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).     

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.     

Harmonisation of Measurement Procedures: how do we get it done?

Clinical laboratory measurement results must be comparable among different measurement procedures, different locations and different times in order to be used appropriately for identifying and managing disease conditions. Harmonisation in the broad sense is the overall process of achieving comparability of results among clinical laboratory measurement procedures that measure the same measurand. The term standardisation is used when comparable results among measurement procedures are based on calibration traceability to SI using a reference measurement procedure of the highest available order. When there is no higher order reference measurement procedure available, and it is unlikely that one can be developed, the term harmonisation refers to any process for achieving comparable results among measurement procedures for an individual measurand.This review explains calibration traceability and focuses on the principles of harmonisation for those measurands for which a reference measurement procedure does not exist. We discuss the value of harmonisation, the importance of commutable reference materials, the barriers to harmonisation that exist today, and conclude with a discussion of a current global effort to improve the state of harmonisation.     

The Joint Committee for Traceability in Laboratory Medicine (JCTLM): a global approach to promote the standardisation of clinical laboratory test results

Clinical laboratories are moving towards global standardisation to produce equivalent test results across space and time. Standardisation allows use of evidence-based medicine, eliminates the need of method-specific reference intervals, decision levels and cut-offs, and can be achieved by application of metrological principles. For example, in vitro diagnostics (IVD) manufacturers can make kit calibrators traceable to internationally recognised reference materials and reference methods.The first step towards standardisation is to identify appropriate reference materials and methods. This has been undertaken by a new international consortium, the Joint Committee for Traceability in Laboratory Medicine (JCTLM), formed in 2002. It brings together experts representing the clinical laboratory profession, government agencies, and manufacturers, to promote international comparability, reliability, and equivalence of measurement results in clinical laboratories for the purpose of improving healthcare. Through the efforts of the JCTLM, manufacturers are able to assign values to kit calibrators with consistency using appropriate higher order reference materials and methods, and traceability flowcharts, according to ISO Standards to ensure accuracy of test results and to promote assay performance harmonisation. Users of assay kits can assess suitability of calibrators on the basis of acceptable reference materials and/or methods identified by the JCTLM. The JCTLM exemplifies the dynamic nature of clinical laboratory medicine, the inherent spirit of cooperation among professionals in this scientific field, and the international desire to strive for the highest level of clinical laboratory practice for the benefit of patients.     

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.     

Testosterone and estradiol assays: current and future trends

Sex steroid measurements for the investigation of endocrine disorders have been fraught with accuracy and imprecision problems since the advent of high throughput, direct assays almost 10 years ago on automated analyzers. Results from testosterone and estradiol measurements at the low end of detectability have suffered the most and there are few automated systems that can accurately measure these steroids in women, children and hypogonadal males on a routine basis. With the advent of mass spectrometry coupled to either gas chromatography or liquid chromatography, an improved approach to the measurement of these steroids has developed that shows promise for accurately and precisely measuring testosterone and estradiol in all patient populations including women and children. These mass spectrometry based methods for the sex steroids have been established as higher order reference method procedures that will resolve the issues of low end sensitivity measurements for these steroids, provide for appropriate standardization and reference materials and align most laboratories in hospital and reference laboratories to generate results that are inter-changeable between laboratories and methods.     

A repeated measures approach to pooled and calibrated biomarker data

Participant-level meta-analysis across multiple studies increases the sample size for pooled analyses, thereby improving precision in effect estimates and enabling subgroup analyses. For analyses involving biomarker measurements as an exposure of interest, investigators must first calibrate the data to address measurement variability arising from usage of different laboratories and/or assays. In practice, the calibration process involves reassaying a random subset of biospecimens from each study at a central laboratory and fitting models that relate the study-specific “local” and central laboratory measurements. Previous work in this area treats the calibration process from the perspective of measurement error techniques and imputes the estimated central laboratory value among individuals with only a local laboratory measurement. In this work, we propose a repeated measures method to calibrate biomarker measurements pooled from multiple studies with study-specific calibration subsets. We account for correlation between measurements made on the same person and between measurements made at the same laboratory. We demonstrate that the repeated measures approach provides valid inference, and compare it to existing calibration approaches grounded in measurement error techniques in an example describing the association between circulating vitamin D and stroke.     

