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.     

Reference materials and commutability

Maintaining accurate laboratory measurements over time is crucial for assuring appropriate patient care and disease management. Accurate results over time and location are achieved by standardising measurements and establishing traceability to a reference system. Reference materials are key components of such reference systems and for establishing traceability. Commutability of reference materials is a critical property to ensure they are fit for use.Commutability is defined as the equivalence of the mathematical relationships between the results of different measurement procedures for a reference material and for representative samples from healthy and diseased individuals. This material characteristic is of special importance for measurement procedures that are optimised for measuring analytes directly in patient samples. The commutability of a reference material is measurement procedure specific and its assessment requires special experimental designs.This review explains the importance of commutability and summarises different experimental approaches described in the literature that have been used to assess the commutability of reference materials in clinical chemistry.     

Certified reference materials (GBW09170 and 09171) of creatinine in human serum

Creatinine is the most widely used clinical marker for assessing renal function. Concentrations of creatinine in human serum need to be carefully checked in order to ensure accurate diagnosis of renal function. Therefore, development of certified reference materials (CRMs) of creatinine in serum is of increasing importance. In this study, two new CRMs (Nos. GBW09170 and 09171) for creatinine in human serum have been developed. They were prepared with mixtures of several dozens of healthy people's and kidney disease patient's serum, respectively. The certified values of 8.10, 34.1 mg/kg for these two CRMs have been assigned by liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) method which was validated by using standard reference material (SRM) of SRM909b (a reference material obtained from National Institute of Standards and Technology, NIST). The expanded uncertainties of certified values for low and high concentrations were estimated to be 1.2 and 1.1%, respectively. The certified values were further confirmed by an international intercomparison for the determination of creatinine in human serum (Consultative Committee for Amount of Substance, CCQM) of K80 (CCQM-K80). These new CRMs of creatinine in human serum pool are totally native without additional creatinine spiked for enrichment. These new CRMs are capable of validating routine clinical methods for ensuring accuracy, reliability and comparability of analytical results from different clinical laboratories. They can also be used for instrument validation, development of secondary reference materials, and evaluating the accuracy of high order clinical methods for the determination of creatinine in human serum.     

Improving the accuracy of chloride measurements through participation in regular external quality assessment programme

BackgroundA chloride test is an integral part of a basic metabolic panel that is essential for the assessment of a patient’s acid-base and electrolyte status. While many methods are available commercially for the routine measurement of chloride, there is a need to address the accuracy and variability among the measurement results, especially with the prevalence of patients seeking treatment across different healthcare providers for alternative opinions.MethodA method based on sector field inductively coupled plasma isotope dilution mass spectrometry (SF-ICP-IDMS) was developed for the measurement of chloride in human serum. The SF-ICP-IDMS method was then used to assign the target values in the Health Sciences Authority (HSA) External Quality Assessment (EQA) Programme to evaluate the results of chloride test from participating clinical laboratories.ResultsThe accuracy of the measurements was evaluated by comparing the results with the certified values of Electrolytes in Frozen Human Serum Certified Reference Materials (SRM 956c and SRM 956d) from the National Institute of Standards and Technology (NIST) at different chloride concentration levels. Over a five-year period from 2014–2018, the number of clinical laboratories which participated in the EQA Programme increased from 23 to 33. Comparison of robust means from the laboratories’ results with our assigned target values revealed a reduction in relative deviation over time. The relationship between the deviation of each brand of clinical analysers and the chloride levels was established, where a larger deviation was uncovered at low chloride concentration. The SF-ICP-IDMS method was further demonstrated to be comparable with methods used by other metrology institutes in an international comparison organised by HSA under the auspice of the Consultative Committee for Amount of Substance – Metrology in Chemistry and Biology (CCQM).ConclusionThe use of metrologically traceable assigned target values enabled the study of method biasness from a small pool of dataset in each of the four brands of clinical analysers in HSA EQA Programme. This work underscores the need to improve the accuracy of chloride measurements by regular participation in an accuracy-based EQA Programme.     

