIC50 90% confidence intervals from 11- to three-point titrations were 10-fold with the previously reported sigma/deltamicro = 1.52 and twofold with our sigma/deltamicro = 0.32. With these normalized parameters, this report provides a common statistical foundation, independent of the assay details, for evaluating the performance of imaging data on any instrument.

Quantification of venlafaxine and O-desmethylvenlafaxine in human serum using HPLC analysis

We describe an isocratic reversed-phase liquid chromatographic method for the determination of venlafaxine (VLX) and its main active metabolite O-desmethylvenlafaxine (ODV) in serum, using haloperidol as internal standard and liquid/liquid extraction for sample preparation. VLX and ODV were separated on a C18 column with a mobile phase of acetonitrile/buffer (30/70, v:v) at 60 degrees C and a flow rate of 1.5 ml/min. The measurement of the native fluorescence signals of the eluted compounds were carried out at 227/300 nm (excitation/emission) without interference from endogenous components in serum. High linearities for VLX and ODV for concentrations between 20 and 500 microg/l were obtained (r = 0.9997). A large spectrum of routinely prescribed drugs did not interfere in the assay. The coefficients of variation for repeatability varied between 5.40% and 5.99% and for reproducibility between 9.43% and 21.63%. Absolute recoveries were more than 52% for both substances.

Simultaneous determination of the tobacco smoke uptake parameters nicotine, cotinine and thiocyanate in urine, saliva and hair, using gas chromatography-mass spectrometry for characterisation of smoking status of recently exposed subjects.

A method using gas chromatography (GC)-mass spectrometry (MS) for the simultaneous determination of the smoke uptake parameters thiocyanate, nicotine and cotinine in human tissues is reported. Nicotine, cotinine and thiocyanate, in combination with a phase-transfer catalyst, were extracted from urine, saliva and hair into dichloromethane (DCM). Thiocyanate was alkylated in the DCM-layer to form a pentafluorobenzyl derivative. The biochemical markers in DCM were directly injected into the GC system and separated on a DB-1MS column using a 9.4 min temperature program. The method was validated in urine and saliva between the limits of quantitation (1.0-15 microg ml(-1) thiocyanate, 0.010-3.0 microg ml(-1) nicotine and cotinine in urine, 0.010-1.0 microg ml(-1) nicotine and cotinine in saliva). The calibration curves were found to be linear (r > 0.996), the within- and between-day accuracy's were 83-120%, the repeatability coefficients of variation were 3-20% and the limits of detection were 0.060 ng ml(-1) thiocyanate and 0.60 ng ml(-1) nicotine and cotinine. The results of the analysis of the biomarkers in the urine of 44 volunteers were used to develop a predictive model for smoking status, using discriminant analysis. The classification model correctly classified 93.2% of cross-validated grouped cases. Saliva samples were used to confirm the results of the classification method.

Direct determination of alkyl phosphates in human urine by liquid chromatography/electrospray tandem mass spectrometry.

A novel and rapid procedure based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of dialkyl phosphates (metabolites of organophosphorus pesticides) in human urine has been developed. After addition of 40 mM tetrabutylammonium acetate, 10 microL of urine sample were directly injected into the LC/MS/MS system. The method was validated yielding calibration curves with correlation coefficients greater than 0.997 and repeatability coefficient of variation (CV) lower than 9%. The accuracy and precision were evaluated by direct injection of spiked samples at 10 and 100 microg/L obtaining recoveries between 78 and 119% with coefficients of variation below 12%. Limits of detection of 1 microg/L for diethyl phosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP) and 2 microg/L for dimethyldithiophosphate (DMDTP) were achieved, all the analytes being detected in negative ion mode. The fragmentation pathway of dialkyl phosphates allowed us the use of an additional transition for confirmation in order to improve their identification in real-world samples. The applicability of the LC/MS/MS method was demonstrated by applying it to the analysis of urine samples of farmers exposed to the organophosphorus pesticide chlorpyrifos. Good correlation between application of the product in the field (citrus orchards), concentration levels of dialkyl phosphates and levels of the chlorpyrifos-specific metabolite (1,3,5-trichloro-2-pyridinol) was obtained.