The role of SSDL in quality assurance in radiotherapy

This paper describes the role of the Polish Secondary Standard Dosimetry Laboratory (SSDL) in quality assurance in radiotherapy by means of providing calibration of ionisation chambers, TLD postal dosimetry audits and end-to-end audits for radiation therapy. A historical review of the methods and results are presented. The influence of the SSDL in Warsaw on radiation protection of patients in Poland is discussed. The International Atomic Energy Agency together with World Health Organisation (IAEA/WHO), through its network of SSDLs around the world, propagates newly developed methods for calibration and auditing. Suitable high quality equipment was provided by the IAEA, as well as special materials and technical support to the SSDL in Warsaw. The activity of the SSDL and the services provided for Polish radiotherapy centres have resulted in a reduction of discrepancies between planned doses and doses delivered to patients. The newly tested IAEA methods of end-to-end on-site dosimetry audits allow for monitoring and improving the quality of IMRT in Poland. The traceability of standards used for the calibration of therapy level dosimeters from Polish radiotherapy centres is assured by the IAEA dosimetry laboratory. The consistency of methods performed in the Polish SSDL with the ISO:17025 norm is supervised by the Polish Centre for Accreditation – a member of International Laboratory Accreditation Cooperation (ILAC), for calibration and testing. Due to the rapid technological development of radiotherapy, special attention has to be paid to newly developed methods for dosimetry auditing and institutions which provide services for assuring radiation safety of patients.     

Prerequisites for use of common reference intervals

The theory of reference values was developed more than 30 years ago, but its application in most clinical laboratories is still incomplete today. This is for several reasons, the most relevant ones being the lack of standardisation of the analytical methods, resulting in method-dependent values, and the difficulty in recruiting the proper number of reference subjects for establishment of reference intervals. With the recent progress in method standardisation the first problem is reducing while the second can be addressed optimally via multicentre collaborative studies that aim to establish common reference intervals. To be effective this approach requires the following prerequisites: 1) the existence of a reference measurement system for the analyte; 2) field methods producing results traceable to the reference system; and 3) a carefully planned multicentre reference interval study. Such a procedure will produce results traceable to the reference measurement system for a large number of reference subjects, under controlled pre-analytical conditions. It will also enable a better understanding of the various sources of population variability, if there is the need for partitioning of a reference interval or if there are any limitations to adopting the established reference intervals on a national or global scale. Once reference intervals are determined, clinical laboratories can adopt a common reference interval provided: 1) the population that the laboratory services is similar to the one studied; 2) methods producing traceable results are used; and 3) analytical quality is within defined targets of precision and bias. Moreover, some validation of the interval using a small sample of reference individuals from the laboratory's population is advisable.     

A new reference material for UV-visible circular dichroism spectroscopy

To obtain accurate and consistent measurements from circular dichroism (CD) instruments over time and from different laboratories, it is important that they are properly calibrated. The characteristics of the available reference materials are not ideal to ensure proper calibration as they typically only give peaks in one or two spectral regions, and often have issues concerning purity and stability. Currently either camphor sulfonic acid or ammonium camphor sulfonate are used. The latter can be an unstable, slightly hygroscopic secondary standard compound with only one characterized CD band. The former is the very hygroscopic primary standard for which only one enantiomer is readily available. We have synthesized a new reference material for CD, Na[Co(EDDS)].H(2)O (EDDS = N,N-ethylenediaminedisuccinic acid) which addresses these problems. It is extremely stable and available in both enantiomeric forms. The CD spectrum of Na[Co(EDDS)].H(2)O has nine distinct peaks between 180 and 599 nm. It thus fulfils the principal requirements for CD calibration chemical standards and has the potential to be used to ensure good practice in the measurement of CD data, providing two spectra of equal magnitude and opposite sign for a given concentration and path length. We have carried out an interlaboratory comparison using this material and show how it can be used to improve CD comparability between laboratories. A fitting algorithm has been developed to assess CD spectropolarimeter performance between 750 and 178 nm. This could be the basis of a formal quality control process once criteria for performance have been decided.     

