Coefficients Of Variation For Reproducibility Research Articles

A reliable enzyme-linked immunosorbent assay for the determining of sesame proteins in raw food ingredients and in processed foods

Sesame is a frequent cause of adverse food reactions in allergic patients. We developed a novel sandwich enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies and a unique extraction buffer for the detection and quantification of sesame proteins in processed foods and in raw food ingredients to clarify the validity of sesame labeling and for precautionary allergen labeling. The developed sandwich ELISA method is highly specific for sesame proteins. The limit of detection (LOD) and limit of quantification (LOQ) are 0.013 µg/g and 0.025 µg/g, respectively. The recoveries for incurred food samples, such as dressing, breads, sauce and pudding, ranged from 67 % to 81 %, while the repeatability and reproducibility coefficients of variation were less than 4.7 % and 4.5 %, respectively. The developed method has applicability for food products and is a reliable tool for the detection of hidden sesame proteins in raw food ingredients and in processed foods.

Simultaneous determination of oxacillin and cloxacillin in plasma and CSF using turbulent flow liquid chromatography coupled to high-resolution mass spectrometry: Application in therapeutic drug monitoring

Cloxacillin and oxacillin are group M penicillins. The therapeutic monitoring of plasma concentrations of these antibiotics and those of their hydroxymethylated metabolites is of great clinical interest, especially in the choice of an adequate dosage allowing an effective treatment while limiting the occurrence of undesirable effects and the development of bacterial resistance. In this context, we conducted this work aiming at developing and validating a method allowing the determination of cloxacillin and oxacillin as well as the identification of their active metabolites in different biological matrices (CSF and plasma) using turbulent flow liquid chromatography coupled to high-resolution mass spectrometry. To do this, we carried out several optimisation tests. Subsequently, we validated our method according to the latest bioanalytical validation recommendations of the European Medicines Agency. The validation results showed that our method is specific and sensitive. We obtained good linearity in the range 0.5 to 100 µg/mL with correlation coefficients above 0.995. The lower limit of quantification was 0.5 µg/mL for each analyte. The method was found to be accurate with repeatability and reproducibility coefficients of variation below 15 %. Our method is also accurate with bias values below 15 %. Recovery values ranged from 87 % to 95 %. Finally, we were able to apply our method to the therapeutic monitoring of the analysed molecules and to identify their active metabolites. Our results suggest that LC-MS shows superiority in the therapeutic monitoring of these antibiotics due to the superiority of specificity shown by this method. This assay method can be routinely used for the daily plasma assays of patients treated with these antibiotics in the context of therapeutic monitoring.

Agreement, repeatability, and reproducibility of quantitative retinal layer assessment using swept-source and spectral-domain optical coherence tomography in eyes with retinal diseases.

To evaluate the agreement and precision of retinal thickness measurements obtained using swept-source optical coherence tomography (SS-OCT) and spectral-domain OCT (SD-OCT) in healthy eyes and eyes with retinopathy. This cross-sectional prospective study involved three DRI-OCT Triton (SS-OCT) and three 3D-OCT-1 Maestro (SD-OCT) devices. One of each device (Maestro and Triton) was paired with a single operator. Healthy subjects and patients with retinal diseases were recruited, with study eye and testing order randomized. At least 3 scans per eye were captured for wide scan (12 mm × 9 mm-Triton and Maestro) and macular cube scan (7 mm × 7 mm-Triton, 6 mm × 6 mm-Maestro). Thickness of the full retina, ganglion cell layer + inner plexiform layer (GCL+), and ganglion cell complex (GCL++) were obtained from wide scan and cube scans. Agreement of the measurements between the Triton and Maestro was evaluated by Bland-Altman analysis and Deming regression for each group. Repeatability and reproducibility were assessed using a two-way random effect analysis of variance (ANOVA) model for each parameter by group. Twenty-five healthy subjects (25 eyes) and 26 patients with retinal diseases (26 eyes), including, but not limited to, age-related macular degeneration, macular hole, and diabetic retinopathy were recruited. Overall, the measurement differences between Triton and Maestro were <6 μm (mean differences of full retina, GCL++, and GCL+ thickness were ≤5.5 μm, 1.3 μm, and 2.8 μm, respectively) and not statistically significant across the parameters. The repeatability and reproducibility estimates indicate high precision in both devices and groups. Across all the parameters, the repeatability limit was ≤7.6 μm for Triton and ≤12.7 μm for Maestro; reproducibility limit was ≤9.2 μm for Triton and ≤14.4 μm for Maestro. In eyes with retinal pathology, the repeatability coefficient of variation (CV)% was ≤2.6% for Triton and ≤3.4% for Maestro; reproducibility CV% was ≤3.3% for Triton and ≤3.5% for Maestro. Both Triton SS-OCT and Maestro SD-OCT provide reliable measurements of retinal thickness in healthy eyes and eyes with retinal diseases. Excellent agreement between the two devices indicates interoperability when testing healthy eyes or eyes with retinal pathology. These findings support the use of thickness measurements from Triton SS-OCT and Maestro SD-OCT in clinical practice.

