Development of a non-competitive immunoassay for cortisol and its application to the analysis of saliva

A general method of performing non-competitive immunoassays for a low-molecular-mass analyte was developed and applied to cortisol determination in saliva samples. The method is based on the use of a “blocking reagent”, which is able to bind to antibody sites not occupied by the analyte, and in a stronger way than the analyte itself. When an enzyme-labelled analyte is added it substitutes the analyte in the antibody complex, but not the blocking reagent. The measured signal is linearly correlated to the concentration of the complex and, consequently, to the analyte concentration. The 3σ limit of detection (LOD, 0.2 nmol l⁻¹) obtained by the above method was 10 times lower than that obtained by the corresponding ELISA. As non-competitive immunoassays reported for small molecules up to now have been no more than just approaches, the suitability of the proposed assay for cortisol quantification in a real matrix was investigated. Human saliva was chosen as a matrix because of the need for very sensitive techniques to determine salivary cortisol content. The matrix effect was offset by performing the calibration experiments in acidic conditions (pH=5.6) and adding 0.1% of bovine serum albumin (BSA) to the buffer. In these conditions, the LOD was 1.4 nmol l⁻¹, which was adequate to measure normal levels of cortisol. Spiked samples were analysed and gave recoveries ranging from about 80 to 120%. Therefore, five subject samples, collected over 18 h showed salivary cortisol concentrations compatible with the circadian variation of reported normal values.     

National External Quality Assessment Schemes for microbiology, parasitology, and virology in Europe

Quality assurance is an important aspect of laboratory management. One of the activities involved is the participation in external quality assessment (EQA) schemes by the clinical laboratory. These EQA schemes should be organised according to well-defined guidelines, such as the ESO/IEC Guide 43. The present work provides an inventory of the EQA schemes organised in Europe. The schemes are related to the fields of bacteriology, parasitology and virology. For each field various surveys have been organised. Data such as the number of participants, frequency of survey and number of samples in a survey are presented. The nature and way of manufacturing the control material is specified.     

External quality assurance programmes in medical laboratories

 Medical laboratories have a long tradition of external quality assessment. Starting from pure quality control of laboratory performances, most schemes have evolved to a powerful tool for improving quality of clinical outcome of results. External quality assurance in medical laboratories not only includes laboratory performance evaluation, but also evaluation of method performance, post-marked vigilance, training and help. In the future, the quality of programmes must further be improved by accreditation of schemes and by using electronic data interchange. Received: 9 December 2000 Accepted: 14 December 2000     

The Swiss External Quality Assessment Scheme in Bacteriology and Mycology 1992–1996

 The Swiss External Quality Assessment Scheme in Bacteriology and Mycology was created in 1980 and has been organised since 1983 by the Department of Medical Microbiology in Zurich. The number of Swiss participants has steadily risen from 66 in 1989 to 92 in 1996. Twelve bacterial and fungal strains are sent to the participants in four despatches, each containing three specimens, per year. Scores are allocated per specimen and range between 0 and 1. Participants with mean scores of ≤0.75 are considered poor performers. The mean scores increased from 0.85 in 1992 to 0.91 in 1996. This improvement can be attributed to the educational effect of the external quality control scheme, since all participants receive a detailed discussion for each specimen together with their individual results. On average, both large University and Cantonal (state) laboratories as well as private laboratories show satisfactory performance. In particular, laboratories officially recognised by the Swiss Federal Office of Public Health (SFOPH) rate better than non-recognised participants. Many small regional hospital laboratories, most of them not SFOPH-recognised, are often among the poor performers. They are often managed by technical staff and lack a trained microbiologist. The recently introduced legislation in Switzerland renders participation in external quality assessment schemes compulsory, and all clinical microbiology laboratories are required to employ qualified microbiologists. This will certainly help to improve the quality standards of all laboratories performing microbiological tests. Received: 13 November 1997 · Accepted: 28 December 1997     

Application of nanofiber‐packed SPE for determination of salivary‐free cortisol using fluorescence precolumn derivatization and HPLC detection

Salivary cortisol has emerged as an easy‐to‐collect biologic marker of stress in many researches. In this study, we present a method for the determination of salivary‐free cortisol using HPLC method with fluorescence precolumn derivatization, which is based on a novel extraction from the strongly acidic medium (fluorescent derivatives of cortisol in sulfuric acid medium) by electrospun polystyrene nanofibers packed SPE. For high‐throughput sample extraction, an array pretreatment device based on nanofibers packed SPE micro‐column was designed. The LOD of cortisol was 0.01 μg/L (S/N=3). The RSDs (n=6) for all analytes were below 8.0%, and the recoveries were 110, 102.4, and 99.4% (n=3) for saliva spiked with 0.1, 10, and 20 μg/L of cortisol, respectively. The proposed method was then successfully applied in the determination of free cortisol in human saliva. The salivary cortisol concentrations in the real samples ranged from 0.22 to 7.45 μg/L. The nanofiber‐packed SPE overcame the low extraction recovery and bad clean‐up effect of the conventional methods, and increased the sensitivity and selectivity of the method.     