Closing the Gaps in Paediatric Reference Intervals: The CALIPER Initiative

Screening, diagnosis and monitoring of paediatric diseases relies on the measurement of a spectrum of disease biomarkers in clinical laboratories to guide important clinical decisions. Physicians rely on the availability of suitable and reliable reference intervals to accurately interpret laboratory test results with data collected during medical history and physical examination. However, critical gaps currently exist in accurate and up-to-date reference intervals (normal values) for accurate interpretation of laboratory tests performed in children and adolescents. These gaps in the available paediatric laboratory reference intervals have the clear potential of contributing to erroneous diagnosis or misdiagnosis of many diseases of childhood and adolescence. Most of the available reference intervals for laboratory tests were determined over two decades ago on older instruments and technologies, and are no longer relevant considering the current testing technology used by clinical laboratories. It is thus critical and of utmost urgency that a more acceptable and comprehensive database be established. There are however many challenges when attempting to establish paediatric reference intervals. Paediatric specimen collection is a major concern for health care providers as it is frequently difficult to obtain sufficient volumes of blood or urine from paediatric patients. Common reference intervals have not been widely implemented due to lack of harmonisation of methods and differences in patient populations. Consequently, clinical laboratory accreditation organisations and licensing agencies require that each laboratory verify or establish reference intervals for each method. To provide such reference intervals requires selection criteria for suitable reference individuals, defined conditions for specimen collection and analysis, method selection to determine reference limits and validation of the reference interval. The current review will provide a brief introduction to the current approach to establishment of reference intervals, will highlight the current gaps in data available in paediatric populations, and review a recent Canadian initiative, CALIPER (Canadian Laboratory Initiative on Paediatric Reference Intervals), to establish a comprehensive database for both traditional and emerging biomarkers of paediatric disease.     

Standardization of insulin and C-peptide – A status report

ObjectiveIt is important that assays used for insulin and C-peptide testing are metrologically traceable and meet predefined quality specifications. Realizing this is the mission statement of the Insulin Standardization Work Group and the C-Peptide Standardization Committee.Materials and methodsBoth groups adopt the traceability concept as described by the International Organization for Standardization. It is based on a higher-order reference measurement system, defining the measurand and système internationale d’unités, and comprising a primary calibrator (highly purified standard materials with certified property values in mass units of milligram), trueness-based reference measurement procedures (ReMPs) and secondary calibrators. Both groups developed already isotope dilution-mass spectrometry candidate ReMPs, investigated different options of secondary calibrators and negotiated with the World Health Organization to obtain the required certified primary calibrators. Meanwhile, the first studies have been done to look into the feasibility of using a reference measurement system for the purpose of establishing traceability of immunoassays, while judging on their quality of performance.ResultsThe studies showed that native sera, assigned with reference measurement values, are the best secondary calibrators. They also demonstrated the feasibility of establishing traceability of immunoassays from a method comparison study with a candidate ReMP and use of the relationship of the results as a recalibration basis.ConclusionBoth groups aim at completing the establishment of the reference measurement system. They intend to provide the in-vitro diagnostic manufacturers with a panel of single-donor sera assigned with ReMP values for recalibration of their assays and to follow-up this process by periodic surveillance.     

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.     

Assessing accuracy and precision for field and laboratory data: a perspective in ecosystem restoration

Unlike most laboratory studies, rigorous quality assurance/quality control (QA/QC) procedures may be lacking in ecosystem restoration (“ecorestoration”) projects, despite legislative mandates in the United States. This is due, in part, to ecorestoration specialists making the false assumption that some types of data (e.g. discrete variables such as species identification and abundance classes) are not subject to evaluations of data quality. Moreover, emergent behavior manifested by complex, adapting, and nonlinear organizations responsible for monitoring the success of ecorestoration projects tend to unconsciously minimize disorder, QA/QC being an activity perceived as creating disorder. We discuss similarities and differences in assessing precision and accuracy for field and laboratory data. Although the concepts for assessing precision and accuracy of ecorestoration field data are conceptually the same as laboratory data, the manner in which these data quality attributes are assessed is different. From a sample analysis perspective, a field crew is comparable to a laboratory instrument that requires regular “recalibration,” with results obtained by experts at the same plot treated as laboratory calibration standards. Unlike laboratory standards and reference materials, the “true” value for many field variables is commonly unknown. In the laboratory, specific QA/QC samples assess error for each aspect of the measurement process, whereas field revisits assess precision and accuracy of the entire data collection process following initial calibration. Rigorous QA/QC data in an ecorestoration project are essential for evaluating the success of a project, and they provide the only objective “legacy” of the dataset for potential legal challenges and future uses.     

