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.     

A Guide to Harmonisation and Standardisation of Measurands Determined by Liquid Chromatography – Tandem Mass Spectrometry in Routine Clinical Biochemistry

Globally, harmonisation in laboratory medicine is a significant project. The relatively new implementation of liquid chromatography coupled with tandem mass spectrometry (LC-MSMS) techniques as routine assays in diagnostic laboratories provides the unique opportunity to harmonise, and in many cases standardise, methods from an early stage. This guide aims to provide a practical overview of the steps required to achieve agreement between LC-MSMS analytical procedures for routine clinical biochemistry diagnostic assays, with particular focus on the harmonisation and standardisation of methods currently implemented.To achieve harmonisation, and where practical standardisation, the approach is more efficient if divided into sequential stages. The suggested division entails: (i) planning and preliminary work; (ii) initial assessment of performance; (iii) standardisation and harmonisation initiative; (iv) establishing common reference intervals and critical limits; (v) developing best practice guidelines; and (vi) performing an ongoing review.The profession has a unique and significant opportunity to bring clinical mass spectrometry-based assays into agreement. Harmonisation of assays should ultimately provide the same result and interpretation for a given patient’s sample, irrespective of the laboratory that produced the result. To achieve this goal, we need to agree on the best practice LC-MSMS methods for use in routine clinical measurement.     

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).     

Regional Variation in Analytical Techniques used in the Diagnosis and Monitoring of Porphyria: a Case for Harmonisation?

Background:The Royal College of Pathologists of Australasia (RCPA) Porphyrin Quality Assurance Program assesses the measurement of urine, faecal, plasma and whole blood porphyrins and their components plus urinary porphobilinogen and delta aminolaevulinic acid and has laboratories enrolled from around the world. It was observed that there was a wide scatter in results submitted to some subsections of the program.Methods:A detailed questionnaire covering the analytical techniques used in the diagnosis of porphyria was sent to all laboratories enrolled in the RCPA Porphyrin Quality Assurance Program. Additionally, self-enrolment data over a five year period was examined for trends/changes in standardisation, reagent sources and analytical technique.Results:Twenty of the 45 laboratories enrolled in the Porphyrin Quality Assurance Program completed the survey, providing a snapshot of the analytical techniques used world-wide. Post survey self enrolment data indicated only little or no noticeable changes to analytical standardisation of techniques despite the continual lack of agreement of results in subsections of the External Quality Assurance program.Conclusions:While some aspects of porphyria testing are relatively consistent between laboratories, other diagnostic techniques vary widely. A wide variety of individualised reference intervals and reporting techniques is currently in use world-wide. While most of the participants in the survey are regional reference centres specialising in the diagnosis of porphyria and, as such, their diagnostic capability is not in question, international guidelines or global harmonisation of analytical techniques should allow better inter-laboratory comparisons to be made, ultimately improving diagnostic accuracy.     

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.     

HbA1c standardisation: history, science and politics

Significant analytical improvements have occurred since glycated haemoglobin (GHb), measured as total HbA(1), was first used in routine clinical laboratories around 1977. Following the publication of the Diabetes Control and Complications Trial (DCCT) study in 1993 the issue of international standardisation became an important objective for scientists and clinicians. The lack of international standardisation led several countries to develop national standardisation programs. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Working Group on Standardisation of HbA(1c) established a true international reference measurement system for HbA(1c) and the successful preparation of pure HbA(1c) calibration material that should lead to further improvements in inter-method and inter-laboratory variability. Reporting of HbA(1c) has been agreed using the units of mmol/mol (IFCC) and percent (National Glycohemoglobin Standardization Program, NGSP).     

The case for common reference intervals

The current paradigm for pathology reference intervals is for each laboratory to determine its own interval for use with each test offered by the laboratory. It is our contention that this approach does not best serve the medical community, especially at a time when electronic databases of health information are being expanded and integrated. We also believe that this approach is not performed well in many laboratories and is excessively expensive in practice. In contrast, we believe that the preferable option is to develop and apply common reference intervals throughout Australia and New Zealand, together with common reporting formats and assay standardisation wherever this is possible.

We are aware that these are neither trivial nor simple issues, however we believe that failure to achieve this goal where technically possible will be a failure of the pathology profession to meet the challenges of the modern health community.