魚油中のドコサヘキサエン酸およびｎ‐３脂肪酸の高分解能プロトン核磁気共鳴スペクトロスコピーによる定量分析 日本油化学会ワーキンググループ（ＤＨＡ／ＮＭＲ小委員会）による合同研究

Seven Japanese laboratories provided with high resolution nuclear magnetic resonance (NMR) equipment undertook a collaborative study for determining docosahexaenoic acid (DHA) and n-3 fatty acids in fish oils with NMR. This study was organized by the DHA/NMR subcommittee of the Japan Oil Chemists’ Society and conducted as a part of an IUPAC collaborative study (Project 3/98 of the commission VI-6 on Oils, Fats, and Derivatives). At each laboratory, examination was made of six blind duplicate fish oils, as follows; refined tuna oil, two extracted tuna oils, extracted bonito oil, extracted salmon oil and extracted sardine oil 9 to 30 mol% DHA and 20 to 35 mol% n-3 fatty acids. To ensure maximum accuracy of determination, signal area measurement and addition of the internal standard solution were taken as critical control points. Repeatability coefficient of variation (CVr) ranged from 0.8 to 2.6% and reproducibility coefficient of variation (CVR) ranged from 1.4 to 3.3% for DHA weight concentration (mg/g); CVr from 0.4 to 2.2% and CVR from 0.7 to 3.1% for DHA (mol%); CVr from 0.2 to 1.1% and CVR from 1.1 to 2.2% for total n-3 fatty acids (mol%). The present method was thus shown to qualify for use as a tentative official method of the Japan Oil Chemists’ Society.

Characterisation of the staphylococcal enterotoxin research method in a dairy product

The staphylococcal enterotoxin research method is a quantitative method, involving a sample preparation and an imunoenzymatic detection. In order to estimate its performance characteristics, a method characterisation is carried out with a single dairy product and for staphyloccal enterotoxin A. Statistical analysis shows that the method is linear over a contamination interval between 0 - 1 ng SEA per gram of cheese. The limit of detection is about 0.01 ng SEA per gram of cheese and the limit of quantification is about 0.04 ng SEA per gram of cheese. The intra laboratory repeatability and intermediate precision are estimated on two levels of contamination. The repeatability coefficient of variation is included between 3.1 % and 6.2 %, the intermediate precision coefficient of variation is included between 4.7 % and 11.0 %.

Image analysis of scaly skin using Dsquame® samplers: technical and physiological validation

An image analysis-based technique for evaluating skin scaliness using Dsquame(R) instant testers is presented, with four surface texture parameters characterizing this condition. Studies on the variability of the technique were carried out to determine the influence of instrumental and physiological factors. Parameters of texture, optical density, number and surface area of squames are calculated from images of obliquely lit Dsquame(R) samples. Repeated measurements were done on artificial and real samples to determine instrumental and sample variability, respectively. In addition, the technique was used in a volunteer study to assess anatomical differences and seasonal variation. For five consecutive measurement days, the artificial samples remained stable, with coefficients of variation for repeatability and reproducibility of less than 8% for all parameters. During the same period, the real samples showed decreases mainly in those parameters of texture and optical density. In the volunteer study, the calf showed significantly higher values (more scaly) in December than July, with no seasonal change in forearm or crow's foot area sites. Assessments of Dsquame(R) instant tester samples using image analysis-derived parameters provides information on the quantity and quality of skin scaliness or flakiness. As such, it is a useful tool for studying desquamation. The technique presented is repeatable and reproducible; however, samples need to be analysed the same day to avoid deterioration problems. Anatomical differences have been demonstrated, with, in addition, the calf showing marked winter/summer changes in comparison to the face and forearm.