External quality assessment schemes for clinical analysis in Russia

 The history and the present condition of external quality assessment (EQA) schemes for clinical laboratories in Russia are described briefly. The creation of EQA programmes started in Russia in the 1980s. Now almost 20 years later these schemes have been transformed. The National External Quality Assessment Scheme ensures quality control in the clinical chemistry sector (more than 2000 laboratories) and is the most powerful scheme in Russia. The (Sistema Vneshnego Kontrola Katchestva, *) (BKK) system, covering about 120 Russian laboratories, and a lot of local regional programmes (mainly in Siberia), is also very active. The purposes and design of the operating programs, reference materials used, algorithms of estimation, modes of result representation and development prospects are considered. The basic obstacle to the development of EQA schemes in Russia is financial restriction.     

Applications of the Internet to PT

The Internet provides laboratory professionals with a unique opportunity to come together as a network and to develop an integrated system for monitoring the quality of testing in their laboratories on a real-time basis. Once established this network could provide a quality and standards promoting infrastructure for catalyzing the standardization process. Through a collaborative sharing of information on performance and through the use of a common database with harmonized test materials, proficiency testing providers can play a pivotal role in developing such a system. The challenge is now up to us.     

Calibration bias in the determination of polynuclear aromatic hydrocarbons in water

 We present the results of an investigative interlaboratory test to determine the reasons for poor agreement between data from different laboratories for the determination of polynuclear aromatic hydrocarbons in water. Received: 2 July 1998 · Accepted: 1 August 1998     

Quality assurance in clinical chemistry laboratories in the UK

 Since the mid-1960s quality assurance in clinical chemistry has progressed from a need to define and improve precision and accuracy in analytical test procedures to an all-embracing process of assuring that the whole process of pre-analytical, analytical and post-analytical phases of handling patient samples is managed effectively and efficiently. Automated and computer-controlled equipment has reduced many of the analytical errors, in particular in imprecision, that were present in manual analysis. New management techniques have been developed to control the quality and appropriateness of results. Developments in internal quality control and external quality assessment procedures have enabled laboratories to continually improve the quality of assays. Laboratory accreditation and external quality assessment scheme accreditation have ensured that peer review and peer pressure have been applied to both laboratory and external quality assessment scheme performance. As the NHS reviews its priorities and places more emphasis on primary care provider demands, hospital laboratories will of necessity assist with near patient testing outside the laboratory. This will provide new challenges to the quality of the service provided. Received: 2 July 1998 · Accepted: 1 August 1998     

An inter-laboratory comparison study for the determination of copper and lead from the wastewater of printed circuit board manufacturing industry in Pakistan

This inter-laboratory comparison study was arranged for 28 laboratories from different public and private sector organizations in Pakistan having wastewater testing capabilities aimed at improving the quality and comparability of test results. This national inter-laboratory study was started in December 2003 and completed in July 2004. Laboratories were invited to analyze the wastewater collected from printed circuit board (PCB) industry for lead and copper contamination. The samples fulfill the criteria for homogeneity and stability as done by the reference laboratory. The results obtained from participating laboratories were analyzed in terms of Hampel Test for outliers, while the performance evaluation of the participating laboratories was done on the basis of Z-score. An assigned value derived from the participant's results was compared with a reference value provided by a reference laboratory. Overall >50% of the participating laboratories have shown good performance in this PT-program     

A validation concept for purity determination and assay in the pharmaceutical industry using measurement uncertainty and statistical process control

 The analytical chemists in process development in the pharmaceutical industry have to solve the difficult problem of producing high quality methods for purity determination and assay within a short time without a clear definition of the substance to be analyzed. Therefore the quality management is very difficult. The ideal situation would be that every method is validated before use. This is not possible because this would delay the development process. A process-type quality development approach with an estimation type fast validation (measurement uncertainty) is therefore suggested. The quality management process consists of the estimation of measurement uncertainty for early project status. Statistical process control (SPC) is started directly after measurement uncertainty estimation and a classical validation for the end of the project. By this approach a process is defined that allows a fast and cost-efficient way of supporting the development process with the appropriate quality at the end of the process and provides the transparency needed in the development process. The procedure presented tries to solve the problem of the parallelism between the two development processes (chemical and analytical development) by speeding up the analytical development process initially. Received: 25 March 1997 · Accepted: 17 May 1997     

饮酒者唾液中乙醇浓度的酶催化光度法测定

研究了醇氧化酶-过氧化氢酶联合的酶促反应的最佳条件,建立了简便测定乙醇的吸光光度法,乙醇浓度在0.0686~5.115 mmol/L范围内符合比尔定律,回归方程为:A=0.00403 0.08585c(mmol/L),相关系数为0.9986,检出限为0.0172 mmol/L(S/N=3),本法所用仪器简单,已用于酒后唾液中乙醇的测定。     

Quality assurance programs for countries in need: a global view from the College of American Pathologists

Providing laboratory external quality assessment (EQA) programs for countries in need requires special considerations not ordinarily part of EQA for laboratories in industrialized countries. Cultural, professional, service and economic factors must be understood and accommodated in order to carry out successful programs. Coordination of worldwide efforts for countries in need requires more resources and planning than have thus far been devoted to the enterprise.     