     

Plasma creatinine in dogs: intra- and inter-laboratory variation in 10 European veterinary laboratories

Background  

There is substantial variation in reported reference intervals for canine plasma creatinine among veterinary laboratories, thereby influencing the clinical assessment of analytical results. The aims of the study was to determine the inter- and intra-laboratory variation in plasma creatinine among 10 veterinary laboratories, and to compare results from each laboratory with the upper limit of its reference interval.     

Standardisation of reference intervals: an Australasian view

The development of regional databases and doctors’ desktop programs that accept pathology results from different laboratories should improve patient care by allowing easy assessment of cumulative data. This has the potential to be unnecessarily confusing unless laboratories contributing to the databases provide standardised results and common reference intervals, where this is valid. The analytical methods that produce significantly different results need to be reported in a manner that avoids inappropriate interpretation.

The process of setting reference intervals requires an organisational structure which enables appropriate intervals to be set taking all relevant factors into account, including the opinions of expert clinicians. There must also be criteria for analytical agreement between the laboratories involved based on comparison studies using patient samples.

A network of QA groups across Australasia, with leadership from the AACB and RCPA, should be formed to share the ongoing work of defining reference intervals (RIs) for common tests, and reviewing them as the testing environment changes with the introduction of new techniques and instruments.

     

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.     

International reference standards in coagulation

Measurement of coagulation factor activity using absolute physico-chemical techniques is not possible and estimation therefore relies on comparative bioassay relative to a reference standard with a known or assigned potency. However the inherent variability of locally prepared and calibrated reference standards can give rise to poor agreement between laboratories and methods. Harmonisation of measurement between laboratories at the international level relies on the availability of a common source of calibration for local reference standards and this is provided by the World Health Organization (WHO) International Standards which define the International Unit for the analyte. This article describes the principles, practices and problems of biological standardisation and the development and use of reference standards for assays of coagulation factors, with particular emphasis on WHO International Standards for both concentrates and plasma.     

Reference intervals

Recommended elements of a process for establishing a reference interval: Define the analyte (measurand) for which the reference interval is being established, the clinical utility, biological variation and major variations in form. Define the method used, the accuracy base, and analytical specificity. Define important pre-analytical considerations together with any actions in response to the interference. Define the principle behind the reference interval (i.e. central 95% etc.). Describe the data source(s), including: number of subjects, nature of subjects, exclusions, pre-analytical factors, statistical measures, outliers excluded and analytical method. Define considerations of partitioning based on age, sex etc. Define the number of significant figures, i.e. the degree of rounding. Define the clinical relevance of the reference limits. Consider the use of common reference intervals. Decision and implementation.     

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.     

Estimation of the Optimal Statistical Quality Control Sampling Time Intervals Using a Residual Risk Measure

Background

An open problem in clinical chemistry is the estimation of the optimal sampling time intervals for the application of statistical quality control (QC) procedures that are based on the measurement of control materials. This is a probabilistic risk assessment problem that requires reliability analysis of the analytical system, and the estimation of the risk caused by the measurement error.

Methodology/Principal Findings

Assuming that the states of the analytical system are the reliability state, the maintenance state, the critical-failure modes and their combinations, we can define risk functions based on the mean time of the states, their measurement error and the medically acceptable measurement error. Consequently, a residual risk measure rr can be defined for each sampling time interval. The rr depends on the state probability vectors of the analytical system, the state transition probability matrices before and after each application of the QC procedure and the state mean time matrices. As optimal sampling time intervals can be defined those minimizing a QC related cost measure while the rr is acceptable. I developed an algorithm that estimates the rr for any QC sampling time interval of a QC procedure applied to analytical systems with an arbitrary number of critical-failure modes, assuming any failure time and measurement error probability density function for each mode. Furthermore, given the acceptable rr, it can estimate the optimal QC sampling time intervals.

Conclusions/Significance

It is possible to rationally estimate the optimal QC sampling time intervals of an analytical system to sustain an acceptable residual risk with the minimum QC related cost. For the optimization the reliability analysis of the analytical system and the risk analysis of the measurement error are needed.     