Capillary electrophoresis in parenteral nutrition control – validation of two analytical methods: Amino acids/glucose/glucose-1-phosphate and K/Na/Ca/Mg

ObjectivesProduction of parenteral nutrition bags (PNBs) involves many nutrients: complete control of the production process decreases the risk of error. This study aimed to develop and validate two analytical methods...

Inter-laboratory validation of liquid chromatography-tandem mass spectrometry multi-mycotoxin determination in animal feed - method transfer from the reference laboratory to regional laboratories.

The results are presented of the inter-laboratory validation of a liquid chromatography-tandem mass spectrometry method for the determination of eight mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1, fumonisin B2, ochratoxin A, toxin T-2, toxin HT-2 and zearalenone) in animal feeds. This study was an essential part of the method's transfer from the National Reference Laboratory to six regional laboratories in Poland working in the official survey of mycotoxins in feed. The laboratories received a batch of standard solutions, blank samples and quality control materials on which to perform analysis with one procedure and different liquid chromatography-tandem mass spectrometry conditions. The validation results show good precision (reproducibility coefficient of variation 3.7-20.5%) and accuracy of the method (recovery 89-120% and trueness 94-103%) and sufficient skills of the laboratory personnel. The study is an example of the successful transfer of the method among laboratories.

Simultaneous real-time PCR detection of nine prevalent sexually transmitted infections using a predesigned double-quenched TaqMan probe panel.

Sexually transmitted diseases are major causes of infertility, ectopic pregnancy, and premature birth. Here, we developed a new multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of nine major sexually transmitted infections (STIs) found in Vietnamese women, including Chlamydia trachomatis, Neisseria gonorrhoeae, Gardnerella vaginalis, Trichomonas vaginalis, Candida albicans, Mycoplasma hominis, Mycoplasma genitalium, and human alphaherpesviruses 1 and 2. A panel containing three tubes × three pathogens/tube was predesigned based on double-quenched TaqMan probes to increase detection sensitivity. There was no cross-reactivity among the nine STIs and other non-targeted microorganisms. Depending on each pathogen, the agreement with commercial kits, sensitivity, specificity, repeatability and reproducibility coefficient of variation (CV), and limit of detection of the developed real-time PCR assay were 99.0%-100%, 92.9%-100%, 100%, <3%, and 8-58 copies/reaction, respectively. One assay cost only 2.34 USD. Application of the assay for the detection of the nine STIs in 535 vaginal swab samples collected from women in Vietnam yielded 532 positive cases (99.44%). Among the positive samples, 37.76% had one pathogen, with G. vaginalis (33.83%) as the most prevalent; 46.36% had two pathogens, with G. vaginalis + C. albicans as the most prevalent combination (38.13%); and 11.78%, 2.99%, and 0.56% had three, four, and five pathogens, respectively. In conclusion, the developed assay represents a sensitive and cost-effective molecular diagnostic tool for the detection of major STIs in Vietnam and is a model for the development of panel detections of common STIs in other countries.

Rapid, Low-Complexity, Simultaneous Bacterial Group Identification and Antimicrobial Susceptibility Testing Performed Directly on Positive Blood Culture Bottles Using Chromogenic Agar.