Statistical evaluation of stability data for assessing variability of an analytical method

 Stability assay results of a stable solid oral dosage form, designated Product X 5 mg, were subjected to a statistical analysis in view of evaluating the intra-team repeatability and the interval-to-interval reproducibility of the analytical method. Analysis of variance and analysis of method capability were performed on stability data obtained from tablets stored in four different packages over a two-year period. Replicate-to-replicate and period-to-period standard deviations of assay results of the active drug ingredient were found to vary among the packages and were up to 1.3 and 1.5% (% assay), thus leading to maximum differences between any two reported average results of up 4.7%. Analysis of method capability showed that up to 51% of the specification range (90–110%) is taken up by variations of the reported average stability results. This large but realistic method variability does not reflect the formation of low-level impurities (ca. 0.1%) required by regulatory agencies. Received: 10 December 1998 · Accepted: 25 January 1999     

Quality assurance in analytical measurement

 The peculiarities of analytical measurement require to check characteristics of the error (its components) of the obtained analysis results to assure the quality of the measurements. This article deals with the various quality assurance procedures and algorithms which are used to check the quality indices, i.e. the accuracy, reproducibility, certainty and repeatability of analytical measurements: These procedures include: laboratory rapid control; Intra-laboratory statistical control (statistical selection control by alternative attribute, statistical selection control by quantity method of periodic check of the analysis procedure for conformity to the specified requirements) and external control (inter-laboratory control checks, inter-laboratory comparison tests, and intra-laboratory control algorithms carried out by the appropriate supervisory body.) in the separately taken laboratory. The respective algorithms, control plans and control requirements, specified according to the different control aims and assurance tasks, enable the quality and certianty of analytical information obtained in laboratories in Russia to be assured. Received: 9 November 1998 / Accepted: 24 November 1998     

Advances in microbiology EQA

External quality assessment for microbiology laboratories has seen many changes over the past ten years. EQA is much less focused on the pure analytic process of identifying single organisms submitted in an artificial medium. Samples are more realistic, and can be used to address clinical relevancy. Samples can be supplemented with a variety of non-traditional challenges to extend testing to include a wider range of the pre- and post-analytic aspects of the laboratory cycle. A look to the future sees a broad range of newer targets, with increasing interest in virology and point-of-care testing. Received: 13 April 2002 Accepted: 24 June 2002     

Validation of the uncertainty evaluation for the determination of metals in solid samples by atomic spectrometry

 Every analytical result should be expressed with some indication of its quality. The uncertainty as defined by Eurachem ("parameter associated with the result of a measurement that characterises the dispersion of the values that could reasonably be attributed to the, . . ., quantity subjected to measurement") is a good tool to accomplish this goal in quantitative analysis. Eurachem has produced a guide to the estimation of the uncertainty attached to an analytical result. Indeed, the estimation of the total uncertainty by using uncertainty propagation laws is components-dependent. The estimation of some of those components is based on subjective criteria. The identification of the uncertainty sources and of their importance, for the same method, can vary from analyst to analyst. It is important to develop tools which will support each choice and approximation. In this work, the comparison of an estimated uncertainty with an experimentally assessed one, through a variance test, is performed. This approach is applied to the determination by atomic absorption of manganese in digested samples of lettuce leaves. The total uncertainty estimation is calculated assuming 100% digestion efficiency with negligible uncertainty. This assumption was tested. Received: 3 November 1997 · Accepted: 2 January 1998     

Quality in clinical laboratory measurements

 Clinical chemistry deals with measurements and observations using samples from patients in order to supply clinicians with information to support their decisions in diagnosis and treatment. The discipline utilizes advanced chemical and biochemical methods and also sophisticated instrumentation which allows a high throughput. Clinical chemistry has a long tradition of quality assessment and improvement. The major tools have been proficiency testing or external quality assessment and internal quality control. Nowadays, total quality management has become widely recognized, and accreditation according to international, regional or national schemes has contributed to the design of quality systems and improvement of the reliability of results from clinical laboratories.     

Proficiency testing in occupational and environmental health – Current practice and international initiatives for harmonisation

An increasing number of proficiency testing schemes (PT schemes) related to occupational and environmental health have been organised. Most schemes emphasise the importance of the validation of analytical results. With regards to harmonisation of the schemes, there are many differences between the schemes at many levels. These include factors concerning their relationship with legislation, national status, type and quality of proficiency testing material, analytical range and priorities for future development. Since differences between PT schemes have been recognised at the European level by organisers of PT schemes and external quality assessment schemes (EQASs) it seems appropriate to reinforce collaboration between scheme organisers in order to improve the quality of analytical performance in occupational and environmental health.