Measurement of serum creatinine--current status and future goals

The first methods for the measurement of creatinine in serum and plasma were published over a century ago. Today, the Jaffe reaction using alkaline picrate remains the cornerstone of most current routine methods, after continuous refinements attempting to overcome inherent analytical interferences and limitations. With the recent introduction of the reporting of estimated glomerular filtration rate (eGFR), inter-laboratory agreement of serum creatinine results has become an important international priority. Expert professional bodies have recommended that all creatinine methods should become traceable to a reference method based on isotope dilution-mass spectrometry (IDMS).It is important that clinical biochemists have a good understanding of the relative performance of routine creatinine methods. Using a new commutable IDMS-traceable reference material (SRM 967), and a validated tandem IDMS assay developed in our laboratory, we assessed the accuracy of nine routine creatinine methods with assistance from other laboratories in our region. Three methods appeared to have patient sample bias that exceeded 5% in the range of creatinine concentrations where eGFR estimations are most important.Companies are currently recalibrating their creatinine assays. This task should be complete in 2007, and then creatinine results for eGFR calculations will require the use of a modified eGFR equation. Laboratories considering calibration changes before this time can seek advice from the Australasian Creatinine Working Group.     

Inter-laboratory comparison of methods to measure androstenone in pork fat

Today, different analytical methods are used by different laboratories to quantify androstenone in fat tissue. This study shows the comparison of methods used routinely in different laboratories for androstenone quantification: Time-resolved fluoroimmunoassay in Norwegian School of Veterinary Science (NSVS; Norway), gas chromatography coupled to mass spectrometry in Co-operative Central Laboratory (CCL; The Netherlands) and in Institut de Recerca i Tecnologia Agroalimentàries (IRTA; Spain), and high-pressure liquid chromatography in Agroscope Liebefeld-Posieux Research Station (ALP; Switzerland). In a first trial, a set of adipose tissue (AT) samples from 53 entire males was sent to CCL, IRTA and NSVS for determination of androstenone concentration. The average androstenone concentration (s.d.) was 2.47 (2.10) μg/g at NSVS, 1.31 (0.98) μg/g at CCL and 0.62 (0.52) μg/g at IRTA. Despite the large differences in absolute values, inter-laboratory correlations were high, ranging from 0.82 to 0.92. A closer look showed differences in the preparation step. Indeed, different matrices were used for the analysis: pure fat at NSVS, melted fat at CCL and AT at IRTA. A second trial was organised in order to circumvent the differences in sample preparation. Back fat samples from 10 entire males were lyophilised at the ALP labortary in Switzerland and were sent to the other laboratories for androstenone concentration measurement. The average concentration (s.d.) of androstenone in the freeze-dried AT samples was 0.87 (0.52), 1.03 (0.55), 0.84 (0.46) and 0.99 (0.67) μg/g at NSVS, CCL, IRTA and ALP, respectively, and the pairwise correlations between laboratories ranged from 0.92 to 0.97. Thus, this study shows the influence of the different sample preparation protocols, leading to major differences in the results, although still allowing high inter-laboratory correlations. The results further highlight the need for method standardisation and inter-laboratory ring tests for the determination of androstenone. This standardisation is especially relevant when deriving thresholds of consumer acceptance, whereas the ranking of animals for breeding purposes will be less affected due to the high correlations between methods.     

Normal Laboratory Reference Intervals among Healthy Adults Screened for a HIV Pre-Exposure Prophylaxis Clinical Trial in Botswana

Introduction

Accurate clinical laboratory reference values derived from a local or regional population base are required to correctly interpret laboratory results. In Botswana, most reference intervals used to date are not standardized across clinical laboratories and are based on values derived from populations in the United States or Western Europe.

Methods

We measured 14 hematologic and biochemical parameters of healthy young adults screened for participation in the Botswana HIV Pre-exposure Prophylaxis Study using tenofovir disoproxil fumarate and emtricitabine (TDF/FTC) (TDF2 Study). Reference intervals were calculated using standard methods, stratified by gender, and compared with the site-derived reference values used for the TDF2 study (BOTUSA ranges), the Division of AIDS (DAIDS) Grading Table for Adverse Events, the Botswana public health laboratories, and other regional references.

