Preparation Of Reference Materials Research Articles

Diagnostic and prognostic significance of biomarkers

In modern laboratory medicine there is a growing tendency for finding out the most specific and most sensitive biomarkers as indicators of a disease and diagnostic means for the follow-up treatment and therapeutical results. Detection of sensitive biomarkers was speeded up by world companies as manufacturers of diagnostic reagents. Today, the determination of some biochemical parameters is performed by immunodetermination which uses monoclonal antibodies and different markers, and thus achieving the desired high specificity of determination. The selections of biomarkers which are the most important for the clinical practice and which will give the best diagnostic information, requires large researches including analytical clinical and economic properties. Thanks to statistical methods, especially to ROC analysis, the so-called cutoff-values are determined. On the basis of these values differentiation between sick people and healthy individuals is possible. In other words, the evaluation of clinical specificity requires also inclusion of patients whose clinical picture reflects the disease for which a marker is tested. The development and choice of biomarkers is also helped by IFCC with suggestion of the best procedures and preparation of reference materials. The use of selected biomarkers must be profitable in respect of the price and diagnostic information. In the paper the principles of selection, use diagnostic and prognostic significance of biomarkers is discussed. The following biomarkers served as examples: cardiovascular (troponin T and I myoglobin, CK-MB mass), bone (deoxypyridinolin, osteocalcin, Cterminal propeptide collagen, type I, osteo-alkaline-phosphase), malignant (PSA) and for pancreas diseases (lipase, PAM).

Electron Probe Microanalysis of Environmental Samples: Preparation of Reference Materials, Correction for Particle Size Effects and Representative Results on Sediments Recovered from Snow, Power Station Fly Ash and Bone Samples

In this paper we report the results of electron probe X‐ray microanalysis (EPMA) that was used to study environmental materials. The mode of preparation and certification of reference samples based on a basaltic glass matrix for environmental applications of EPMA is described. These samples were prepared containing scandium, strontium and barium (from 0.03 up to 7% m/m) and an evaluation was made of homogeneity, their stability to local heating, followed by analysis by independent methods for certification of composition. Matrix correction procedures for the EPMA technique have been developed for particles having a size commensurable with the volume of X‐ray generation. An analytical equation for the size factor is proposed and two techniques for selecting optimum conditions for the analysis of environmental samples by EPMA are reported. These procedures provided satisfactory results when utilized in analysing sediments recovered from snow, coal fly ash and the bones of animals and fish, results from which can be used as indicators for evaluating the pollution level of the lower atmosphere, surface and ground water, as well as revealing pollution mechanisms.

Development of Reference Materials for Paralytic Shellfish Poisoning Toxins

A project was undertaken to develop mussel reference materials that were certified for their mass fractions of saxitoxin and decarbamoyl-saxitoxin. Fifteen laboratories from various European countries participated. Three of these had major responsibility for substantial parts of the work and overall coordination of the project. The project involved 4 main activities: (1) procurement and characterization of calibrants; (2) improvement of analytical methodology; (3) preparation of reference materials, including homogeneity and stability studies; (4) 2 interlaboratory studies and a certification exercise. The joint activities resulted in 3 homogeneous and stable reference materials: 2 lyophilized mussel materials with and without naturally incurred paralytic shellfish poisoning (PSP) toxins, and a saxitoxin enrichment solution. The reference materials were certified with respect to their saxitoxin and decarbamoyl-saxitoxin content. The lyophilized mussel material with PSP toxins (CRM 542) contained <0.07 mg saxitoxin x 2HCl/kg and 1.59 +/- 0.20 mg decarbamoyl-saxitoxin x 2HCl/kg. The lyophilized mussel material without PSP toxins (CRM 543) contained <0.07 mg saxitoxin x 2HCl/kg and <0.04 mg decarbamoyl-saxitoxin x 2HCl/kg. The certified value of the saxitoxin mass fraction in the saxitoxin enrichment solution (CRM 663) was 9.8 +/- 1.2 microg/g.

Purification of human adenosine deaminase for the preparation of a reference material

The goal was to optimise a purification procedure of adenosine deaminase from human erythrocytes for the preparation of a European Reference Material. Adenosine deaminase was purified from human erythrocytes with a specific activity of 4.46 μkat/mg of protein and a catalytic concentration of 133 μkat/l. The isolation and purification procedure involved ion-exchange chromatography (STREAMLINE™ DEAE), and two purine riboside affinity chromatographies. The purified enzyme exhibits a single band in SDS–PAGE with a molecular weight of 41 600 g/mol, and three bands in PAGE, isoelectric focusing and two-dimensional electrophoresis with pI 4.7, 4.85 and 5.0.

The IAEA programme and perspectives for environmental radionuclide monitoring and preparation of reference materials

The recent achievements of and perspectives for the IAEA programme on measurements of radionuclides in the environment are reviewed. The programme includes sampling and measurement campaigns for the assessment of the radiological situation in potentially contaminated regions and the establishment of a world-wide network to assist the IAEA in carrying out those assessments. Furthermore the programme includes the different actions pertaining to the improvement of the quality of radioanalytical measurements in the Member States. In addition to training and assistance in the implementation of quality systems, the programme includes the development, compilation, review and dissemination of radioanalytical methods, the organisation of interlaboratory comparisons and proficiency tests and the preparation, characterisation and distribution of reference materials.

National and international veterinary reference laboratories for infectious diseases.

Reference laboratories play an increasingly important role in the harmonisation of laboratory diagnostic tests and the standardisation of veterinary vaccines. This is particularly important in building confidence between international trading partners. The authors review aspects of the organisation, designation and support of reference laboratories for infectious diseases of animals and discuss the principal activities which such laboratories would normally perform. These activities include advice and consultancy, publications and communication, training, research, disease surveillance, maintenance of culture collections, evaluation of reference methods, preparation of reference materials and organisation of inter-laboratory comparisons.

Preparation of reference materials for carbon and oxygen stable isotopes of carbonates

Preparation of reference materials for carbon and oxygen stable isotopes of carbonates

An international interlaboratory study for the production of a sewage sludge certified reference material for routine use in inorganic quality control

Two existing interlaboratory schemes are used to certify a sewage sludge reference material. Data from the analysis (As, Cd, Cr, Cu, Pb, Hg, Ni and Zn) of the material using routine procedures, US EPA and Netherlands NEN, plus INAA and IDMS and an interlaboratory comparison in Eastern Europe have been examined. The Dutch results show significantly lower analytical results for As for the graphite furnace technique compared to HG-AAS, probably due to matrix-effects. The differences between the Dutch and USA results for Cr and Ni suggest that the USEPA 3050A digestion method for dissolution of the metals is less aggressive for this material compared to aqua regia destruction. For international comparability of analytical data it is essential to harmonize method dependent dissolution/extraction techniques. The preparation of reference materials which mimic real samples and certification by ILS result in useful materials for quality control.

Experience of the National Institute of Metrology (INM) in development and certification of reference materials used in the metrological assurance of clinical laboratory measurements

In order to provide quality assurance in the field of clinical chemistry, required by European Standards, the Romanian national metrological infrastructure has to be improved. The main objectives of this important activity are metrological assurance of concentration measurements, the assessment of the uncertainty, and the traceability of measurements. Within this framework, reference materials play an important role. The experience of INM in the preparation, certification and use of reference materials and in metrological assurance of clinical laboratory measurements (especially when absorption photometric devices are used) is described. The combined uncertainty associated with chemical preparation of these reference materials is estimated.

Production of CTC-containing porcine reference materials.

The European Commission (EC) established the Standards, Measurements and Testing programme for the preparation of Reference Materials (RMs) as an aid to harmonise testing for veterinary drug residues throughout the European Union (EU). The production of chlortetracycline (CTC)-free and CTC-incurred pig tissues as candidate RMs is described. High performance liquid chromatography (HPLC) with fluorescence detection of CTC and 4-epi-CTC was used for all tissue analyses. A pilot study revealed that incurred CTC residues were stable in pig kidney, liver and muscle lyophilised powders during storage for 10 weeks at -70, -20 and +37 degrees C, obviating the need for addition of a stabiliser (thimerosal). In the main study, 500 vials each of CTC-free and CTC-incurred kidney, liver and muscle were produced. Target concentrations in the CTC-incurred lyophilised tissue powders were 750-1500, 500-1000 and 300-600 micrograms kg-1 for kidney, liver and muscle, respectively. Following lyophilisation, the mean +/- s concentrations of CTC in the incurred positive RMs were 1,315 +/- 56.9, 765 +/- 35.3 and 378 +/- 16.8 micrograms kg-1 for kidney, liver and muscle respectively. Residual moisture in the RMs ranged from 1.6 +/- 0.53% for muscle to 3.0 +/- 0.50% for liver. Between-vial homogeneity for incurred powders was determined for 20 vials of each material, which had been removed at regular intervals during the filling process. Relative standard deviations (RSDs) for kidney, liver and muscle were 4.3, 4.6 and 4.4% respectively, being within the interassay RSD of the method and indicating that mixing was effective. Stability of powders stored at -18, 4, 20 and 37 degrees C was assessed over a period of 79 weeks. No measurable degradation occurred over this time period at any of the storage temperatures. It is concluded that these candidate RMs are homogenous, stable and are suitable for certification.