The use of positive blood culture bottles for direct disk diffusion susceptibility testing (dDD), together with chromogenic culture limited to groups of pathogens for antimicrobial susceptibility testing interpretation may provide a means for laboratories-in-development to introduce rapid abbreviated blood culture testing. We assessed the performance of dDD on Chromatic MH agar using contrived positive blood culture bottles and compared findings with current standard practice. Furthermore, we characterized the growth of 24 bacterial and 3 yeast species on Chromatic MH agar with the aid of rapid spot tests for same-day identification. The coefficient of variation for reproducibility of dDD of four reference strains in 4 to 10 replicates (238 data points) ranged from 0% to 16.3%. Together with an additional 10 challenge isolates, the overall categorical agreement was 91.7% (351 data points). The following bacteria were readily identifiable: cream/white Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes; turquoise Streptococcus agalactiae, enterococci, Listeria monocytogenes; mauve Escherichia coli, Shigella sonnei, Citrobacter freundii; dark-blue Klebsiella and Enterobacter; green Pseudomonas aeruginosa; and brown Proteus. Clear colonies were seen with Salmonella, Acinetobacter, Burkholderia, and Yersinia enterocolitica (turns pink). Our study suggests that Chromatic MH for dDD may show promise as a rapid, clinically useful presumptive method for overnight simultaneous identification and antimicrobial susceptibility testing. However, there is a need to optimize the medium formulation to allow the recovery of Streptococcus pneumoniae and Haemophilus influenzae.

Second‐generation fully automated Elecsys cerebrospinal fluid immunoassays demonstrate high precision, reproducibility, and sample stability suitable for clinical use to aid Alzheimer’s disease diagnosis

AbstractBackgroundBeta‐amyloid(1–42) (Abeta42) and phosphorylated tau 181 (pTau) cerebrospinal fluid (CSF) biomarker analysis is recommended alongside cognitive evaluation to aid Alzheimer’s disease (AD) diagnosis. For clinical use, CSF biomarker assays should demonstrate high precision/reproducibility and sample stability. This study evaluated precision, reproducibility, and sample stability of second‐generation fully automated Elecsys® CSF immunoassays.MethodFrozen/pooled CSF and PreciControl samples were analyzed using Elecsys β‐Amyloid(1–42) CSF II/Phospho‐Tau (181P) CSF immunoassays on cobas e 601 (Roche Diagnostics International Ltd). pTau/Abeta42 ratios were calculated. Precision was evaluated at one site per Clinical and Laboratory Standards Institute (CLSI) EP02‐A3 (two runs/day in duplicate over 21 days; n=84). Reproducibility was determined per CLSI EP05‐A3 over five days using a single lot across three sites (site‐to‐site)/three lots at one site (lot‐to‐lot). Coefficients of variation (CVs)/standard deviations (SDs) for repeatability/intermediate precision/reproducibility were calculated using variance component analysis. Sample stability was determined from fresh CSF, collected per manufacturer's instructions. Baseline measurements were taken &lt;6 hours after lumbar puncture and follow‐up measurements (from stored aliquots) at defined intervals for ≤8 days at room temperature (RT; 25°C; n=13), ≤15 days at 6°C (n=14), and ≤13 weeks at ‐20°C (n=25). Storage stability was determined from percent recoveries/ordinary linear regression.ResultRepeatability CVs were &lt;2.4% and &lt;1.9% for Abeta42 and pTau immunoassays, respectively, and &lt;2.4% for pTau/Abeta42 ratio. Intermediate precision CVs were &lt;3.3% and &lt;3.7% for Abeta42 and pTau immunoassays, respectively, and &lt;3.9% for pTau/Abeta42 ratio. Site‐to‐site reproducibility CVs were &lt;5.1% and &lt;3.6% for Abeta42 and pTau immunoassays, respectively, and &lt;6.5% for pTau/Abeta42 ratio (Table 1). For the Abeta42 immunoassay, lot‐to‐lot reproducibility CVs were &lt;5.4%, except for one sample pool (mean concentration: 322 pg/mL; SD: 34.1 pg/mL; Table 2). Lot‐to‐lot reproducibility CVs were &lt;3.6% for the pTau immunoassay and &lt;6.0% for pTau/Abeta42 ratio. At maximum sample storage duration (8 days at RT/15 days at 2−8°C), median recoveries of 95%/98% were observed for Abeta42 and 97%/99% for pTau immunoassays. Stability assessment at ‐20°C is ongoing.ConclusionThe Elecsys immunoassays demonstrated high precision/reproducibility, supporting clinical use in AD diagnosis. Conservative recommendations for sample storage are 5 days at RT/14 days at 2−8°C.