Results

Out of 2533 screened participants, 1786 met eligibility criteria for participation in study and were included in the analysis. Our reference values were comparable to those of the Botswana public health system except for amylase, blood urea nitrogen (BUN), phosphate, total and direct bilirubin. Compared to our reference values, BOTUSA reference ranges would have classified participants as out of range for some analytes, with amylase (50.8%) and creatinine (32.0%) producing the highest out of range values. Applying the DAIDS toxicity grading system to the values would have resulted in 45 and 18 participants as having severe or life threatening values for amylase and hemoglobin, respectively.

Conclusion

Our reference values illustrate the differences in hematological and biochemical analyte ranges between African and Western populations. Thus, the use of western-derived reference laboratory values to screen a group of Batswana adults resulted in many healthy people being classified as having out-of-range blood analytes. The need to establish accurate local or regional reference values is apparent and we hope our results can be used to that end in Botswana.     

Accuracy of popular automatic QT Interval algorithms assessed by a 'Gold Standard' and comparison with a Novel method: computer simulation study

Background

Accurate measurement of the QT interval is very important from a clinical and pharmaceutical drug safety screening perspective. Expert manual measurement is both imprecise and imperfectly reproducible, yet it is used as the reference standard to assess the accuracy of current automatic computer algorithms, which thus produce reproducible but incorrect measurements of the QT interval. There is a scientific imperative to evaluate the most commonly used algorithms with an accurate and objective 'gold standard' and investigate novel automatic algorithms if the commonly used algorithms are found to be deficient.

Methods

This study uses a validated computer simulation of 8 different noise contaminated ECG waveforms (with known QT intervals of 461 and 495 ms), generated from a cell array using Luo-Rudy membrane kinetics and the Crank-Nicholson method, as a reference standard to assess the accuracy of commonly used QT measurement algorithms. Each ECG contaminated with 39 mixtures of noise at 3 levels of intensity was first filtered then subjected to three threshold methods (T1, T2, T3), two T wave slope methods (S1, S2) and a Novel method. The reproducibility and accuracy of each algorithm was compared for each ECG.

Results

The coefficient of variation for methods T1, T2, T3, S1, S2 and Novel were 0.36, 0.23, 1.9, 0.93, 0.92 and 0.62 respectively. For ECGs of real QT interval 461 ms the methods T1, T2, T3, S1, S2 and Novel calculated the mean QT intervals(standard deviations) to be 379.4(1.29), 368.5(0.8), 401.3(8.4), 358.9(4.8), 381.5(4.6) and 464(4.9) ms respectively. For ECGs of real QT interval 495 ms the methods T1, T2, T3, S1, S2 and Novel calculated the mean QT intervals(standard deviations) to be 396.9(1.7), 387.2(0.97), 424.9(8.7), 386.7(2.2), 396.8(2.8) and 493(0.97) ms respectively. These results showed significant differences between means at >95% confidence level. Shifting ECG baselines caused large errors of QT interval with T1 and T2 but no error with Novel.

Conclusion

The algorithms T2, T1 and Novel gave low coefficients of variation for QT measurement. The Novel technique gave the most accurate measurement of QT interval, T3 (a differential threshold method) was the next most accurate by a large margin. The objective and accurate 'gold standard' presented in this paper may be useful to assess new QT measurement algorithms. The Novel algorithm may prove to be more accurate and reliable method to measure the QT interval.     

Significant figures

* For consistency of reporting the same number of significant figures should be used for results and reference intervals. * The choice of the reporting interval should be based on analytical imprecision (measurement uncertainty).     

Accreditation and the use of validated/recognised methods to analyse human semen

Accreditation of laboratories who perform diagnostic semen analysis in Australia and New Zealand is a requirement of the healthcare system. Within the accreditation process laboratories are required to set ISO standards within their policies and procedures. In order to achieve their aims, laboratories need to be able to measure a number of defined semen parameters both accurately and repetitively, especially around the lower limit of the reference intervals. The methods documented in the WHO-manual are used almost universal as the laboratory standard. Some laboratories incorporate minor method variations into their procedures. As part of the ISO requirements all variations require validation using internally approved processes that are documented and that incorporate appropriate statistical analysis and comparison of results. Validation is an ongoing process and regular review is essential. Evidence of the validation must be available for review by external auditors during accreditation. Where any validated variant method returns results that are significantly different to any method within the WHO-manual, the laboratory needs to develop its own, in-house reference interval for that method.