Development of a reference material using methamphetamine abusers' hair samples for the determination of methamphetamine and amphetamine in hair

In the present study, we developed a reference material (RM) using authentic hair samples for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) in human hair. MA abusers' hair samples were collected, homogenized and finally bottled. The concentration of each bottle was determined using two extraction methods, agitation with 1% HCl in methanol at 38 degrees C and ultrasonication with methanol/5M HCl (20:1), followed by gas chromatography/mass spectrometry (GC-MS) after derivatization with trifluoroacetic anhydride (TFAA). Both analytical procedures were fully validated and their extraction efficiency was compared. The homogeneity of analytes was evaluated and their property values were determined with their uncertainties. The two methods were acceptable to analyze MA and AP in human hair through the validation and comparative studies using spiked and authentic hair samples as well as NIST SRM 2379 certified reference material. Satisfying homogeneity was reached for MA and AP in the prepared RM. Finally, a human hair RM containing MA and AP is prepared at the level of 7.64+/-1.24 and 0.54+/-0.07 ng/mg, respectively. This material can be useful in forensic laboratories for internal quality control and external quality assurance.     

Design considerations for proteomic reference materials

In order to improve the repeatability, comparability, and accuracy of MS-based proteomic measurements, there has been considerable international effort to develop appropriate reference materials. Although the majority of reference materials are developed to support measurement quality of routine assays, the development of reference materials for a diverse and changing research field such as proteomics represents unique challenges. In order to define common measurement components and common features of typical proteomic samples, the metrology underpinning proteomics must be considered due to the diversity and changing nature of the field. Reference materials can then be designed around common aspects in order to produce reference materials with the broadest applicability. Reference materials are needed to support both qualitative and quantitative proteomic measurements, involving different design considerations. Consensus and validated statistical approaches to describe the confidence in qualitative measurement, such as protein identification, needs to be established. Common sources of measurement bias also need to be considered in proteomic reference material design.     

Development of a bead-based aptamer/antibody detection system for C-reactive protein

A multiplexing bead-based platform provides an approach for the development of assays targeting specific analytes for biomonitoring and biosensing applications. Multi-Analyte Profiling (xMAP) assays typically employ a sandwich-type format using antibodies for the capture and detection of analytes of interest, and the system permits the simultaneous quantitation of multiple targets. In this study, an aptamer/antibody assay for the detection of C-reactive protein (CRP) was developed. CRP is an acute phase marker of inflammation whose elevated basal levels are correlated with an increased risk for a number of pathologies. For this assay, an RNA aptamer that binds CRP was conjugated to beads to act as the capture agent. Biotinylated anti-CRP antibody coupled to fluorescently labeled streptavidin was used for quantification of CRP. The detection limit of the CRP assay was 0.4 mg/L in diluted serum. The assay was then used to detect spiked CRP samples in the range of 0.4 to 10 mg/L in diluted serum with acceptable recoveries (extrapolated values of 70–130%), including that of a certified reference material (129% recovery). The successful incorporation of the CRP aptamer into this platform demonstrates that the exploration of other aptamer–target systems could increase the number of analytes measurable using xMAP-type assays.     

Traceability, reference systems and result comparability

The standardisation of measurements is of high priority in laboratory medicine, its purpose being to achieve closer comparability of results obtained using routine measurement procedures. At present, there is international cooperation in developing reference measurement systems (reference methods, reference materials, and reference laboratory networks) for analytes of clinical significance. These reference systems will reduce, wherever possible, measurement uncertainty and promote the comparability of results. The implementation of measurement traceability through the reference system provides one of the most important tools that supports the standardisation process in laboratory medicine. It aims to achieve result comparability regardless of the measurement procedure (test kit) and the clinical laboratory where analyses are carried out. The aim of this review is to discuss some concepts related to the achievement of standardisation by the implementation of a metrologically-correct measurement system and to provide some examples that illustrate the complexity of this approach and the impact of these activities on patient care.     

A proteomics performance standard to support measurement quality in proteomics

The emergence of MS-based proteomic platforms as a prominent technology utilized in biochemical and biomedical research has increased the need for high-quality MS measurements. To address this need, National Institute of Standards and Technology (NIST) reference material (RM) 8323 yeast protein extract is introduced as a proteomics quality control material for benchmarking the preanalytical and analytical performance of proteomics-based experimental workflows. RM 8323 yeast protein extract is based upon the well-characterized eukaryote Saccharomyces cerevisiae and can be utilized in the design and optimization of proteomics-based methodologies from sample preparation to data analysis. To demonstrate its utility as a proteomics quality control material, we coupled LC-MS/MS measurements of RM 8323 with the NIST MS Quality Control (MSQC) performance metrics to quantitatively assess the LC-MS/MS instrumentation parameters that influence measurement accuracy, repeatability, and reproducibility. Due to the complexity of the yeast proteome, we also demonstrate how NIST RM 8323, along with the NIST MSQC performance metrics, can be used in the evaluation and optimization of proteomics-based sample preparation methods.     

