Josep M. Queraltó

Need for revisiting the concept of reference values.

Abstract The reference values concept has been adopted by health care professionals, including clinical chemists, laboratory scientists, and clinicians and simultaneously by all the official organizations in charge of the establishment of legislation. But the estimation of reference limits, and the evaluation of biological variability need to be improved at the level of the procedures, which are currently too long and too expensive and not feasible easily for all laboratories. The procedures for obtaining reference values, if we follow the original documents, are complex, and that is the main reason that clinical chemists or diagnostic kit manufacturers have not used them systematically. There is clearly a need that scientific societies and international organizations propose practical recommendations: 1) Recommendations to describe methods linked to systematic error. · How to transfer reference limits from one laboratory to another laboratory using different methods? · Should we determine reference limits for each method? · How can we differentiate bias due to the populations from these due to the method? Clear collaborations with manufacturers involved in kits and diagnostic systems are needed. 2) Practical recommendations linked to the reference population. · How to transfer reference limits from one laboratory to another laboratory using different methods? · Should we determine reference limits for each method? · How can we differentiate bias due to the populations from these due to the method? Clear collaborations with manufacturers involved in kits and diagnostic systems are needed. · How to select a homogenous population? (Careful recommendations on the choice between healthy individuals, blood donors and individuals hospitalised for other diseases should be given.) · How to estimate ethnic differences? · How to define the exclusion and inclusion criteria according to quantity? · How to deal with the question of reference limits for unstable periods, aging or old people particularly, when the difference between aging and disease is very difficult to define? 3) Practical recommendations on the statistical methods to be used. · How to transfer reference limits from one laboratory to another laboratory using different methods? · Should we determine reference limits for each method? · How can we differentiate bias due to the populations from these due to the method? Clear collaborations with manufacturers involved in kits and diagnostic systems are needed. · How to select a homogenous population? (Careful recommendations on the choice between healthy individuals, blood donors and individuals hospitalised for other diseases should be given.) · How to estimate ethnic differences? · How to define the exclusion and inclusion criteria according to quantity? · How to deal with the question of reference limits for unstable periods, aging or old people particularly, when the difference between aging and disease is very difficult to define? · How to make a good choice of the interquantile interval? Should we use and present only the centiles 2.5 or 97.5, or on the contrary should we give other centiles in addition, for example 5, 10, 75, 80, 85, 90? 4) Practical recommendations linked to the use of the concept of the reference values. · How to transfer reference limits from one laboratory to another laboratory using different methods? · Should we determine reference limits for each method? · How can we differentiate bias due to the populations from these due to the method? Clear collaborations with manufacturers involved in kits and diagnostic systems are needed. · How to select a homogenous population? (Careful recommendations on the choice between healthy individuals, blood donors and individuals hospitalised for other diseases should be given.) · How to estimate ethnic differences? · How to define the exclusion and inclusion criteria according to quantity? · How to deal with the question of reference limits for unstable periods, aging or old people particularly, when the difference between aging and disease is very difficult to define? · How to make a good choice of the interquantile interval? Should we use and present only the centiles 2.5 or 97.5, or on the contrary should we give other centiles in addition, for example 5, 10, 75, 80, 85, 90? · How to make this concept more concrete and to have official definitions which are better understandable and not only abstract? · How to demonstrate the value of using simultaneously reference limits and decision limits, and what does each of these limits bring to results interpretation? · How to improve the presentation of the results? How to give more information on biological variability in the laboratory data, taking into account the scientific validity of their determination? Should we use new information techniques and new communication systems for reaching these objectives? The responses to all these questions could only be provided if there is a concerted effort at the international level. Practical recommendations should be given, which would be very useful for a better understanding and use of reference values by laboratory scientists and clinicians.

