Implementation of reference systems in laboratory medicine.

In laboratory medicine, meaningful measurements are essential for the diagnosis, risk assessment, treatment, and follow-up of patients; therefore, methods applied in diagnostic measurements must be accurate, precise, specific, and comparable among laboratories (1 ). A given analytical measurement has only one true result, and the reliability of a measurement lies both in the result obtained and in the performance of a well-defined measurement procedure. Inadequate or incorrect analytical performance has consequences for the patient, the clinician, and the healthcare system. Poor-quality laboratory results may lead to incorrect interpretation by the physician, to a wrong diagnosis, and hence to treatment that impairs, or at least does not help, the patient’s situation. Measurement procedures in laboratory medicine should not be based on consensus but must follow the established rules of metrology as outlined in the International Vocabulary of Basic and General Terms in Metrology (VIM) (2 ). According to VIM, metrology includes all aspects of measurements in whatever fields of science or technology they occur. One key element of metrology is the traceability of a test result to the International System (SI), which ensures comparable results for different measurements of the same analyte in the same sample. “Traceability” is defined as the property of the result related to national or international standards through an unbroken chain of comparisons, each of which has stated uncertainties (2 ). The importance of these metrologic principles is described in an International Organization for Standardization (ISO)/European Committee for Standardization (CEN) standard (3 ). These rules must be followed if results of diagnostic measurements are to be comparable and true wherever in the world they are performed. Given the increased mobility of patients, comparable (true) test results are essential for a rational and cost-effective diagnostic approach. To reach these goals, international and regional organizations such as the ISO, the CEN, the IFCC, the International Union for Pure and Applied Chemistry (IUPAC), the International Council for Standardization in Hematology, and the NCCLS have agreed on a metrologically sound Reference System. In laboratory medicine, a Reference System consists of a network of reference laboratories that use Reference Methods and Certified Reference Materials (CRMs) for optimal measurement of analytes in various biological matrices (4–8). Reference laboratories, analytical centers of competence, perform measurements with the greatest competence and are considered expert institutions for quantifying certain well-defined analytes, using the best, internationally agreed-on measurement procedure. Their main responsibility is to assign target values to Reference Materials, using the best analytical method available. In addition, they establish for so-called routine methods the extent of associated analytical bias in comparison with primary methods, with established Reference Methods, or if no Reference Method has been developed, with designated comparison methods. Thus, these laboratories establish through a chain of comparisons the traceability of routine methods and their respective biases. According to the International Centre for Metrology, isotope dilution with mass spectrometry, coulometry, gravimetry, titrimetry, and determination of freezingpoint depression are primary methods, yielding results in SI units (moles) without requiring reference to a standard (9 ). However, these kinds of methods are applicable only for the measurement of elements and of exactly defined metabolites, which is not the case for many of the analytes used for clinical diagnosis. In laboratory medicine, the exact definition of the analyte, its biological and clinical function, and the influence of the matrix are crucial elements when establishing a Reference Method. An analyst first must know what to measure before deciding by which means the analyte (enzyme, protein, isoform, or metabolite) can be measured. These two steps usually are formalized by an expert committee. The Reference Method (the highest possible level in the metrologic hierarchy) needs to be specific for the defined analyte, and the chemical, biochemical, or immunological reaction used in the method must be well defined and completely described. Moreover, a statement of the uncertainty of the measurement must be included. These methods must be reproducible in time and space, which means that if the description of the method is followed, the true values obtained in a certain sample must be within the described uncertainty. When based on the most up-to-date knowledge, Reference Methods are considered “Definitive Methods”. In cases where no Reference Method is available for an analyte, a so-called designated comparison method can be established according to the principles mentioned above. After extensive analytical investigations, such methods might evolve into Reference Methods. Accordingly, a designated comparison method can be a candidate for a Reference Method, but it should not be treated as an alternative to an existing Reference Method that is considered troublesome or too expensive (10 ). The overall objective in the hierarchy of measurement procedures is to achieve the highest possible analytical quality. Within this Reference System, biological CRMs play a key role, because the analyte concentration in a patient’s sample is measured by comparing its signal with the signal given by the standard/calibrator. CRMs are either primary or secondary matrixed reference materials. CRMs carry a value for the analyte as measured with a defined uncertainty by a primary method or a Reference Method by reference laboratories. The starting points in the preparation of a CRM are the pure or purified analytes from human origin and the matrix. In the case of proteins, recombinant preparations for which the structure, amino acid composition, and degree of glycosylation have been established can also be used. The values are assigned to the pure analyte by Definitive Methods and transferred to Opinion