Concentrations of iodine determined by pre-irradiation combustion and neutron activation analysis in powdered grass as a function of particle size

Pre-irradiation combustion of a sample to liberate iodine, trapping the iodine on charcoal and quantifying the element by neutron activation analysis (NAA), has proven to be a reliable technique for the determination of iodine in biological samples. This method has been applied to the determination of iodine content of a powdered grass material that was prepared by another laboratory for use as an in-house quality assurance material. Analyses of aliquots of the material yielded results that ranged from 0.45 to 0.96 μg/g of iodine, so additional studies were undertaken in order to determine material homogeneity. Visual inspection indicated that the material was inhomogeneous with respect to particle size. To determine whether various sizes of particles contained different amounts of iodine, the material was sieved into four differently sized fractions and a portion of each fraction was analyzed. Variations in iodine concentration as a function of particle size were observed. The fraction containing particles of sizes greater than 350 μm, the woody and fibrous plant parts, contained the least iodine (0.43 ± 0.09 μg/g, average ± expanded uncertainty), while iodine concentrations in the subsequent three fractions increased with decreasing particle size with a maximum concentration of 0.89 ± 0.13 μg/g for particles less than 75 μm. These results support conclusions of many researchers that trace element concentrations are inhomogeneously distributed in various cells and structural parts, and emphasize the importance of achieving uniform particle sizes during sample grinding and blending in the preparation of reference materials.

Reference materials and analytical standards to stimulate improved laboratory performance: Experience from the external quality assessment scheme for trace elements in biological samples

The performance of a large number of laboratories measuring trace elements in biological fluids has been monitored over many years by examination of their results in the Guildford external quality assessment scheme. Specific experiences of the UK trace elements reference laboratories have been used to stimulate improvements in performance of other participants in the scheme. The key features of these initiatives were: specially prepared reference materials, used as internal quality control specimens within a common procedure, contributed to accuracy control; proposed standards of satisfactory and unacceptable analytical performance associated with a new system for scoring; regular non-threatening open discussion of performance with interested colleagues. The impact of these features is illustrated with reference to measurements of Al and Zn in serum and Pb in whole blood.

Analytical requirements and standardization of CA 15—3

The CA 15-3 assay could play, probably in combination with cytokeratin markers, a role in the detection of early recurrence and in monitoring therapy in metastatic breast cancer patients. The CA 15-3 assay is, however, not useful for early detection of breast cancer and has no prognostic significance. The analyte is not clearly defined and a primary reference material cannot be synthesized. A reference measurement procedure is not available and many commercial assays use the original Centocor IRMA as such. The comparability of different CA 15-3 assays is not sufficient according to the results of EQAS. The general recommendations of analytical requirements for tumour marker determinations specified by the working group "Quality Control and Standardization" (Hamburger Symposia on Tumour Markers) includes intra-assay and inter-assay precision, drift, carry-over, dilution effect and high-dose hook effect. From a medical point of view reference limits should be established. Specificity/sensitivity profiles should preferentially be represented by ROCcurves. Confounding factors should be analyzed. To improve the clinical value of CA 15-3, the following needs for improvement have been identified: > Standardization of response criteria during treatment; > Definition of significant increase at recurrence; > Investigation of the biological variance during follow-up; > Definition of the analyte; > Preparation of reference material; > Development of an accepted reference method; > Integration of different international activities and > Optimizing decision-making criteria and clinical application.

Interactive programme for evaluation of circular analyses (IPECA)

A PC-based interactive programme is described which is designed to help in suggesting the best estimate of the true value of an analyte content from results of collective studies aiming at deriving consensus values and/or reference material preparation by employing combined statistical and analytical considerations. The Grubbs, Dixon, Huber tests, and the coefficients of skewness and curtosis tests are used for outlier detection, the Bartlett, Cochran, and the standard error tests are employed for testing variance homogeneity testing and/or variance outliers identification, while the normality of results distribution is tested according to the Kolmogoroff-Smirnoff-Lilliefors and Shapiro-Wilk tests. One-way analysis of variance (ANOVA) is employed to test differences among means of results obtained in different conditions (laboratories, analytical methods, etc.) and to calculate the overall mean and its confidence interval accordingly. Points for an analytical discussion are given which should be considered prior to a decision whether a result of a trace element determination, identified as an outlier from statistical reasons, should be rejected.

Trace element standards for the electron microanalyser using layered and ion-implanted materials

The merits and limitations of thin surface film deposition and ion implantation for preparing reference materials for quantitative analysis of trace elements using wavelength-dispersive X-ray spectrometry with the electron probe microanalyser are illustrated and discussed. The objective is to generate X-ray intensities typical of elements present at trace levels and to relate the measured intensities to the mass concentration of these elements when assumed to be homogeneously distributed within bulk materials. For this purpose, X-ray intensity calculations based on the use of analytical expressions for both the depth–ionization and the depth–concentration distributions, respectively, are presented. It will be demonstrated that gold-layered and materials ion implanted with gold can be advantageously used to validate the experimental approaches used for X-ray intensity measurements in the presence of distortions in the shape of an X-ray peak and the underlying continuous emission distribution, and also to test the algorithms and the physical parameters used in the analytical descriptions of the ionization and concentration profiles as a function of depth. The contribution of layered and ion-implanted compounds to a better understanding of surface and bulk charging phenomena in strongly insulating specimens is also presented.

Standardization of reagents and methods used in cytological and histological practice with emphasis on dyes, stains and chromogenic reagents.

The need for the standardization of reagents and methods used in the histology laboratory is demonstrated. After definitions of dyes, stains, and chromogenic reagents, existing standards and standards organizations are discussed. This is followed by practical instructions on how to standardize dyes and stains through the preparation of reference materials and the development of chromatographic methods. An overview is presented of the problems concerned with standardization of the Romanowsky-Giemsa stain for cytological and histological application. Finally, the problem of how to convince routine dye and stain users of the need for standardization in their histology laboratories is discussed.

Reference materials and environmental specimen banking

The preservation and protection of the environment has become a major challenge of our times since this has to be compatible with the ecological needs. The concept of Environmental Specimen Banking (ESB) acquires importance in providing the data base for deeper insight into ecochemical processes. Methodological development and preparation of reference materials (RMs) are also essential to such studies. Systematic efforts are to be made to acquire a good number of reference materials to be useful for analysis of toxic as well as essential components. Our experience has shown that it is possible to procure worthy and scientifically valuable specimens from soils and leafy food substances. Exploring preparation of suitable specimens from a country such as India might be a worthy exercise since it is possible to obtain clean specimens in this part of the world. Analytical data for determination of some trace and major elements in prepared RMs are presented.

Ammonium Acetate Extracts and Their Analysis for the Speciation of Metal Ions in Soils and Sediments

Abstract The use of ammonium acetate (1 mol/l at pH 7) extraction of soils and sediments for the speciation of metal ions is briefly discussed. Because the sensitivity of flame atomic absorption spectrometry (FAAS) is insufficiently sensitive for the determination of many of the heavy metals in ammonium acetate extracts of unpolluted, and even in some polluted soils, the use of electrothermal atomic absorption spectrometry (ETAAS) was studied. A general procedure, using graphite furnace atomisation and the “universal” matrix modifier, palladium, was developed, that was sufficiently sensitive for the determination of Cd, Cr, Cu, Ni, Pb and Zn even in unpolluted soils. The concentration of zinc, however, will almost always be high enough for determination of FAAS and this method is to be preferred to ETAAS for this element. While, for Cr, Cu, Ni and Pb, direct calibration with external standard solutions is practicable, it is necessary to use the standard additions calibration method for cadmium, to avoid matrix interference effects. The standard additions technique is recommended, however, because it compensates for real differences in the operating parameters of different instruments set to the same nominal values. This is particularly important for interlaboratory comparisons or for certification analyses in the preparation of reference materials.

Improvements in the determination of vitamins in food through intercomparisons and preparation of RMs for vitamin analysis within the BCR1 programme

In order to improve the methods for the determination of vitamins in food for nutritional purposes, the Commission's Community Bureau of Reference (BCR) has initiated a comprehensive research programme consisting of intercomparisons of methods to identify and eliminate sources of error and the preparation of reference materials (RMs). Six food RMs have been prepared to date including brussels sprouts, mixed vegetables and pigs' liver (all in the lyophilised form), vitamin enriched milk powder, wholemeal flour and margarine. The first five materials have been packaged into heat sealable, aluminium-laminate sachets under an inert atmosphere; margarine is a canned product. The initial homogeneity results have indicated no detectable signs of inhomogeneity for the vitamins/RMs investigated. Stability testing has monitored both short-term stability at elevated temperatures (+25 to 40°C, 8 weeks) and long-term stability −30 to +20°C, 36 months). The former was used to evaluate the effect of adverse shipment conditions on vitamin stability. Vitamins C and B1, two of the more labile vitamins, have been found to be stable for up to 4 weeks at +25°C and 8 weeks at +37°C in brussels sprouts (RM 431) and wholemeal flour (RM 122), respectively.

CII: What is it?

The CII, a group of 30 laboratories, has for its aims the preparation of reference materials from agricultural or environmental sources for chemical analysis and the development of reference analytical methods. The tasks and organization of the CII are presented.