Use of thermography for quantitative studies of spatial and temporal variation of stomatal conductance over leaf surfaces

This paper describes a new approach to the calibration of thermal infrared measurements of leaf temperature for the estimation of stomatal conductance and illustrates its application to thermal imaging of plant leaves. The approach is based on a simple reformulation of the leaf energy balance equation that makes use of temperature measurements on reference surfaces of known conductance to water vapour. The use of reference surfaces is an alternative to the accurate measurement of all components of the leaf energy balance and is of potentially wide application in studies of stomatal behaviour. The resolution of the technique when applied to thermal images is evaluated and some results of using the approach in the laboratory for the study of stomatal behaviour in leaves of Phaseolus vulgaris L. are presented. Conductances calculated from infrared measurements were well correlated with estimates obtained using a diffusion porometer.     

Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: I. Without exogenous activation

There has been an increasing need in genetic toxicology to progress from strictly qualitative tests to more quantitative tests. This, in turn, has increased the need to develop better quality assurance and comparative bioassay methods. In this paper, two laboratories tested 10 Salmonella mutagens in order to determine the usefulness of selected chemicals as potential reference materials to calibrate the Salmonella assay. If variance within a bioassay is sufficiently low and the rankings of the compounds are of acceptable consistency, the chemicals later could be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The results demonstrated that the chosen chemicals (with the possible exception of dimethylcarbamylchloride) provide such consistent results in the Salmonella mutagenicity bioassay that they can be used for semi-quantitative calibration and as possible bioassay controls, special audit chemicals, and potentially as reference standards in comparative bioassay efforts. Reference standards, whether used as audit materials or in comparative bioassays, must be used concurrently with the test substances of interest; used without bias; used in a standardized, highly controlled bioassay; and be tested across an appropriate dose range. The study also shows that when these compounds are used as reference standards much care must be given to the number and spacing of doses if highly reproducible slope values are to be generated. We recommend use of a pilot test to establish a dose range for definitive tests and the placement of doses for the definitive tests within the first half of the linear dose-response curve. For appropriate comparisons, one should replicate the tests using the defined dose range and analyze the results in a non-biased statistical manner.     

The IRMM—International Measurement Evaluation Program (IMEP)

The aim of the IRMM—International Measurement Evaluation Program (IMEP) is to test out a possible realization of international measurement comparability for field laboratories through traceability of their measurements to the SI Unit for amount of substance: the mole. In IMEP-3, 10 different trace elements, B, Ca, Cd, Cu, Fe, K, Li, Pb, Rb, and Zn, were determined in a synthetic and a natural water by participating laboratories using their routine methods and graphically compared (in coded form) to certified values, established by IRMM and NIST using an isotope-specific method (Isotope Dilution Mass Spectrometry, Neutron Activation Analysis). The number of participants was 70; 64 laboratories have reported results. The results show a spread of more than 50% asymmetrically around the certified value. The Youden graphs allow evaluation of the overall performance of the laboratories in the IMEP-3 round.

     

Introduction to standardization of laboratory results

A fundamental goal of laboratory medicine is that laboratory results will be comparable or standardized and be independent of the laboratory where the testing was performed. Routine measurement procedures that are traceable to the same system of reference standards should produce numerical values for clinical samples that are comparable regardless of time, place, or laboratory generating the result. Standardization of laboratory measurements is key to providing accurate and reliable results from investigational studies and for optimal patient care.     

Quality assessment of measurement

Assessing the quality of measurements is of interest to organizers of external quality assessment schemes (EQAS, or proficiency testing schemes), laboratory analysts and managers, users of laboratory results and other agencies. Scheme organizers run test programmes, define standards of acceptable and non-acceptable performance, and interact with participants and oversight authorities. Laboratory personnel are responsible for the quality management system and to choose whether to accept the standards set by scheme organizers or to adopt their own. Users receive and act upon the laboratory results.Schemes within the same analytical sector are often organized very differently causing contradictory assessment of performance. The Network of EQAS in occupational and environmental laboratory medicine established collaborative projects designed to enhance assessment of measurement quality and to improve the reliability of laboratory results.To address the issue of variations in assessing the quality of measurements, and in response to comments from some participants, standards derived from biological variation, rather than on the analytical performance of participants have been developed. Evaluation of test materials with respect to homogeneity and stability, and work on methods to give the assigned value to test materials, has also been undertaken. Following from these projects, further collaboration is planned which will provide better quality assessment of measurement to scheme participants and their users.     

Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation

Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements.