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Accreditation and Quality Assurance: Journal for Quality, Comparability and Reliability in Chemical Measurement (v.18, #2)
Considerations for the development and application of control materials to improve metagenomic microbial community profiling by Jim F. Huggett; Thomas Laver; Sasithon Tamisak; Gavin Nixon; Denise M. O’Sullivan; Ramnath Elaswarapu; David J. Studholme; Carole A. Foy (pp. 77-83).
Advances in DNA sequencing technology provide the possibility to analyse and characterize the genetic material from microbial populations (the microbiome) as a whole. Such comprehensive analysis of a microbiome using these ‘metagenomic’ approaches offers the potential to understand industrial, clinical and environmental microbiology to a level of detail that is unfeasible using conventional molecular or culture-based methods. However, the complexity offered by metagenomic analysis is also the weakness of this method and poses considerable challenges during analytical standardisation. In this manuscript, we discuss options for developing control materials for metagenomic analysis and describe our preliminary work investigating how such materials can be used to assist metagenomic measurements. The control materials we have developed demonstrate that, when performing 16S rDNA sequencing, different library preparation methods (incorporating adapters before and after the PCR) and small primer mismatches can alter the reported metagenomic profile. These findings illustrate that metagenomic analysis can be heavily biased by the choice of method and underpin the need for control materials that can provide a useful tool in informing choice of protocol for accurate analysis.
Keywords: Metagenomics; Microbes; Microbiome; Molecular; Next generation sequencing; Standards; Microbial-profiling; NGS
Comparison of biased and unbiased estimators of variances of qualitative and semi-quantitative results of testing by Tamar Gadrich; Emil Bashkansky; Ilya Kuselman (pp. 85-90).
Unbiased estimators of within-laboratory and between-laboratory (or within reference material unit and between-unit) variances of results of qualitative and semi-quantitative testing are formulated and discussed. Qualitative and semi-quantitative test results were treated as binary nominal and ordinal values, respectively, in framework of the newly developed ordinal analysis of variance (ORDANOVA). It is shown that the difference of the unbiased and the biased estimators of a within-laboratory variance does not exceed 5 %, when the number of replicate tests in a laboratory is larger than 20. Such a difference is increasing when the replicate number is decreasing, not depending on the number of laboratories and the between-laboratory variation, since both the unbiased and the biased estimators are based on the averaged within-laboratory variances. The difference of the unbiased and the biased estimators of the between-laboratory variance depends not only on the number of replicates, but also on the number of laboratories and on the ratio of the contributions to the total variance (the between-laboratory variance and the averaged within-laboratory variance). This difference does not exceed 5 %, when the number of replicates and the number of laboratories are larger than 20 and the ratio of the between-laboratory to the averaged within-laboratory variances does not yield 1. For a limited size of experiment (smaller numbers of replicates and laboratories), the difference is increasing with the size decreasing and can be significant.
Keywords: Analysis of variance; Unbiased estimator; Ordinal property; Nominal property; Interlaboratory comparison; Homogeneity study; Reference materials
Using high-performance quantitative NMR (HP-qNMR®) for certifying traceable and highly accurate purity values of organic reference materials with uncertainties <0.1 % by Michael Weber; Christine Hellriegel; Alex Rück; Robert Sauermoser; Jürg Wüthrich (pp. 91-98).
Quantitative nuclear magnetic resonance (qNMR) in combination with metrological weighing is optimised to demonstrate the power of the qNMR measurement method. It is shown that with 1H-qNMR it is possible to certify the purity of organic reference materials (expressed as mass fraction) with relative expanded uncertainties of <0.1 % for a 95 % confidence interval (k = 2). Following well-defined selection criteria, a set of twelve different chemical compounds is evaluated and certified to serve as internal references for 1H-qNMR measurements. A series of comparison measurements is made amongst a subset of the selected compounds. The purity of maleic acid is determined by six different 1H-qNMR measurement series, and all results show full consistency. All the six mean values are covered within the range of ±0.05 %. In two more measurement series, four different nuclei are analysed within the same sample against one calibrator. Even with non-optimised signal intensity ratios and varying signal pattern, a high consistency was obtained. Therefore, the validity and robustness of 1H-qNMR measurement results are demonstrated. 1H-qNMR measurement results are directly traceable to a variety of internationally accepted primary reference materials, and therefore, traceability to SI units is obtained. All experiments are performed under ISO/IEC 17025 and ISO Guide 34 accreditation.
Keywords: Quantitative NMR; qNMR; Purity determination; Traceability; Certification; Uncertainty; Organic reference materials; CRM
Certification of a low value electrolytic conductivity solution using traceable measurements by Isabel Cristina Serta Fraga; Jéssica Corrêa Lopes; Sidney Pereira Sobral; Carla Matos Ribeiro (pp. 99-104).