Common reference intervals for aspartate aminotransferase (AST), alanine aminotransferase (ALT) and γ-glutamyl transferase (GGT) in serum: results from an IFCC multicenter study

Abstract Background: Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and γ-glutamyl transferase (GGT) measurements are important for the assessment of liver damage. The aim of this study was to define the reference intervals (RIs) for these enzymes in adults, paying attention to standardization of the methods used and careful selection of the reference population. Methods: AST, ALT and GGT were measured with commercial analytical systems standardized to the IFCC-recommended reference measurement systems. Three centers (two in Italy and one in China) measured their own freshly collected samples; one of these centers also measured frozen samples from the Nordic Countries RI Project and from a Turkish center. RIs were generated using non-parametric techniques from the results of 765 individuals (411 females and 354 males, 18–85 years old) selected on the basis of the results of other laboratory tests and a specific questionnaire. Results: AST results from the four regions (Milan, Beijing, Bursa and Nordic Countries) were statistically different, but these differences were too small to be clinically relevant. Likewise, differences between the upper reference limits for genders was only 1.7 U/L (0.03 μkat/L), allowing a single RI of 11–34 U/L (0.18–0.57 μkat/L) to be defined. Interregional differences were not statistically significant for ALT, but partitioning was required due to significant gender differences. RIs for ALT were 8–41 U/L (0.13–0.68 μkat/L) for females and 9–59 U/L (0.15–0.99 μkat/L) for males, respectively. The upper reference limits for GGT from the Nordic Country population were higher than those from the other three regions and results from this group were excluded from final calculations. The GGT RIs were 6–40 U/L (0.11–0.66 μkat/L) for females and 12–68 U/L (0.20– 1.13 μkat/L) for males, respectively. Conclusions: For AST and ALT, the implementation of common RIs appears to be possible, because no differences between regions were observed. However, a common RI for GGT that is applicable worldwide appears unlikely due to differences among populations. Clin Chem Lab Med 2010;48:1593–601.

A controlled trial of daily versus thrice-weekly buprenorphine administration for the treatment of opioid dependence.

This study was aimed at determining whether thrice-weekly administration of buprenorphine is as effective as daily administration for treating opioid dependence. A total of 60 treatment-seeking opioid addicts were randomly assigned to take buprenorphine tablets sublingually either every day (8 mg) or thrice-weekly (16 mg on Mondays and Wednesdays and 24 mg on Fridays) over the course of a 12-week, double-blind, parallel trial. The buprenorphine dosing schedule had no significant effect on treatment retention. The rates of opioid-positive urine tests were significantly higher among those subjects who were given buprenorphine thrice weekly (58.5%) than among those who took it daily (46.6%). An analysis of the completers did not detect a significant effect of buprenorphine dosing schedule. The results obtained in our clinical trials indicate the advisability of daily doses of buprenorphine, at least at the beginning of a maintenance programme.

The theory of reference values: an unfinished symphony

Abstract The history of the theory of reference values can be written as an unfinished symphony. The first movement, allegro con fuoco, played from 1960 to 1980: a mix of themes devoted to the study of biological variability (intra-, inter-individual, short- and long-term), preanalytical conditions, standardization of analytical methods, quality control, statistical tools for deriving reference limits, all of them complex variations developed on a central melody: the new concept of reference values that would replace the notion of normality whose definition was unclear. Additional contributions (multivariate reference values, use of reference limits from broad sets of patient data, drug interferences) conclude the movement on the variability of laboratory tests. The second movement, adagio, from 1980 to 2000, slowly develops and implements initial works. International and national recommendations were published by the IFCC-LM (International Federation of Clinical Chemistry and Laboratory Medicine) and scientific societies [French (SFBC), Spanish (SEQC), Scandinavian societies…]. Reference values are now topics of many textbooks and of several congresses, workshops, and round tables that are organized all over the world. Nowadays, reference values are part of current practice in all clinical laboratories, but not without difficulties, particularly for some laboratories to produce their own reference values and the unsuitability of the concept with respect to new technologies such as HPLC, GCMS, and PCR assays. Clinicians through consensus groups and practice guidelines have introduced their own tools, the decision limits, likelihood ratios and Reference Change Value (RCV), creating confusion among laboratorians and clinicians in substituting reference values and decision limits in laboratory reports. The rapid development of personalized medicine will eventually call for the use of individual reference values. The beginning of the second millennium is played allegro ma non-troppo from 2000 to 2012: the theory of reference values is back into fashion. The need to revise the concept is emerging. The manufacturers make a friendly pressure to facilitate the integration of Reference Intervals (RIs) in their technical documentation. Laboratorians are anxiously awaiting the solutions for what to do. The IFCC-LM creates Reference Intervals and Decision Limits Committee (C-RIDL) in 2005. Simultaneously, a joint working group IFCC-CLSI is created on the same topic. In 2008 the initial recommendations of IFCC-LM are revised and new guidelines are published by the Clinical and Laboratory Standards Institute (CLSI C28-A3). Fundamentals of the theory of reference values are not changed, but new avenues are explored: RIs transference, multicenter reference intervals, and a robust method for deriving RIs from small number of subjects. Concomitantly, other statistical methods are published such as bootstraps calculation and partitioning procedures. An alternative to recruiting healthy subjects proposes the use of biobanks conditional to the availability of controlled preanalytical conditions and of bioclinical data. The scope is also widening to include veterinary biology! During the early 2000s, several groups proposed the concept of ‘Universal RIs’ or ‘Global RIs’. Still controversial, their applications await further investigations. The fourth movement, finale: beyond the methodological issues (statistical and analytical essentially), important questions remain unanswered. Do RIs intervene appropriately in medical decision-making? Are RIs really useful to the clinicians? Are evidence-based decision limits more appropriate? It should be appreciated that many laboratory tests represent a continuum that weakens the relevance of RIs. In addition, the boundaries between healthy and pathological states are shady areas influenced by many biological factors. In such a case the use of a single threshold is questionable. Wherever it will apply, individual reference values and reference change values have their place. A variation on an old theme! It is strange that in the period of personalized medicine (that is more stratified medicine), the concept of reference values which is based on stratification of homogeneous subgroups of healthy people could not be discussed and developed in conjunction with the stratification of sick patients. That is our message for the celebration of the 50th anniversary of Clinical Chemistry and Laboratory Medicine. Prospects are broad, enthusiasm is not lacking: much remains to be done, good luck for the new generations!