Development of a new procedure for the determination of the reactivity of brandies used in wine fortification

The main purpose of this work was to describe and validate a new methodology for the determination of a brandy reactivity index (BRI) towards flavan-3-ol compounds in wines (catechins and condensed tannins) and to correlate this index with the amount and type of aldehydes present in those brandies. This new method consists of two main reactions: a) the ability of aldehydes to react with C8 and/or C6 positions in the phloroglucinol ring of a catechin present in excess (A ring); b) further reaction of the remaining free catechin with p-dimethylaminocinamaldehyde (DMACA) yielding to the formation of a blue compound that can be quantified by Visible spectroscopy at 640 nm. The impact of different experimental conditions such as reagent concentrations and reaction time on BRI were also evaluated. The method was validated through the determination of repeatability (intra-day variability) and reproducibility. The repeatability was considered acceptable with a CV of 11.87 %. The analysis of the reproducibility variance, S2R (11.59), the reproducibility limit R (9.5) and the reproducibility coefficient of variation, CVR (15.25 %) postulates BRI methodology to be reliable and robust.Using the developed methodology, the BRI of fourteen different commercial brandies and some pure standards aldehydes were determined. The aldehydes present in brandies, and the content of total aldehydes were determined by GC-MS and then correlated with the BRI. In general, it was observed a direct correlation between the BRI and the concentration of total aldehydes, in particular acetaldehyde.

Myocardial fibrosis quantification methods by cardiovascular magnetic resonance imaging with contrast-enhancement in patients with Fabry disease

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): National Agency for Academic Exchange (NAWA). Background. Presence and extent of late gadolinium enhancement (LGE) in patients with Fabry disease (FD) is a predictor of adverse cardiac events. However there is no gold standard method to quantify the amount of myocardial fibrosis in cardiac magnetic resonance imaging (CMR). Purpose. The aim of this study was to establish the most reliable and reproducible technique for quantifying LGE in patients with FD. Methods. 68 patients with FD (40% male, 40 ± 16 years old) treated in our outpatient clinic, who underwent CMR with administration of 0.2 mmol of gadobutrol per kilogram of body weight between December 2012 and March 2019 were enrolled into the study. Presence of LGE was described in 25 patients (37%). Twenty patients underwent CMR with the same LGE sequence (Philips 1.5 Tesla, two-dimensional multi-breath-hold inversion recovery sequence) and were enrolled into further analysis. LGE quantifications were performed using gray-scale thresholds with 2, 3, 4, 5 and 6 standard deviations (SD) above the mean signal intensity for the remote myocardial tissue, full width at half maximum method (FWHM), visual assessment with threshold (VAT) and fully manual method (MM; Philips IntelliSpace Portal 10). LGE quantifications were done twice by the same observer in different time periods and once by another observer. Intraclass correlation coefficient (ICC), Bland- Altman analysis and coefficient of variation (CV) were used to assess intra- and interobserver reliability and reproducibility. Results. The mean quantity of fibrosis mass (in gram) in all studied patients was: 35.5 ± 18.7 at 2SD, 21.0 ± 12.8 at 3SD, 12.7 ± 8.5 at 4SD, 8.0 ± 5.7 at 5SD, 5.3 ± 4.1 at 6SD, 1.9 ± 1.8 at FWHM, 8.6 ± 7.4 at VAT and 9.1 ± 6.1 at MM. Intra-observer reliability of almost all studied LGE quantification methods was excellent, with a range of ICCs from 0.90 for 6SD to 0.95 for VAT, with one exception for FWHM, which had good intraobserver reliability (ICC 0.84; all P &amp;lt; 0.05). Interobserver reliability was excellent for VAT (ICC 0.94) and good for all other LGE quantifications methods (range of ICCs from 0.76 for MM to 0.87 for 5SD, all P &amp;lt; 0.05). 5SD had the lowest CV (6%) for intraobserver reproducibility and 2SD and VAT for interobserver reproducibility (35% and 38%). FWHM had the highest CV for both intra- and interobserver reproducibility (63% and 94%, accordingly). Conclusions 1. All studied methods of LGE quantification in patients with FD and presence of myocardial fibrosis have good to excellent intra- and interobserver reliability. 2. The total amount of LGE differs in studied LGE quantifications methods. Therefore in clinical practice it is important to report which technique of LGE quantification was used and choose the same for CMR-follow up. 3. FWHM might be avoided for LGE quantification in patients with FD due to the highest intra- and interobserver variability in comparison to other available techniques.

Quantitative MRI: Defining repeatability, reproducibility and accuracy for prostate cancer imaging biomarker development

IntroductionQuantitative MRI (qMRI) parameters have been increasingly used to develop predictive models to accurately monitor treatment response in prostate cancer after radiotherapy. To reliably detect changes in signal due to treatment response, predictive models require qMRI parameters with high repeatability and reproducibility. The purpose of this study was to measure qMRI parameter uncertainties in both commercial and in-house developed phantoms to guide the development of robust predictive models for monitoring treatment response. Materials and methodsADC, T1, and R2* values were acquired across three 3 T scanners with a prostate-specific qMRI protocol using the NIST/ISMRM system phantom, RSNA/NIST diffusion phantom, and an in-house phantom. A B1 field map was acquired to correct for flip angle inhomogeneity in T1 maps. All sequences were repeated in each scan to assess within-session repeatability. Weekly scans were acquired on one scanner for three months with the in-house phantom. Between-session repeatability was measured with test-retest scans 6-months apart on all scanners with all phantoms. Accuracy, defined as percentage deviation from reference value for ADC and T1, was evaluated using the system and diffusion phantoms. Repeatability and reproducibility coefficients of variation (%CV) were calculated for all qMRI parameters on all phantoms. ResultsOverall, repeatability CV of ADC was <2.40%, reproducibility CV was <3.98%, and accuracy ranged between −8.0% to 2.7% across all scanners. Applying B1 correction on T1 measurements significantly improved the repeatability and reproducibility (p<0.05) but increased error in accuracy (p<0.001). Repeatability and reproducibility of R2* was <4.5% and <7.3% respectively in the system phantom across all scanners. ConclusionRepeatability, reproducibility, and accuracy in qMRI parameters from a prostate-specific protocol was estimated using both commercial and in-house phantoms. Results from this work will be used to identify robust qMRI parameters for use in the development of predictive models to longitudinally monitor treatment response for prostate cancer in current and future clinical trials.

Volume and surface area of Holstein dairy cows calculated from complete 3D shapes acquired using a high-precision scanning system: Interest for body weight estimation

Three-dimensional (3D) imaging is a solution for monitoring morphology and growth of dairy cows, but it can also estimate indicators such as body volume, surface area and body weight. A 3D full-body scanning device was used to scan 64 lactating Holstein cows from March-June 2018. The cows were individually and automatically weighed at a static weighing station (mean ± standard deviation = 673 ± 65 kg). These measured weights were compared to those predicted from regression models based on volume, area or morphological traits determined from 177 3D images. Since some images were truncated due to cow movement or technical problems, we developed additional regression models to reconstruct total volume or area. The accuracy of volume and area measurements was first tested on an inert cylindrical form (coefficients of variation (CVs) < 0.72%). The CVs for repeatability and reproducibility of the method of calculating volume and area from truncated images were 0.17% and 3.12%, respectively. Cow volume and area ranged from 0.61 to 0.96 m3 and 5.80 to 8.32 m2 respectively. Five regression models were developed to estimate cow body weight. Their coefficients of determination ranged from 0.82 to 0.93 with prediction errors of ca. 3% (20 kg) and 4% (29 kg) as a function of volume and area, respectively. The device and the method, evaluated and validated in this study, offer the possibility to use new indicators such as body volume and area in precision livestock farming.

