Comparison of pencil-type ionization chamber calibration results and methods between dosimetry laboratories

A comparison of calibration results and procedures in terms of air kerma length product, P_KL, and air kerma, K, was conducted between eight dosimetry laboratories. A pencil-type ionization chamber (IC), generally used for computed tomography dose measurements, was calibrated according to three calibration methods, while its residual signal and other characteristics (sensitivity profile, active length) were assessed. The results showed that the “partial irradiation method” is the preferred method for the pencil-type IC calibration in terms of P_KL and it could be applied by the calibration laboratories successfully. Most of the participating laboratories achieved high level of agreement (>99%) for both dosimetry quantities (P_KL and K). Estimated relative standard uncertainties of comparison results vary among laboratories from 0.34% to 2.32% depending on the quantity, beam quality and calibration method applied. Detailed analysis of the assigned uncertainties have been presented and discussed.     

Quality assessment of human mitochondrial DNA quantification: MITONAUTS, an international multicentre survey

Mitochondrial DNA quantification by qPCR is used in the context of many diseases and toxicity studies but comparison of results between laboratories is challenging. Through two multigroup distributions of DNA samples from human cell lines, the MITONAUTS group anonymously compared mtDNA/nDNA quantification across nine laboratories involved in HIV research worldwide. Eight of the nine sites showed significant correlation between them (mean raw data R(2)=0.664; log(10)-transformed data R(2)=0.844). Although mtDNA/nDNA values were well correlated between sites, the inter-site variability on the absolute measurements remained high with a mean (range) coefficient of variation of 71 (37-212) %. Some variability appeared cell line-specific, probably due to chromosomal alterations or pseudogenes affecting the quantification of certain genes, while within cell line variability was likely due to differences in calibration of the standard curves. The use of two mtDNA and two single copy nDNA genes with highly specific primers to quantify each genome would help address copy number variants. Our results indicate that sample shipment must be done frozen and that absolute mtDNA/nDNA ratio values cannot readily be compared between laboratories, especially if assessing cultured cell mtDNA content. However, within laboratory and relative mtDNA/nDNA comparisons between laboratories should be reliable.     

Development and validation of an EI-GC/MS method for the determination of sertraline and its major metabolite desmethyl-sertraline in blood

A sensitive and specific GC/MS method for the determination of sertraline and its main metabolite desmethyl-sertraline in whole blood has been developed, optimized and validated. Sample preparation included solid-phase extraction of both analytes and their derivatization with heptafluorobutyric anhydride (HFBA). Protriptyline was used as internal standard for the determination of both analytes. Limits of detection and quantification for both sertraline and desmethyl-sertraline were 0.30 and 1.00 μg/L, respectively. The calibration curves were linear within the dynamic range of each analyte (1.00-500.0 μg/L) with a correlation coefficient (R(2)) exceeding 0.991. Extraction efficiency ranged from 90.1(± 5.8)% to 95.4(± 3.0)% for sertraline, and from 84.9(± 8.2)% to 107.7(± 4.4)% for desmethyl-sertraline. The precision for sertraline and desmethyl-sertraline was between 3.6-5.5% and 4.7-7.2%, respectively, while the accuracy was in the range of -6.67% to 2.20% and -6.33% to 2.88% for sertraline and desmethyl-sertraline, respectively. The method was applied to real blood samples obtained from patients that follow sertraline treatment and also in cases of forensic interest. The developed method can be used in routine every day analysis by clinical and forensic laboratories, for pharmacokinetic studies, for therapeutic sertraline monitoring or for the investigation of forensic cases where sertraline is involved.     

Estimates of measurement uncertainty from proficiency testing schemes, internal laboratory quality monitoring and during routine enforcement examination of foods

AIMS: To determine the levels of measurement uncertainty (MU) obtained in proficiency testing and routine microbiological analyses of foods and to compare these with estimates of MU obtained for results of analyses obtained in collaborative interlaboratory studies of microbiological methods. METHODS AND RESULTS: Raw data submitted by participants in the Food Examination Proficiency Assessment Scheme were obtained from the Central Science Laboratory (York). Internal quality monitoring data were obtained from Health Protection Agency (HPA) laboratories, together with data from routine food examinations undertaken in HPA laboratories. The data sets were analysed to determine the relative standard deviations of reproducibility (RSD(R)), based on log(10) colony count values, and thence the relative measures of expanded uncertainty. Analysis of proficiency test data showed extreme values of RSD(R) up to +/-30% depending upon the organism, the laboratory and the method of examination. RSD(R) values on routine samples averaged around +/-12% but ranged up to +/-41% in a few instances. Internal quality assessments for different organisms ranged up to +/-27%, depending upon the particular organism and examination procedure. The results show little difference in uncertainty for counts obtained using different plating systems (e.g. pour plates, spread plates or spiral plating) on the same dilutions of the same food samples. The data are compared with estimates of microbiological uncertainty derived in interlaboratory studies. CONCLUSIONS: The estimates of uncertainty ranged widely, both within and between laboratories, and appeared to bear little relationship to the foodstuff under examination. The extent of MU associated with many routine microbiological examinations is generally no worse than those produced in inter-laboratory trials, although notable exceptions were seen. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the levels of MU may have wide impact on the establishment of international standard methods for microbiological examination of foods and the ability to set realistic microbiological criteria.     

On the use of EBT3 film for relative dosimetry of kilovoltage X ray beams

EBT3 films were evaluated for relative dosimetry in water, in the energy range of therapeutic kV X ray beams. A film batch was calibrated in air for all nine beam qualities of a clinical unit (XStrahl 200). Monte Carlo (MC) simulations using MCNP v.6 facilitated the calculation of the film absorbed dose (f), and beam quality (k_bq) energy dependences in air. Results were found in agreement with corresponding data in the literature. Film samples from the same batch were irradiated in water along the central beam axis for each beam quality. Experimental percentage depth dose (PDD) results obtained using calibration data in air showed quality and depth dependent differences from corresponding MC simulations. These differences increased beyond film dosimetry uncertainty (<3.3%), reaching up to 8% at increased depth. The observed differences reduced only slightly when spectral variation as a function of measurement point was accounted for, using photon effective energy. PDD measurements and corresponding MC results facilitated the determination of f and k_bq in water. Results showed that the origin of the observed differences between experimental and MC PDD results is the difference between film response in air and water, as a result of radiation field perturbation from the film oriented along the central beam axis. This implies a directional dependence of film response which necessitates that the angular distribution of photons impinging on the film is the same in the calibration and measurement geometries.     

Interlaboratory comparison of the dicentric chromosome assay for radiation biodosimetry in mass casualty events

This interlaboratory comparison validates the dicentric chromosome assay for assessing radiation dose in mass casualty accidents and identifies the advantages and limitations of an international biodosimetry network. The assay's validity and accuracy were determined among five laboratories following the International Organization for Standardization guidelines. Blood samples irradiated at the Armed Forces Radiobiology Research Institute were shipped to all laboratories, which constructed individual radiation calibration curves and assessed the dose to dose-blinded samples. Each laboratory constructed a dose-effect calibration curve for the yield of dicentrics for (60)Co gamma rays in the 0 to 5-Gy range, using the maximum likelihood linear-quadratic model, Y = c + alphaD + betaD(2). For all laboratories, the estimated coefficients of the fitted curves were within the 99.7% confidence intervals (CIs), but the observed dicentric yields differed. When each laboratory assessed radiation doses to four dose-blinded blood samples by comparing the observed dicentric yield with the laboratory's own calibration curve, the estimates were accurate in all laboratories at all doses. For all laboratories, actual doses were within the 99.75% CI for the assessed dose. Across the dose range, the error in the estimated doses, compared to the physical doses, ranged from 15% underestimation to 15% overestimation.     

Rate of Pyrite Bioleaching by Thiobacillus ferrooxidans: Results of an Interlaboratory Comparison

Ten laboratories participated in an interlaboratory comparison of determination of bioleaching rates of a pyrite reference material. A standardized procedure and a single strain of Thiobacillus ferrooxidans were used in this study. The mean rate of bioleaching of the pyrite reference material was 12.4 mg of Fe per liter per h, with a coefficient of variation (percent relative standard deviation) of 32% as determined by eight laboratories. These results show the precision among laboratories of the determination of rates of pyrite bioleaching when a standard test procedure and reference material are used.     

Round robin investigation of methods for the recovery of poliovirus from drinking water.

Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Round robin investigation of methods for the recovery of poliovirus from drinking water

Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

An international comparability study on quantification of total methyl cytosine content

Various methods have been developed for quantitative analysis of DNA methylation. However, there is currently no reference analysis system regarding DNA methylation with which other analytical approaches can be compared and evaluated. A standard measurement system that includes reference methods and reference materials may improve comparability and credibility of data obtained from different analytical environments. In an effort to establish a standard system for measurement of DNA methylation, the Korea Research Institute of Standards and Science (KRISS) coordinated an international comparison study among different national metrology institutes. An initial stage of the study involved an intercomparison regarding quantitative measurement of total methyl cytosine contents in artificially constructed DNA samples. The measurement principle involved measurement of dNMP contents following enzymatic hydrolysis of DNA samples. Results of the study showed good comparability among four of five participants and close agreement with reference values assigned by the coordinating laboratory. Conflicting data from one participant may have resulted from incomplete hydrolysis of samples due to use of insufficient amounts of enzymes. These results indicate that comparable and accurate results can be obtained from different measurement environments if digestion conditions are controlled appropriately and valid calibration systems are employed.     

High-performance liquid chromatography determination of ketone bodies in human plasma by precolumn derivatization with p-nitrobenzene diazonium fluoroborate

We developed and validated a sensitive and convenient high-performance liquid chromatography (HPLC) method for the specific determination of ketone bodies (acetoacetate and d-3-hydroxybutyrate) in human plasma. p-Nitrobenzene diazonium fluoroborate (diazo reagent) was used as a precolumn derivatization agent, and 3-(2-hydroxyphenyl) propionic acid was used as an internal standard. After the reaction, excess diazo reagent and plasma proteins were removed by passing through a solid-phase cartridge (C₁₈). The derivatives retained on the cartridge were eluted with methanol, introduced into the HPLC system, and then detected with UV at 380 nm. A calibration curve for acetoacetate standard solution with a 20-μl injection volume showed good linearity in the range of 1 to 400 μM with a 0.9997 correlation coefficient. For the determination of d-3-hydroxybutyrate, it was converted to acetoacetate before reaction with the diazo reagent by an enzymatic coupling method using d-3-hydroxybutyrate dehydrogenase and lactate dehydrogenase. A calibration curve for d-3-hydroxybutyrate standard solution also showed good linearity in the range of 1.5 to 2000 μM with a 0.9988 correlation coefficient. Analytical recoveries of acetoacetate and d-3-hydroxybutyrate in human plasma were satisfactory. The method was successfully applied to samples from diabetic patients, and results were consistent with those obtained using the thio-NAD enzymatic cycling method used in clinical laboratories.     

Immobilised activated sludge based biosensor for biochemical oxygen demand measurement

A biochemical oxygen demand (BOD) sensor, based on an immobilised mixed culture of microorganisms in combination with a dissolved oxygen electrode, has been developed for the purpose of on-line monitoring of the biological treatment process for waste and wastewater. The sensor was designed for easy replacement of the biomembrane, thereby making it suitable for short-term use. The drawbacks of activated sludge based sensor, such as short sensor lifetime, were thereby circumvented. The sensor BOD measurements were carried out in the kinetic mode using a flow injection system, resulting in 25 s for one measurement followed by 4–8 min recovery time. Based on the results of normalised sensor responses, the OECD synthetic wastewater was considered to be a more suitable calibration solution in comparison with the GGA solution. Good agreement was achieved between the results of the sensor BOD measurement and those obtained from BOD₅ analysis of a wastewater sample from a food-processing factory. Reproducibility of responses using one sensor was below ±5.6% standard deviation. Reproducibility of responses using different sensors was within acceptable bias limits, viz. ±15% standard deviation.     

Interlaboratory reproducibility of selective reaction monitoring assays using multiple upfront analyte enrichment strategies

Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays. Furthermore, an important caveat of SRM-based assays for proteins is that many low-abundance analytes require some type of enrichment before detection with MS. This adds a level of complexity to the procedure and the potential for irreproducibility increases, especially across different laboratories with different operators. The purpose of this study was to test the interlaboratory reproducibility of SRM assays with various upfront enrichment strategies and different types of clinical samples (representing real-world body fluids commonly encountered in routine clinical laboratories). Three different, previously published enrichment strategies for low-abundance analytes and a no-enrichment strategy for high-abundance analytes were tested across four different laboratories using different liquid chromatography-SRM (LC-SRM) platforms and previously developed SOPs. The results demonstrated that these assays were indeed reproducible with coefficients of variation of less than 30% for the measurement of important clinical proteins across all four laboratories in real world samples.     

Use of the LA 640 for a simple method of measuring the concentration of muscle lactate

A simple technique was elaborated for the measurement of muscle lactate concentration. It was tested on samples of muscle vastus lateralis taken by the Bergstr?m (1962) needle biopsy technique at the end of 20 min exercise bouts corresponding to 60-70% of Vo2 max. The biopsies were freshly frozen in liquid nitrogen, powdered and weighed in a cryostat at -20 degrees C. The extraction were made by saponine and the lactate measured in the saponine solution by an electrochemical-enzymatic method (LA 640). The results concern: the time of taking the biopsies and the freezing time (27 +/- 11 s and 34 +/- 9 s respectively); the accuracy of weighing (inducing a 1% uncertainty in the final result); a comparison of the saponine extraction with the perchloric acid extraction and a checking of the extraction capacity of the former; the accuracy of the whole measurement (the mean relative confidence limits are +/- 8-10%; the reproducibility of the technique through measurement of the variation coefficient (18%) calculated on measurements performed at a 15 days interval in 6 trained subjects. The discussion of the results and their comparison with those of the literature lead to the conclusion that the described method is suitable for muscle lactate measurements.     

Evaluation of uncertainty in quantitative real-time PCR

Quantitative real-time PCR is one of the newer methods for measurement of the amount of nucleic material in biological systems. However, reliable measurement requires an appropriate estimation of uncertainty and this paper has developed the uncertainty budget associated with this procedure using as an example, data from a quantitative real-time PCR method for the enumeration of Campylobacter jejuni. This uncertainty is relatively large and for instance, a measured result of 151 units of DNA would have a 95% confidence interval of +/-84 units of DNA with the main sources of uncertainty being the measurement of the threshold cycle (Ct) value, the predicted DNA content of the unknown sample from the calibration line and the molar absorbance value for DNA.     

Requirements for reference (calibration) laboratories in laboratory medicine

In addition to reference measurement procedures and reference materials, reference or calibration laboratories play an integral role in the implementation of measurement traceability in routine laboratories. They provide results of measurements using higher-order methods, e.g. isotope dilution mass spectrometry and may assign values to materials to be used for external quality assessment programs and to secondary reference materials. The requirements for listing of laboratories that provide reference measurement services include a statement of the metrological level or principle of measurement, accreditation as a calibration laboratory according to ISO 15195 and the participation in a proficiency testing system (regular inter-laboratory comparisons) for reference laboratories. Ring trials are currently conducted for thirty well-defined measurands and the results are made available to all laboratories. Through the use of reference laboratory services that are listed by the Joint Committee for Traceability in Laboratory Medicine there is the opportunity to further promote traceability and standardisation of laboratory measurements.     

应用ELISA和ELFA定量检测牛奶中葡萄球菌肠毒素A方法的建立

探讨了Tecra SEs SET(ELISA法)和Vidas SET2(ELFA法)两种葡萄球菌肠毒素定性检测试剂盒用于定量检测牛奶中葡萄球菌肠毒素A(SEA)的可行性。根据GB/T27404-2008《实验室质量控制规范食品理化检测》的要求,对两种方法应用于定量检测时的检出限、校正曲线范围、相关系数和加标回收率指标进行了分析比较。实验结果显示,Tecra SEs SET对牛奶中SEA的检出限为0.79 ng/mL,校正曲线范围为0.79~10 ng/mL,相关系数r=0.997,SEA加标浓度为0.80、2.5和10 ng/mL时的回收率分别为110%、81%和100%。Vidas SET2的检出限为0.09 ng/mL,校正曲线范围为0.09~1.0 ng/mL,相关系数r=0.998,SEA加标浓度为0.1、0.25和1.0 ng/mL时的回收率分别为90%、95%和104%。上述结果结合对阳性样品的检测表明:这两种定性检测试剂盒能满足牛奶中SEA定量检测的要求。     

Quantification of growth hormone in serum by isotope dilution mass spectrometry

Interassay variation of antibody-based routine tests hampers comparability of measurement results for growth hormone (GH) between different laboratories and decision making in clinical practice. Here it is demonstrated that quantification of GH by isotope dilution mass spectrometry (IDMS) constitutes a way to obtain precise and reliable results that can be referred to in evaluation of performance of commercial test kits. With the IDMS method developed, tryptic cleavage products YSFLQNPQTSLCFSESIPTPSNR (T6) and LEDGSPR (T12) of GH are quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using isotopically labeled forms of the peptides as internal standards. The GH cleavage fragments are obtained by whole serum tryptic proteolysis and then extracted from the resulting mixture by semipreparative reversed-phase LC followed by strong cation exchange chromatography. Analysis of blank serum spiked with recombinant 22-kDa GH at different concentration levels would result in a mean recovery of 101.6%, a standard deviation (SD) of 2.5%, a combined uncertainty (u_c) of 3.0%, and a limit of quantification (LOQ) of 1.7 μg/L when quantifying T6 as a GH-derived fragment, whereas recovery = 100.7%, SD = 2.4%, u_c = 2.5%, and LOQ = 2.7 μg/L were found with T12. The potential to acquisition of reference values is exemplified by application to serum materials used in a recent quality assessment exercise for routine laboratories.     

Estimating Uncertainty in Ecosystem Budget Calculations

Ecosystem nutrient budgets often report values for pools and fluxes without any indication of uncertainty, which makes it difficult to evaluate the significance of findings or make comparisons across systems. We present an example, implemented in Excel, of a Monte Carlo approach to estimating error in calculating the N content of vegetation at the Hubbard Brook Experimental Forest in New Hampshire. The total N content of trees was estimated at 847 kg ha⁻¹ with an uncertainty of 8%, expressed as the standard deviation divided by the mean (the coefficient of variation). The individual sources of uncertainty were as follows: uncertainty in allometric equations (5%), uncertainty in tissue N concentrations (3%), uncertainty due to plot variability (6%, based on a sample of 15 plots of 0.05 ha), and uncertainty due to tree diameter measurement error (0.02%). In addition to allowing estimation of uncertainty in budget estimates, this approach can be used to assess which measurements should be improved to reduce uncertainty in the calculated values. This exercise was possible because the uncertainty in the parameters and equations that we used was made available by previous researchers. It is important to provide the error statistics with regression results if they are to be used in later calculations; archiving the data makes resampling analyses possible for future researchers. When conducted using a Monte Carlo framework, the analysis of uncertainty in complex calculations does not have to be difficult and should be standard practice when constructing ecosystem budgets.