Dietary supplement Standard Reference Materials

A new category of Standard Reference Materials (SRMs) based on dietary supplements is under development by the National Institute of Standards and Technology (NIST), with certified values for organic constituents and selected trace elements. These materials are provided primarily for use in method development and as control materials. The SRMs will assist manufacturers of dietary supplements in characterizing raw materials for potency, authenticity, and contamination or adulteration. In addition, the SRMs will assist in assessment of consistency and quality in finished products. The goal of this ongoing effort is to provide tools to the dietary supplement industry and measurement communities that will lead to improved quality of dietary supplements, and ultimately reduce public health risks that could potentially be associated with these products.     

Determination of trace elements in porcine brain by inductively coupled plasma-mass spectrometry,electrothermal atomic absorption spectrometry,and instrumental neutron activation analysis

Methods have been developed for the analyses of trace metals in various areas of porcine brains, (temporal, parietal, frontal cortex, both right and left hemispheres). Determinations were carried out using inductively coupled plasma-mass spectrometry (ICP-MS) and electrothermal atomic absorption spectrometry (ETAAS). The elements investigated were Li, Mn, Cu, Zn, Cd, Hg, and Pb by ICP-MS and Cu, Cd, and Mn by ETAAS. For determination by ICP-MS, a method of standard additions calibration coupled with internal standards was used, and for ETAAS, standard additions calibrations were prepared. The accuracy of all methods was determined using NIST and IAEA certified reference material. A small number of pig brains were analyzed by instrumental neutron activation analysis for Cr, Co, Cs, Fe, Rb, Se, Sc, Sb, and Zn using the comparator method of analysis. Four separate NIST standard reference materials have been used to examine the validity of the comparator method.     

Traceability in clinical enzymology

The primary goal of standardisation for measurements of catalytic concentrations of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments and laboratory where the assay is carried out. In order to pursue this objective, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has established reference systems for the most important clinical enzymes. These systems are based on three requirements: a) reference measurement procedures that are extensively evaluated and carefully described; b) certified reference materials; and c) a network of reference laboratories operating in a highly controlled manner. Using these reference systems and the manufacturer's standing procedures, industry can assign traceable values to commercial calibrators. Clinical laboratories, which use routine procedures with validated calibrators to measure human specimens, can finally obtain values which are traceable to higher-order reference procedures. These reference systems constitute the structure of the traceability chain to which the routine methods can be linked via an appropriate calibration process, provided that they have a comparable specificity (i.e. they are measuring the same quantity).     

Design and implementation of a collaborative study of the mutagenicity of complex mixtures in Salmonella typhimurium.

In 1987, the International Programme on Chemical Safety (IPCS) in collaboration with the U.S. Environmental Protection Agency (U.S. EPA) and the U.S. National Institute of Standards and Technology (U.S. NIST) initiated an international collaborative study of the mutagenicity of complex environmental mixtures in the Ames Salmonella typhimurium mutation assay. The objectives of this study were: (1) to estimate the inter- and intra-laboratory variability associated with the extraction of mixtures for bioassay, (2) to estimate the inter- and intra-laboratory variability associated with the Salmonella typhimurium bioassay when applied to complex mixtures, and (3) to determine whether standard reference complex mixtures would be useful in mutagenicity studies and to evaluate whether reference or certified mutagenicity values determined from this collaborative study should be reported. The complex mixtures used in this study were selected from standard reference materials (SRMs) which had previously been issued by the U.S. NIST as SRM 1597 (coal tar), SRM 1649 (diesel particulate matter) and SRM 1650 (urban air particulate matter) with certified values for polycyclic aromatic hydrocarbons. These SRM complex mixtures are available to scientists as reference standards for analytical chemistry research and are under consideration as SRMs for mutagenicity studies of complex environmental mixtures. This paper briefly describes the final study design, protocol, selection of the complex mixtures, and implementation of this international study.     