Repeatability and reproducibility of longitudinal relaxation rate in 12 small-animal MRI systems

BackgroundMany translational MR biomarkers derive from measurements of the water proton longitudinal relaxation rate R1, but evidence for between-site reproducibility of R1 in small-animal MRI is lacking. ObjectiveTo assess R1 repeatability and multi-site reproducibility in phantoms for preclinical MRI. MethodsR1 was measured by saturation recovery in 2% agarose phantoms with five nickel chloride concentrations in 12 magnets at 5 field strengths in 11 centres on two different occasions within 1–13 days. R1 was analysed in three different regions of interest, giving 360 measurements in total. Root-mean-square repeatability and reproducibility coefficients of variation (CoV) were calculated. Propagation of reproducibility errors into 21 translational MR measurements and biomarkers was estimated. Relaxivities were calculated. Dynamic signal stability was also measured. ResultsCoV for day-to-day repeatability (N = 180 regions of interest) was 2.34% and for between-centre reproducibility (N = 9 centres) was 1.43%. Mostly, these do not propagate to biologically significant between-centre error, although a few R1-based MR biomarkers were found to be quite sensitive even to such small errors in R1, notably in myocardial fibrosis, in white matter, and in oxygen-enhanced MRI. The relaxivity of aqueous Ni2+ in 2% agarose varied between 0.66 s−1 mM−1 at 3 T and 0.94 s−1 mM−1 at 11.7T. InterpretationWhile several factors affect the reproducibility of R1-based MR biomarkers measured preclinically, between-centre propagation of errors arising from intrinsic equipment irreproducibility should in most cases be small. However, in a few specific cases exceptional efforts might be required to ensure R1-reproducibility.

Establishment of glycated albumin unit conversion equation from the standardized value (mmol/mol) to the routinely used value (%).

To promote glycated albumin standardization, the Committee on Diabetes Mellitus Indices, Japan Society of Clinical Chemistry published the recommended reference method and is supplying the reference standard material (JCCRM 611). In this study, we evaluated the basic performance of 'standardized enzymatic method' that is traceable to JCCRM 611 and established a unit conversion formula from standardized glycated albumin value (mmol/mol) to the routinely used glycated albumin value (%). To evaluate the accuracy and within-run reproducibility of the 'standardized enzymatic method', JCCRM 611 was measured 20 times. To establish the unit conversion formula, serum specimens with known HbA1c concentrations were collected, and their glycated albumin (%) and glycated albumin (mmol/mol) were measured. The accuracy of glycated albumin value of the 'standardized enzymatic method' was 100.8-103.0%. The within-run reproducibility CV (coefficient of variation, %) of glycated albumin value was 0.6-0.9%. In total, 240 serum specimens were collected, and the distribution of their HbA1c values was HbA1c ≤ 4.9% ( n = 23), 5.0-5.9% ( n = 50), 6.0-6.9%, ( n = 44), 7.0-7.9% ( n = 40), 8.0-8.9% ( n = 37) and ≥9.0% ( n = 46). The unit conversion equation was defined as: GA (%) = 0.05652 × GA (mmol/mol)-0.4217 ( r = 0.999). Glycated albumin (%) calculated by the equation was in close agreement (96.6-104.2%) with the actual measured glycated albumin (%). The glycated albumin unit conversion formula was established from mmol/mol to %. This formula provides an easy method to convert the unit and is expected to be useful for the standardization of glycated albumin measurement.

Circulating Myeloid-Derived Suppressor Cell Subsets in Patients with Colorectal Cancer – Exploratory Analysis of Their Biomarker Potential

Myeloid-derived suppressor cells (MDSCs) contribute to tumor escape from host immune surveillance and to tumor progression by producing tumor-promoting factors. We focused on clinical and analytical MDSCs-related issues as potential biomarkers and immune regulators involved in tumor progression. We analyzed 10 patients with advanced colorectal carcinoma (CRC) with (M1 subgroup) or without (M0 subgroup) distant metastases at diagnosis. Peripheral blood was collected at diagnosis prior to treatment and subsequently 12 months after therapy initiation. Using multicolor flow cytometry MDSC subsets were evaluated. Monocytic MDSCs (M-MDSCs) were detected as CD45+ CD11b+ CD33+ HLA-DRlow/ CD14+ CD15-, granulocytic MDSCs (CD33hi PMN-MDSC) were detected as CD45+ CD11b+ CD33hi HLA-DRlow/ CD14 CD15+. For analytical and preanalytical studies, random fresh blood specimens predominantly from cancer patients were analyzed. Levels of circulating M-MDSCs were not associated with metastatic disease within advanced CRC patients. Levels of circulating CD33hi PMN-MDSCs were elevated in patients with distant metastases compared to T3 M0 subgroup. Circulating M-MDSCs increased upon treatment initiation in 9 out of 10 patients. CD33hi PMN-MDSCs substantially dropped upon treatment initiation in 5 out of 10 patients and substantially increased in 2 out of 10 patients. Analytical part showed that absolute and relative counts within each MDSC subset are correlated. Coefficient of variation (CV) for repeatability was 6-11% for M-MDSCs and 25-44% for CD33hi PMN-MDSCs. CV for reproducibility was higher with 8-22% for M-MDSCs and 35-79% for CD33hi PMN-MDSCs demonstrating that delay in measurement of MDSCs in whole blood specimen may distort quantification of circulating MDSC subsets. The quantification of MDSC subsets is substantially dependent on the type of specimen examined and its preanalytical processing. Exploratory analysis of M-MDSCs and CD33hi PMN-MDSCs in CRC patients revealed different dynamics of M-MDSC and CD33hi PMN-MDSC subsets in the context anti-cancer treatment. Key words: myeloid-derived suppressor cells - preanalytics - colorectal cancer - flow cytometry - immune monitoring.

Alcohol Determination of Malt-Based Beverages by Rapid Distillation

ABSTRACTThe objective of the study was to assess a rapid distillation unit as an alternative to standard distillation (Beer-4A). A rapid distillation unit (Vapodest 200 by Gerhardt Analytical Systems) was selected as an alternative for its ability to rapidly distill using the power of steam. The NIR/density method (Beer-4G) is quite suitable for measuring the alcohol content of beer; however, flavored malt beverages present a challenge. Deviations between results by the NIR/density method versus results by distillation can occur due to flavor additions and/or high alcohol content. A more accurate way to measure alcohol in this type of product is to first perform a standard distillation (Beer-4A). Unfortunately, this method is time consuming and labor intensive. A precision study was performed to determine repeatability and reproducibility. Three analysts distilled five malt-based products including both beer and flavored malt beverages with alcohol levels ranging from 4.2 to 14.4% (v/v). Repeatability and reproducibility coefficients of variation for the determination of alcohol for this range were 0.05–0.23% and 0.11–0.19%, respectively. In addition, results from Beer-4A and rapid distillation were assessed using a paired t test for alcohol levels ranging from 3.6 to 14.4% (v/v). The P value of 0.30 indicated that there was not a statistically significant difference between the means of the data sets at a 95% confidence level.

Development of astandard protocol for quantitative polymerase chain reaction to detect adenovirus 36, which is associated with obesity.

It has been previously reported that adenovirus 36 (Ad36) infection is associated with obesity in humans and other animals. However, there is no clinically available standard protocol to detect Ad36 DNA. In this study, we developed amethod for quantitative and rapid detection of Ad36 DNA. Using aTaqMan probe quantitative polymerase chain reaction (qPCR), we identified that the E3 and E4orf1 regions specifically detect Ad36 DNA, because these regions did not show cross reactivity with other types of adenoviruses. The limit of detection was 379 copy/ml and 384 copy/ml for E3 and E4orf1 regions of Ad36, respectively. The %CV (coefficient of variation) for reproducibility of the assay using adenovirus reference material ranged from 1.07-13.02. After we developed the standard protocol to detect Ad36 DNA, we used mouse as asurrogate model to confirm its clinical applicability. We administered Ad36 to mice via intranasal and oral routes, with intraperitoneal administration as the positive control, to analyze the effect of infection route. Ad36 DNA could be detected in lungs, liver, pancreas, and epididymal fat tissue after intraperitoneal injection, whereas it was found only in lungs after intranasal injection. No Ad36 DNA was detectable in any tested organ after oral injection. This indicates that the main route of infection with Ad36 is intranasal, suggesting that Ad36 is arespiratory virus. The standard protocol for qPCR developed in this study is useful for clinical detection of Ad36 DNA. Keywords: adenovirus 36; real-time PCR; obesity.

Reprint of: l-Cysteine determination in embryo cell culture media using Co (II)-phthalocyanine modified disposable screen-printed electrodes

Thiol-containing compounds such as l-cysteine have been demonstrated to play an important role in metabolism and cellular growth, acting as powerful antioxidants. Consequently, their analytical determination in biological media has received a considerable amount of attention. In this work, an electrochemical sensor for the accurate electroanalytical determination of l-cysteine is proposed, based upon a Co(II)-phthalocyanine bulk modified disposable screen-printed graphite electrode (CoPc-SPE). Cyclic (CV) and Square Wave (SWV) voltammetry experiments have demonstrated an excellent electrocatalytic activity towards the electrochemical oxidation of l-cysteine using CoPc-SPEs within optimum neutral or basic pH. Moreover, the SWV response of l-cysteine is found to exhibit a linear range of 2.6–200μM, with a low limit of detection of 4μM (S/N=3) and a sensitivity of 0.78μAcm−2μM−1. Coefficient of variation for reproducibility and repeatability of l-cysteine determination using the CoPc-SPE are 3 and 0.4%, respectively. The effect of inherent interferences such as naturally occurring amino acids, cystine and cysteic acid has been also evaluated. Finally, the applicability of the l-cysteine electrochemical sensor based upon CoPc-SPEs has been successfully demonstrated for the first time for the assessment of l-cysteine in a complex embryo cell culture medium.

Large-scale performance evaluation of Accu-Chek inform II point-of-care glucose meters

Background: The aim of this study was to report the experience of large-scale performance evaluation of 238 Accu-Chek Inform II point-of-care (POC) glucose meters in a single medical setting.Methods: The repeatability of 238 POC devices, the within-site imprecision of 12 devices, and the linearity of 49 devices were evaluated using glucose control solutions. The glucose results of 24 POC devices and central laboratory were compared using patient samples.Results: Mean concentration of control solutions was 2.39 mmol/L for Level 1 and 16.52 mmol/L for Level 2. The pooled repeatability coefficient of variation (CV) of the 238 devices was 2.0% for Level 1 and 1.6% for Level 2. The pooled within-site imprecision CV and reproducibility CV of the 12 devices were 2.7% and 2.7% for Level 1, and 1.9%, and 1.9% for Level 2, respectively. The test results of all 49 devices were linear within analytical measurement range from 1.55–31.02 mmol/L. The correlation coefficient for individual POC devices ranged from 0.9967–0.9985. The total correlation coefficient for the 24 devices was 0.998.Conclusions: The Accu-Chek Inform II POC blood glucose meters performed well in terms of precision, linearity, and correlation evaluations. Consensus guidelines for the large-scale performance evaluations of POC devices are required.

L-Cysteine determination in embryo cell culture media using Co (II)-phthalocyanine modified disposable screen-printed electrodes

Thiol-containing compounds such as l-cysteine have been demonstrated to play an important role in metabolism and cellular growth, acting as powerful antioxidants. Consequently, their analytical determination in biological media has received a considerable amount of attention. In this work, an electrochemical sensor for the accurate electroanalytical determination of l-cysteine is proposed, based upon a Co(II)-phthalocyanine bulk modified disposable screen-printed graphite electrode (CoPc-SPE). Cyclic (CV) and Square Wave (SWV) voltammetry experiments have demonstrated an excellent electrocatalytic activity towards the electrochemical oxidation of l-cysteine using CoPc-SPEs within optimum neutral or basic pH. Moreover, the SWV response of l-cysteine is found to exhibit a linear range of 2.6–200μM, with a low limit of detection of 4μM (S/N=3) and a sensitivity of 0.78μAcm−2μM−1. Coefficient of variation for reproducibility and repeatability of l-cysteine determination using the CoPc-SPE are 3 and 0.4%, respectively. The effect of inherent interferences such as naturally occurring amino acids, cystine and cysteic acid has been also evaluated. Finally, the applicability of the l-cysteine electrochemical sensor based upon CoPc-SPEs has been successfully demonstrated for the first time for the assessment of l-cysteine in a complex embryo cell culture medium.