Usefulness of EC4 Essential Criteria for Quality Systems of Medical Laboratories as Guideline to the ISO 15189 and ISO 17025 Documents

Abstract Many medical laboratories have made a start with the introduction of quality management systems. However, it is still not clear against which standards such systems should be measured. The existing ISO and CEN standards do not cover essential aspects of medical laboratories. The publication of the EC4 Essential Criteria has stimulated the development of the ISO/Draft International Standard 15189. This standard seems adequate for our type of laboratories. However, it is not easy to read. The EC4 Essential Criteria could well serve as a guide, covering additional aspects, e.g. on total quality management and budget management as required in the EFQM model, that are not (yet) included in the ISO standard. In the present article the EC4 Essential Criteria are cross-referenced with two new international ISO standards, ISO/FDIS 15189 and ISO/FDIS 17025, the latter being the successor of ISO guide 25 and EN 45000. Both new ISO documents are in compliance with the new ISO 9000:2000 standard.

Intraindividual reference values.

Abstract When a biological quantity examination exhibits a high degree of individuality, developing a strategy for interpreting these values in an individual context can be a useful alternative. Time-series analysis is the appropriate statistical framework to build a model for explanation of the behaviour of laboratory information and to forecast future values. The key concepts in this approach are autocorrelation and withinperson variance. Unfortunately, the powerful tools provided by time-series analysis require many observations, a requisite difficult to meet in every day practice. However, introducing some restrictions in the autocorrelation parameter of the most reliable model, the first order autocorrelation model, and using the average within-person variance from a selected population, it is possible to build predictive reference intervals for an individual, based on only few observations. The most common case is the minimum time series: when there are just two observations. The statistical significance of the change from a previous observation is a problem that arises from both quality control (delta checks) and the interpretative diagnostic fields (reference change limit). Applying the same restrictive criteria, it is possible to develop specific limits for a difference between consecutive observations based on a within-person variance selected from the distribution of variances found in a sample of similar individuals.

Reference change value for HbA1c in patients with Type 2 diabetes mellitus

Abstract We estimated reference change value and steady state disease biological variation of glycohemoglobin (HbA1c from serial measurements in 47 clinically stable type diabetes patients whose home-measured capillary glucose was stable throughout the study. Whole blood HbA1c assays were performed by turbidimetric inhibition immunoassay. The analytical imprecision (coefficient of variation) was 7.1% and 5.0% for control materials with HbA1c of 5.1% and 10.7%, respectively (n=152). The sampling interval was 6 months. Patients were classified into three groups (good, acceptable and poor control) according to the American Diabetes Association (ADA) clinical practice recommendations of 1999 based on HbA1c reference interval. Steady state disease biological variation for each control group was 7.9%, 5.4% and 3.9%. HbA1c absolute reference change value was 1.42, 1.50 and 1.37%, or as relative reference change value, 29.1%, 24.4% and 17.8%, respectively. The analytical goal, defined as one-half of the biological variation, was lower than 3.9% for well-controlled patients.

