Requirements for reference (calibration) laboratories in laboratory medicine

In addition to reference measurement procedures and reference materials, reference or calibration laboratories play an integral role in the implementation of measurement traceability in routine laboratories. They provide results of measurements using higher-order methods, e.g. isotope dilution mass spectrometry and may assign values to materials to be used for external quality assessment programs and to secondary reference materials. The requirements for listing of laboratories that provide reference measurement services include a statement of the metrological level or principle of measurement, accreditation as a calibration laboratory according to ISO 15195 and the participation in a proficiency testing system (regular inter-laboratory comparisons) for reference laboratories. Ring trials are currently conducted for thirty well-defined measurands and the results are made available to all laboratories. Through the use of reference laboratory services that are listed by the Joint Committee for Traceability in Laboratory Medicine there is the opportunity to further promote traceability and standardisation of laboratory measurements.     

Proposition pour la maîtrise de la phase pré-analytique selon la norme NF EN ISO 15189

In spite of the evolutions of the practices of care of the samples intended for the laboratory medicine, this pre analytical phase still remains the source of the most numerous errors in laboratory testing. These conflicts between the clinical situation of the patient and analysis results are mostly attributable in defects of control of the pre analytical procedures. These abnormalities can represent until 93% of the errors met during the process of biological diagnosis. The ascendancy of the human factor in the realization of this phase precedent the analysis is such as the complete elimination of the errors attributable to the laboratory remains very difficult. To improve this situation, the compulsory accreditation of the laboratories Medicine departments according to the standard ISO 15189 was imposed. Indeed the normative aspect of the compulsory requirements is inevitably going to decrease the risks by establishing a strict conduct, in each of the stages of the process. We present in this article a didactic and practical reading, mainly of chapters 4.7 and 5.4 of the ISO 15189 standard.     

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.     

Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories

Samples of allograft musculoskeletal tissue are cultured by bacteriology laboratories to determine the presence of bacteria and fungi. In Australia, this testing is performed by 6 TGA-licensed clinical bacteriology laboratories with samples received from 10 tissue banks. Culture methods of swab and tissue samples employ a combination of solid agar and/or broth media to enhance micro-organism growth and maximise recovery. All six Australian laboratories receive Amies transport swabs and, except for one laboratory, a corresponding biopsy sample for testing. Three of the 6 laboratories culture at least one allograft sample directly onto solid agar. Only one laboratory did not use a broth culture for any sample received. An international literature review found that a similar combination of musculoskeletal tissue samples were cultured onto solid agar and/or broth media. Although variations of allograft musculoskeletal tissue samples, culture media and methods are used in Australian and international bacteriology laboratories, validation studies and method evaluations have challenged and supported their use in recovering fungi and aerobic and anaerobic bacteria.     

Towards Harmonisation of Critical Laboratory Result Management - Review of the Literature and Survey of Australasian Practices

Timely release and communication of critical test results may have significant impact on medical decisions and subsequent patient outcomes. Laboratories therefore have an important responsibility and contribution to patient safety. Certification, accreditation and regulatory bodies also require that laboratories follow procedures to ensure patient safety, but there is limited guidance on best practices. In Australasia, no specific requirements exist in this area and critical result reporting practices have been demonstrated to be heterogeneous worldwide.Recognising the need for agreed standards and critical limits, the AACB started a quality initiative to harmonise critical result management throughout Australasia. The first step toward harmonisation is to understand current laboratory practices. Fifty eight Australasian laboratories responded to a survey and 36 laboratories shared their critical limits. Findings from this survey are compared to international practices reviewed in various surveys conducted elsewhere. For the successful operation of a critical result management system, critical tests and critical limits must be defined in collaboration with clinicians. Reporting procedures must include how critical results are identified; who can report and who can receive critical results; what is an acceptable timeframe within which results must be delivered or, if reporting fails, what escalation procedures should follow; what communication channels or systems should be used; what should be recorded and how; and how critical result procedures should be maintained and evaluated to assess impact on outcomes.In this paper we review the literature of current standards and recommendations for critical result management. Key elements of critical result reporting are discussed in view of the findings of various national surveys on existing laboratory practices, including data from our own survey in Australasia. Best practice recommendations are made that laboratories are expected to follow in order to provide high quality and safe service to patients.     