Reliable measurement results of electrolytic conductivity (EC), in particular for low values, must be metrologically traceable and be based on a realistic measurement uncertainty budget. The use of certified reference materials (CRMs) can help to achieve this goal. This paper presents results from all stages of the certification of an EC CRM with a conductivity of 1.5 μS cm−1, including the preparation of the batch solution and the evaluation of the homogeneity and stability of the bottled CRM. An uncertainty budget is presented for the CRM, including the main contributions from each of these sources. The CRM batch remained stable within its certified uncertainty for more than 1 year.
Keywords: Primary low electrolytic conductivity; Certified reference material; Traceable measurements
Need and use of reference materials in a comprehensive countrywide monitoring of radionuclides by Beata Varga; Sandor Tarjan (pp. 105-114).
The reliability of measurement results is essential for forming a common database of a laboratory network, because a well-maintained and consistent database is the crucial point of countrywide monitoring. Reference materials are important tools in realizing some aspects of quality assurance; they are especially useful in harmonizing work within the network. Among usual types of reference materials in radio analytics solutions, matrix reference materials and some special reference objects are used. All classes of reference materials should be used depending on the purpose of the demonstration of quality, even in-house reference materials. Interlaboratory measurement comparison and performance evaluation programs play important quality assurance role in radio analytical laboratories. Fortunately, nowadays, the main task is to determine a very low radioactivity concentration in the environment; therefore, pre-concentration is necessary. Generally, the radionuclide bearing natural materials collected from sites where there had been sufficient time for natural processes to redistribute various chemically different species of radionuclides are more reliable reference materials than spiked materials—the main difference is the chemical bounding which is crucial from the point of view of the bioavailability. The need of reference material is summarized according to the intended use, like quality control, measurement validation, and instrument calibration.
Keywords: Reference material; Monitoring of radionuclides; Quality of the results
Outline for the revision of ISO Guide 35 by Angelique Botha; Steve Ellison; Thomas Linsinger; Adriaan van der Veen (pp. 115-118).
The production of reference materials (RMs) is a key activity for the improvement and maintenance of a worldwide coherent measurement system. As detailed in ISO Guide 33, RMs with different characteristics are used in measurements, such as calibration, quality control and method validation, as well as for the assignment of values to other materials. Currently, ISO Guide 35 is in its third edition after it was revised in 2006. The Guide was developed to support best practices in the value assignment to specified properties of Certified Reference Materials (CRMs). This Guide gives general guidance and explains concepts to assist the understanding and development of valid methods to assign values to the properties of a reference material, including the evaluation of their associated measurement uncertainties, and the establishment of their metrological traceability. From the outcome of a systematic review of ISO Guide 35 among the members of ISO/REMCO, the ISO Committee on Reference Materials, it followed that there is a need for revising the current edition of ISO Guide 35. The mandate for the revision is focused on editorial updates to explain the concepts in more detail. It is not envisaged that major technical changes will be introduced. This paper explains the approach and rationale for the revision of ISO Guide 35 and invites comments from the users of the current edition of ISO Guide 35.
Keywords: ISO Guide 35; Reference materials; Value assignment; Homogeneity; Stability
How should we proceed with the standardization in case of allergen determination? by M. Lacorn; T. Weiss; U. Immer (pp. 119-125).
Allergens cannot at present be measured with the trueness that attends the analysis of well-characterized chemical entities such as mycotoxins where the analytes are defined and identical in sample and calibrator. By nature, allergens are nearly always a mixture of different proteins with different mass fractions of each single protein. Allergens are furthermore in a commodity with different total protein contents in the presence of an unknown matrix. To circumvent these difficulties, reference materials for allergens would be useful but until now not realized due to biological variabilities of the materials, and the contradiction between medical issues and declaration of foods due to legislation. Examples for standardization efforts are given which are (1) peanut where a common reference material not intended for allergen determination is used and (2) gluten/gliadin where the extraction procedure, the antibody and a calibrator are standardized but proficiency tests are not fit-for-purpose due to inhomogeneity of the testing material. Furthermore, the analytical minimum requirements and the declaration of egg-white proteins, caseins and lysozyme in wine are reviewed. To show one negative example for standardization efforts, milk is discussed. The possibilities and limits of reference materials for the standardization of allergen determinations are discussed.
Keywords: Reference materials; Gluten; Milk; Egg; Calibrator; Antibodies; Allergens
Thirteenth international symposium on biological and environmental reference materials (BERM-13)
by Aleš Fajgelj (pp. 127-128).