Investigating essential and toxic elements in Antarctic macroalgae using a green analytical method

A systematic study for the determination of essential and toxic elements (such as As, Cd, Cu, Mn, Ni, Pb, V and Zn) in Antarctic macroalgae species (Desmarestia anceps, Iridaea cordata, Palmaria decipiens and Pyropia endiviifolia) was performed. For this purpose, a green sample preparation method combined with a high-sensitivity detection technique (inductively coupled plasma mass spectrometry) was used. By using the microwave-assisted digestion combined with ultraviolet radiation (MW-UV) method, 700 mg of macroalgae were digested using a diluted HNO₃ solution (2 mol L^?1). The accuracy was evaluated by analysis of certified reference materials of aquatic plant (BCR 060) and apple leaves (NIST 1515). Agreement with reference or informed values for all analytes ranged from 94 to 106%, except for As and Mo in BCR 060. The results were also compared with those obtained by the reference method, which did not present a significant difference (t test, 95% confidence level). Based on the results, analysed samples showed considerable variations in regards to the concentrations of Zn, Ni and Mn. Moreover, a relatively high concentration of As in Desmarestia anceps and Pyropia endiviifolia was observed. The results for most of the analytes in Antarctic macroalgae were in agreement with other studies that have been published in literature. In addition, the proposed method was suitable for determining toxic and essential elements in Antarctic macroalgae reducing reagent consumption and waste generation, which is in agreement with the green chemistry recommendation.     

水的饱和辛醇溶液水分标准物质的研制

生物燃料的国家标准规定了产品的技术指标和相应的检测方法。水的饱和辛醇溶液的水分标准物质,用于生物燃料水分测量时仪器的校准和方法的验证,能够保障测量结果的准确可靠和等效一致。该标准物质采用卡尔·费休库仑法、卡尔·费休容量法和定量核磁共振等三种不同原理的方法定值。通过方法研究和改进,实现了库仑法和容量法的一致;通过引入新的核磁共振方法,提高了结果的准确性。最终标准物质的水分量值为4.76％,扩展不确定度为0.09％。     

Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA

The measurement of soluble cytokines and other analytes in serum and plasma is becoming increasingly important in the study and management of many diseases. As a result, there is a growing demand for rapid, precise, and cost-effective measurement of such analytes in both clinical and research laboratories. Multiplex bead array assays provide quantitative measurement of large numbers of analytes using an automated 96-well plate format. Enzyme-linked immunosorbent assay (ELISAs) have long been the standard for quantitative analysis of cytokines and other biomarkers, but are not well suited for high throughput multiplex analyses. However, prior to replacement of ELISA assays with multiplex bead array assays, there is a need to know how comparable these two methods are for quantitative analyses. A number of published studies have compared these two methods and it is apparent that certain elements of these assays, such as the clones of monoclonal antibodies used for detection and reporting, are pivotal in obtaining similar results from both assays. By careful consideration of these variables, it should be possible to utilize multiplex bead array assays in lieu of ELISAs for studies requiring high throughput analysis of numerous analytes.     

Microwave-assisted acid extraction methodology for trace elements determination in mastic gum of Pistacia lentiscus using inductively coupled plasma atomic emission spectrometry

Introduction – To ensure food safety, accurate knowledge of the levels of several trace elements is necessary. This is also true for natural products of plants and resins used for human consumption or therapeutic treatment, like the mastic gum of Pistacia lentiscus. The rapid analysis of gum and resin matrices is a challenge because there are problems with the decomposition of such complicated matrices. Objective – To develop an efficient multielemental analytical method for the determination of trace elements and to compare different procedures for analyte extraction when microwave‐assisted digestion is applied. Methodology – The inductively coupled plasma atomic emission spectrometric (ICP‐AES) technique was applied and the optimum ICP conditions like radiofrequency power, argon flow rate and nebuliser sample uptake flowrate were found. The microwave‐assisted procedure was compared with that with conventional heating. Since mastic and resinous materials are difficult for dissolution and extraction of trace element, influential acid mixtures containing hydrofluoric acid proved to be capable of quantitative extraction of the analytes. Results – The digestion of mastic resin or similar matrices is significantly facilitated by using microwave radiation instead of conventional heating since the obtained recovery for several analytes is much higher. It was proved that the acid mixture of HCl–HNO₃–HF was the most efficient for complete sample digestion and recovery of the analytes. Conclusions The performance characteristics of the developed method were evaluated against certified reference material and the method was proved reliable and applicable to the analysis of mastic gum and possibly to similar resinous matrices. Copyright © 2010 John Wiley & Sons, Ltd.     