Verification of quantitative analytical methods in medical laboratories

BackgroundGlobally, all medical laboratories seeking accreditation should meet international quality standards to perform certain specific tests. Quality management program provides disciplines targeted to ensure that quality standards have been implemented by a laboratory in order to generate correct results. The hallmark of the accreditation process is method verification and quality assurance. Before introducing a new method in your laboratory, it is important to assess certain performance characteristics that reflect the concept of method verification.MethodsIn this review, we illustrated how to verify the performance characteristics of a new method according to the recent guidelines. It includes an assessment of precision, trueness, analytical sensitivity, detection limits, analytical specificity, interference, measuring range, linearity, and measurement uncertainty.ConclusionsAlthough the presence of several updated guidelines used to determine the performance characteristics of new methods in clinical chemistry laboratories, the real practice raised several concerns with the application of these guidelines which in need for further consideration in the upcoming updates of these guidelines.     

Validation of qualitative chromatographic methods: strategy in antidoping control laboratories

An experimental approach for the validation of chromatographic qualitative methods and its application in an antidoping control laboratory is described. The proposed strategy for validation of qualitative methods consists of the verification of selectivity/specificity, limit of detection (LOD), extraction recovery and repeatability (intra-assay precision). A one-day assay protocol, based on the analysis of five blank samples obtained from different sources and four replicates of control samples at two different concentrations of the analytes, has been defined to evaluate the validation parameters. The following evaluation criteria have been applied: absence of interfering substances at the retention time of the analytes in the blank samples to check the selectivity/specificity of the method, the LOD recommended by international sports authorities has to be attained, and for repeatability, the relative standard deviation should be <25% for the low concentration control sample and <15% for the high concentration control sample. Qualitative screening procedures are able to detect a great number of analytes so that extraction and analysis conditions are always a compromise for the different analytes. For this reason, no minimum acceptance criteria have been defined for data of extraction recoveries. The proposed protocol has been used for the validation of the screening and confirmation qualitative methods included in the scope of the accreditation of an antidoping control laboratory according to ISO quality standards.     

Définition des intervalles de référence et des seuils décisionnels chez les sujets âgés: cas de l’évaluation de l’état nutritionnel

Malnutrition is highly prevalent in the elderly. Today, there is no gold standard for the diagnosis of malnutrition. Screening tools, anthropometrical and laboratory tests can be performed. The development of quality assurance and the publication of the International Standard specifically developed for medical laboratories (ISO 15189) make necessary to know the analytical specifications for imprecision, inaccuracy and individual variations for each parameter. The reference and the cutoff values must be chosen in agreement with the populations that are screened. In this article, we focus on biochemical markers which could be recommended to evaluate malnutrition in the elderly and we summarize the analytical and biological variations; finally, we suggest appropriate comments that could be associated with laboratory results.     

Current methods for stallion semen cryopreservation: a survey

Various factors affect the success of AI with frozen-thawed semen in horses. Stallion variability is thought to be one of the major factors, but semen processing and evaluation techniques, thawing protocols, packaging systems and timing of insemination are far from standardized among laboratories. Our objective was to survey current methods for stallion semen cryopreservation used commercially around the world. From the answers to the questions in the survey, we attempted to provide an overview of procedures that are standard as well as those that are used by only few laboratories and to review critically the efficacy of these procedures. Twenty-five questionnaires were sent to individuals or laboratories in 14 countries that were i.v. involved in freezing stallion semen for commercial purposes. Questionnaires were returned from 10/14 countries with 21/25 (84%) of the addresses responding. From the responses, it became evident that most of prefreezing, freezing and thawing and post-thawing processing procedures were far from standardized. The great variety of procedures makes it difficult to accept any of them as reliable. In order to increase the credibility of AI technology in the horse, laboratories need to standardize processing methods as well as the record-keeping systems. In addition, it is evident that no group of research mares is large enough to provide meaningful fertility data. It is therefore imperative to have multicentered collaborative studies to record and disseminate information about methods and the corresponding fertility rate. to gain valuable information and be able to compare different protocols.     

Integration of future biotechnologies into the equine industry

There has and will continue to be reproductive techniques available that have a positive impact upon the equine breeding industry. This review focuses on semen technologies that have been developed or are in the process of being developed. The use of fluorescent dyes and flow cytometry has provided the researcher and clinician with powerful tools to evaluate several sperm attributes. These procedures have been utilized to evaluate sperm viability, acrosome status, mitochondrial status, DNA integrity and stages of capacitation. Flow cytometry allows several sperm attributes to be evaluated on thousands of spermatozoa in a matter of seconds. Development of procedures for insemination of mares with relatively small numbers of spermatozoa has the potential to change how stallions and their semen are managed. This review discusses the use of insemination of fresh, frozen and sex-sorted spermatozoa in relatively small numbers compared with conventional insemination technologies. The recent acceptance of frozen-thawed semen by many of the major breed registries has stimulated an increase in research on frozen semen. Many of the studies have focused on identifying damage during the freezing and thawing process. Numerous studies also have been conducted to modify freezing extenders so that the sperm are protected during the freezing and thawing process. The production of in vitro-produced embryos is extremely limited in the horse due to the failure of in vitro fertilization. However, intracytoplasmic sperm injection (ICSI) has been used for the production of foals from stallions that have less than typical sperm numbers or from stallions that have died and a limited quantity of frozen semen is available. This technique has been used by several laboratories to produce embryos in vitro. The breeder and veterinarian now have access to techniques that allow assessment of semen quality, improvement of procedures for freezing and thawing and insemination of mares with fewer numbers of spermatozoa. It is likely that the next decade will also produce tremendous advances in semen technologies that can be utilized in the horse industry.     

Concepts in the validation of neurochemical methods: the proper generation and use of statistics

Nearly every advance made in the study of neurotransmission has resulted from the development of new analytical methods or from the application of such methods to neurochemical problems in new ways. Each investigator places extremely high dependence on the laboratory method from which the data are gathered. It is therefore vital that these methods be proven valid when first selected and when used throughout the experiments being conducted. The process of method validation has flourished and been refined in the field of laboratory medicine. Methods are primarily validated by their accuracy and reproducibility in determining the analyte of interest in the tissue(s) to be studied. It is important that standards of performance be established that will allow objective decisions to be made when methods are tested for these characteristics. Performance standards for several neurotransmitters are suggested. Studies performed to collect method performance data are presented. From these data, statistics can be generated that help to estimate analytical errors. Guidelines for the proper generation and use of these statistics are discussed. Use of these validation approaches should be expanded in the whole of neurochemistry, which should enrich the data gathered within a laboratory and improve the harmonization between laboratories.     

德国医院透明管理制度与标准解读

在美国JCI及国际ISO质量认证体系逐步进入我国后，德国KTQ评审也以其科学的方式和精确的标准得到了国内的广泛关注，文章将从KTQ评审机构、KTQ评审方法、KTQ评审标准、KTQ信息发布及监管几个方面对德国医院评审制度进行解读，以期为我国医院评审提供借鉴。     

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.     

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.     

Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects

Whenever gene expression is being examined, it is essential that a normalization process is carried out to eliminate non-biological variations. The use of reference genes, such as glyceraldehyde-3-phosphate dehydrogenase, actin, and ribosomal protein genes, is the usual method of choice for normalizing gene expression. Although reference genes are used to normalize target gene expression, a major problem is that the stability of these genes differs among tissues, developmental stages, species, and responses to abiotic factors. Therefore, the use and validation of multiple reference genes are required. This review discusses the reasons that why RT-qPCR has become the preferred method for validating results of gene expression profiles, the use of specific and non-specific dyes and the importance of use of primers and probes for qPCR as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. The conflicts arising in the use of classical reference genes in gene normalization and their replacement with novel references are also discussed by citing the high stability and low stability of classical and novel reference genes under various biotic and abiotic experimental conditions by employing various methods applied for the reference genes amplification.     

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).     

Can external quality control improve pig AI efficiency?

External quality control programmes carried out by central laboratories have been long established in human andrology with the aim of enhancing the accuracy and reproducibility of semen assessment. Compared to human, demands on boar semen assessment in AI stations are more complex, with the need both to identify boars with poor ejaculate quality and to monitor individual boar differences for semen storage. Additionally, appropriate assessment serves as a control instrument to ensure the security and efficiency of semen processing. Despite current limitations regarding the ability of sperm assays to estimate the potential fertility of males, it is evident that boar fertility is related to certain conventional semen tests, e.g. sperm morphology. In central studies carried out on stored semen from 11 AI stations, flow cytometric assessment of plasma and acrosome membrane integrity proved to be more sensitive in detecting sperm damage associated with ageing and temperature stress as compared to light microscopy. Membrane integrity of stored semen differed between AI stations indicating significant influences of semen processing on sperm quality. Thus external control of semen quality in reference laboratories may be useful to monitor the efficiency of internal semen quality control in individual AI stations, to identify males with lower semen quality and/or poor response to semen storage, and to verify the precision of sperm counting. The possibility that central laboratories with sufficient resources may be able to identify functionally different responding sperm subpopulations for better estimation of fertility is discussed. Ideally, external quality control schemes for AI stations would comprise application of validated tests with high relevance for fertility (including bacterial status), analysis of semen processing on the AI station, and training courses for laboratory personnel.     

Determining sample size for the morphological assessment of sperm

Morphologic assessment of spermatozoa is an integral component in the analysis of semen. Whether a technician rapidly screening semen quality at a commercial stud, a veterinarian performing breeding soundness examinations, a clinician at a reference andrology laboratory providing auditing or diagnostic services, or a researcher evaluating morphology as a part of a fertility study, it is important to make an informed decision regarding the number of spermatozoa to include in the morphology assessment. Application of basic statistical principles such as the nature of proportions, level of confidence in an observed value, and the interaction of sample size with precision, can and should be used in the decision process. This paper outlines in detail the application of these statistical principles in relation to the morphologic assessment of spermatozoa. Guidelines on how these principles can be utilized in practical situations are discussed. Additionally, methodologies for comparison of results within and between laboratories (an area easily prone to misinterpretation) are reviewed. It is hoped that through the use of these fundamental statistical principles, this paper will bring clarity and delineation to the science of quantifying the morphology of spermatozoa.     

Design and positioning of a reference information model for clinical laboratories

Clinical laboratories produce on a daily basis huge amounts of data, i.e., test results of chemical determinations. The production and the use of these data should be extremely well structured. This and the urge to improve quality was the reason to design a reference information model for clinical laboratories, abbreviated as RILA. A software toolbox facility was used to the design. RILA can be used in any clinical laboratory, but it can also be made laboratory situation-specific. This is done through the implementation of location-specific and detailed information concerning procedures, instruments, capacities, etc. By doing this, the interaction with the laboratories environment has to be defined as well. Certain application areas such as total quality management, workflow analysis and cost analysis can be facilitated through the implemented reference information model. Furthermore, RILA has to be positioned at a proper location with regard to the existing hospital and laboratory automation modules.     

Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI)

The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.