Relative sensitivity of combined standard uncertainty to changes induced in uncertainty components by A. C. Baratto; I. L. Bezerra (pp. 129-135).
This work provides a straightforward and rigorous solution to the problem of evaluating and expressing relative changes in combined uncertainty resulting from relative changes in the uncertainty components. The acquired information is essential to optimize the measurement method, indicating the most significant components to work with. This approach works for any discrete relative change in the uncertainties of the inputs, whether positive or negative, and was developed for the cases of independent and correlated quantities. Examples of both cases are treated. The results agree with that provided by direct calculation and are consistent with the ideas in EA 4/16. We propose the use of the expression “sensitivity of combined standard uncertainty to changes in uncertainty components” as a uniform quantitative reference to the individual importance of the components that form the combined uncertainty. We also discuss the reason to avoid the use of the expression “contribution of an uncertainty component to the combined uncertainty.”
Keywords: Importance of uncertainty components; Relative contribution; Relative sensitivity of combined uncertainty; Administration of uncertainty budget
Influence of desorption and sorption of water on the purity of perfluorooctanoic acid by Nobuyasu Hanari; Nobuyasu Itoh; Taichi Yamazaki; Naomi Fujiki; Ryoko Iwasawa; Masahiko Numata (pp. 137-142).
The composition of impurities in perfluorooctanoic acid (PFOA) was measured and determined to be notably different between lots. Since the purity of PFOA varied, the accuracy of prepared calibration solution concentrations was reduced. In this study, factors influencing the purity were investigated using a Karl Fischer titrator and a LC/MS to support the development of reference materials. The results showed variances of water and isomer/homologue contents under different ambient conditions during sample handling. Moreover, these variances, especially for water, greatly affected the purity of PFOA (0.95–0.99 kg kg−1). Therefore, PFOA of reliable purity should be used for the preparation of calibration solutions.
Keywords: Purity; Perfluorooctanoic acid (PFOA); Water; Desorption; Sorption; KFT; LC/MS
Assessment of reproducibility and uncertainty of food microbiology methods: statistical approach of a multi-site laboratory by Giuliana Blasi; Annalisa Petruzzelli; Donatella Ottaviani; Stefano Fisichella; Enrico Di Raimo; Andrea Valiani; Marinella Capuccella; Stefania Scuota; M. Naceur Haouet (pp. 143-148).
The validation of food microbiology methods and assessment of measurement uncertainty are required for laboratories operating EN ISO/IEC 17025 accreditation systems. This paper aims to describes the statistical approach adopted by a multi-site laboratory to satisfy these requirements and to routinely check the performance of the methods. A validation protocol was performed, reflecting a great variability of experimental conditions, represented by the period of time during which determinations were made, the different laboratories concerned, the large number of technicians involved, the differences in the level of contamination of the matrix analyzed and the operating conditions (equipment, reagents, culture media, environmental conditions, etc.). Despite the very high variability of the experimental conditions, the values of repeatability and reproducibility obtained for the methods were lower than those stated in the respective regulations. In addition, the top-down approach adopted in this study is to be considered effective overtime and allows to randomize all variable factors, including the effects of sensitivity and specificity, in order to simulate the conditions of real reproducibility. A specific Excel spreadsheet was also developed for the routine expression of analytical results. This spreadsheet represents a very useful tool for operators to calculate and routinely check the results, as well as to assess the uncertainty of measurement. This particular quality assessment system is enabling the multi-site laboratory to implement the accuracy, reliability and comparability of the analytical results and to ensure the analytical control of data output.
Keywords: Multi-site laboratory; Reproducibility; Uncertainty; Food microbiology methods
Characterization of reference materials: proposal for a simplification of the options listed in ISO Guide 34 by Thomas P. J. Linsinger; Hendrik Emons (pp. 149-152).
ISO Guide 34 and 35 list 4 approaches for the characterization of reference materials. We argue that an approach using “one method of demonstrable accuracy, performed by a network of competent laboratories” will provide method-specific values. Furthermore, as it is impossible to objectively decide whether a method is a reference method or not, the approach using “two or more independent reference methods in one or several laboratories” should be combined with the approach “two or more methods of demonstrable accuracy, performed by a network of competent laboratories.” We therefore propose to replace the four characterization approaches by three in the ongoing revision of ISO Guide 35.
Keywords: CRM; Certified reference materials; Accreditation; ISO Guide 35; ISO Guide 34
Report on the 35th meeting of ISO/REMCO
by Hendrik Emons; Angelique Botha; Stéphane Sauvage (pp. 153-155).
What is the meaning of the concept ‘consistency’ in (chemical) measurement?
by Paul De Bièvre (pp. 157-158).