EC4 European syllabus for post-graduate training in clinical chemistry. Version 2--1999. European Communities Confederation of Clinical Chemistry, EC4 Register Commission.

Abstract In modern medicine the undeniable value and indispensability of scientific investigations are now universally recognized both for diagnostic purposes and monitoring of disease and in basic epidemiology. The direct treatment of patients is an undeniable task of doctors in medicine. Progress in laboratory science is largely the result of contributions by scientists with an adequate education and specialisation in the field, i.e. by clinical chemists. Clinical laboratory science has developed on a broad front throughout the European Community, resulting in significant differences in what constitutes a national clinical chemistry service in each state. Clinical chemistry is the medical discipline devoted to obtain, explore and employ chemical knowledge and chemical methods of investigation, in order to procure knowledge about normal and abnormal chemical processes in man. These processes are studied on a general level, in order to get insight into human health and disease, and on a patient-specific level for diagnostic or monitoring purposes. The delimitation of clinical chemistry varies from country to country, since there is no sharp boundary to haematology, immunology, molecular biology and microbiology. One of the main tasks of the clinical chemist is direction and supervision of a laboratory department in a hospital or health service (public or private), where his role involves bridging the gap between rapidly developing laboratory science and technology and the growing knowledge on characteristics of disease. He must possess fundamental biochemical knowledge and have the ability to use this knowledge most appropriately as applied to clinical requirements, i.e. diagnosis of disease and planning and monitoring of therapy. Apart from providing a competent laboratory service, the clinical chemist must be able to function as a consultant to his clinical colleagues and liaise with them in the interpretation of laboratory results. His advice and professional consultation have at least three aspects, i.e. choosing the most appropriate laboratory investigation in a certain case, ensuring that the analyses are performed in the best possible way and correctly reported and, finally, providing information and (most important) interpretation on the significance and consequences of the laboratory data obtained. As the results of laboratory investigations and the consultation of the clinical chemist have a direct and important influence on the treatment of the patient, it is to the benefit of the public that the profession of the clinical chemist is duly regulated.

The European Register for Specialists in Clinical Chemistry and Laboratory Medicine: guide to the Register Version 2-2003 and procedure for re-registration.

Abstract The European Communities Confederation of Clinical Chemistry and Laboratory Medicine (EC4) opened a Register for European Chemists in 1997. The operation of the Register is undertaken by a Register Committee (EC4RC). During the last 5 years more than 1400 clinical chemists entered the register. In this article an update of the first Guide to the Register is given, based on the experience of 5 years of operation and the development of the discipline. The registration is valid for 5 years. In a second part the procedure and the conditions for re-registration are presented.

Genetic Mutations in a Spanish Population with Chronic Pancreatitis

Background/Aims: Mutations in the PRSS1 and the SPINK1 genes have variably been associated with alcohol-related, idiopathic and hereditary chronic pancreatitis (CP). The aim of this study was to determine for the first time the significance of PRSS1, SPINK1 mutations and genetic variants of AAT in a group of Spanish patients with CP. Methods: 104 consecutive patients with CP were included, as well as 84 healthy control subjects. The R122H and N29I mutations in the PRSS1 gene, the N34S mutation in the SPINK1 gene and PiS and PiZ mutations in the AAT gene were analyzed by RFLP-PCR methods. Results: No R122H mutation was found in the PRSS1 gene, and N29I mutation was detected in 7.7% of CP patients. A N29I mutation was observed in 3.9% of patients with alcohol-related pancreatitis (ACP). A total of 5.8% of CP patients were identified with the N34S mutation. Genotype MS, SS and MZ were detected in 18.3, 3.8 and 1.3% of CP patients, respectively. Conclusion: The percentage of N29I mutations in ACP patients was higher than that reported in other studies, while the percentage of N34S and AAT mutations in ACP and idiopathic CP patients was similar.