Vanadium levels in urine and cystine levels in fingernails and hair of exposed and normal persons

Vanadium was determined by radiochemical neutron activation analysis (RNAA) with proven accuracy in urine of workers occupationally exposed to vanadium-rich dust in a vanadium pentoxide production plant, and values in the range of 3.02–762 ng/mL (median 33.0 ng/mL) were found. In a control group consisting of administrative workers of the plant, urinary vanadium levels were found in the range of 1.05–53.4 ng/mL (median 2.53 ng/mL), whereas in an another control group of occupationally nonexposed persons, these values amounted to 0.066–0.489 ng/mL (median 0.212 ng/mL). Accuracy of the results was tested by analysis of reference material IAEA A-13 Animal Blood and NIST SRM-1515 Apple Leaves, and very good agreement was found with literature and the NIST certified values, respectively. Unlike urine, no significant differences were found for cystine levels in fingernails and hair of exposed and control persons.     

Miniaturized bioanalytical systems: enhanced performance through liposomes

Biorecognition-element labeled liposomes are simple and versatile tools used to amplify signals for the detection of analytes of environmental, clinical, food safety, and national security interest. Relying on measurement of encapsulated species via electrochemical or spectroscopic techniques, or properties inherent to liposomes themselves (such as mass, refractive index, or charge), many advances have been made in both bench-scale and microfluidic applications. Some of these measurement techniques are inherently sensitivity limited, but through the inclusion of liposomes, reduced limits of detection potentially broaden the utility towards otherwise challenging levels of analytes. Other advances took advantage of the hydrophobic environment required by many biorecognition elements to expand the target selectivity range or utilized the amphipathic nature of the lipid bilayer to provide enhanced separation capabilities. Novel handling approaches included wavelength-specific release of contents encapsulated within thermosensitive liposomes or application of electric fields to move, concentrate, and strategically lyse liposomes. These and other topics are discussed in terms of either present incorporation or adaptation to microfluidic devices.     

Transition metal abundances in microbial carbonate: a pilot study based on in situ LA-ICP-MS analysis

The ability to recognize the former existence of microbes as well as the biological origin of marine precipitates, such as putative microbialites, is crucial for understanding the development and history of early life on Earth. Increasingly, such rocks hold keys to understanding the geochemical evolution of the oceans and linked Earth systems. Vital trace elements previously have received relatively little attention as clues to the origin of carbonate rocks, and low abundance transition elements in particular, have been difficult to analyse in carbonate matrices for technical reasons. We have used laser ablation–inductively coupled plasma–mass spectroscopy for the in situ measurement of a broad suite of vital transition metals in Late Devonian reefal limestones that contain coeval microbialite (the calcimicrobe Renalcis), stromatoporoid sponge skeleton, early marine cement, and later diagenetic cement. Comparative experiments conducted in two different ion extraction modes determined theoretical detection limits for transition elements on NIST reference material SRM 612. Analyses of NIST glasses SRM 614 and 616 demonstrate accuracy relative to previously published data. On that basis we have identified significant enrichment of the vital elements V, Sn, Cu and Zn within the Renalcis. The stromatoporoid skeleton by contrast is enriched only in V. Earliest cements, which also may have been mediated to some degree by microbial biofilms on the basis of their morphology, show a much smaller degree of enrichment, and later cements show no enrichment, with the exception of Zn, which is concentrated in the latest cement. Fine particulate carbonate sediments (micrite) show variable metal enrichments that are attributable to varying contributions from detrital siliciclastic contamination. Renalcis was also enriched above the sponge and cements in regards to Mn, Cd, Co, and possibly Cr, but at less robust levels. Molybdenum and Sb were found not to be enriched in the Renalcis, and Ni, although clearly very low in concentration, could not be evaluated owing to its high detection limit. We additionally were able to identify specific zones of contamination in Renalcis encountered as the laser drilled deeper into the carbonate. Time resolved analysis allows exclusion of such contaminants from integration into the results. Successful application of the new technique will now allow us to assess metal uptake in ancient carbonates with implications for interpreting the biogenicity of putative microbialites.