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Based on the original recommendation of the Expert Panel on the Theory of Reference Values of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC-LM), updated guidelines were recently published under the auspices of the IFCC-LM and the Clinical and Laboratory Standards Institute (CLSI). This article summarises these new proposals: (1) defining more precisely the terminology, which is often confusing, noticeably concerning the terms of reference limits and decision limits; (2) showing the different steps for determining reference limits according to the original procedure and the conditions which should be respected and (3) proposing a simple methodology allowing to the Clinical Laboratories to satisfy the needs of the Regulation and Standards. The updated document proposes to verify if published reference limits are applicable to the Laboratory involved. Finally the strengths and limits of the revised recommendations (noticeably the selection of the reference population, the maintenance of the analytical quality, the choice of the statistical methodology, etc.) will be briefly discussed.

The reticulocyte count is a good index of erythropoietic activity. Automated methods, mainly flow cytometry, have made the counting easier, more accurate and reproducible. The data presented describe the counting of reticulocytes in 1219 apparently healthy subjects of both sexes who came for a periodic health examination. Their ages varied from 4 to over 60 years. The reticulocytes were counted with a flow cytometer (Sysmex R-3000) with Auramine-O as fluorochrome. There was no statistical difference between boys and girls aged 4-19 years. However, the reticulocyte count was significantly higher in men than in women over 20 years of age. The reticulocyte count was affected neither by the menstrual cycle in girls at puberty, nor by oral contraceptives and the menopause in women. No effect of moderate smoking was observed in either sex. Reference limits of the reticulocyte counts (percentage and absolute value) according to age and sex and the three types of reticulocytes are provided.

Based on the original recommendation of the Expert Panel on the Theory of Reference Values of the International Federation of Clinical Chemistry (IFCC) dating back to the 1980s, updated guidelines were recently published under the auspices of the IFCC and the Clinical Laboratory Standard Institute (CLSI). Whereas the theory and the fundamentals of the reference value concept (definition, selection of reference individuals, preanalytical and analytical requirements, analysis of reference values) have not been modified, the updated guidelines add valuable improvements (transference, validation and verifying reference intervals). However, certain limitations remain which will be discussed in the present article.

Background: Reference intervals for serum creatinine remain relevant despite the current emphasis on the use of the estimated glomerular filtration rate for assessing renal function. Many studies on creatinine reference values have been published in the last 20 years. Using criteria derived from published IFCC documents, we sought to identify universally applicable reference intervals for creatinine via a systematic review of the literature.Methods: Studies were selected for inclusion in the systematic review only if the following criteria were met: (a) reference individuals were selected using an “a priori” selection scheme, (b) preanalytical conditions were adequately described; (c) traceability of the produced results to the isotope dilution–mass spectrometry (IDMS) reference method was demonstrated experimentally, and (d) the collected data received adequate statistical treatment.Results: Of 37 reports dealing specifically with serum creatinine reference values, only 1 report with pediatric data and 5 reports with adult data met these criteria. The primary reason for exclusion of most papers was an inadequate demonstration of measurement traceability. Based on the data of the selected studies, we have collated recommended reference intervals for white adults and children.Conclusion: Laboratories using methods producing traceable results to IDMS can apply the selected reference intervals for serum creatinine in evaluating white individuals.

This document is based on the original recommendation of the Expert Panel on the Theory of Reference Values of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), updated guidelines were recently published under the auspices of the IFCC and the Clinical and Laboratory Standards Institute (CLSI). This document summarizes proposals for recommendations on: (i) The terminology, which is often confusing, noticeably concerning the terms of reference limits and decision limits. (ii) The method for the determination of reference limits according to the original procedure and the conditions, which should be used. (iii) A simple procedure allowing the medical laboratories to fulfill the requirements of the regulation and standards. The updated document proposes to verify that published reference limits are applicable to the laboratory involved. Finally, the strengths and limits of the revised recommendations (especially the selection of the reference population, the maintenance of the analytical quality, the choice of the statistical method used…) will be briefly discussed.

Serum C-reactive protein (CRP) concentration was determined for 3605 subjects using an immunonephelometric assay improved to provide greater sensitivity. Subjects were from 5 to 75 years old and belonging to 1003 nuclear families recruited from the Stanislas Coh o rt Study between January 1994 and August 1995. Sample values for CRP ranged from 0.17 mg/l to 100 mg/l. Geometric means (mean − SD; mean + SD) were in the 5–14 years old group 0.37 (0.17–1.07) mg/l, in the 15–28 years old group 0.47 (0.17–1.38) mg/l and in the 29–75 years old group 0.98 (0.34–2.85) mg/l. For women, the geometric means were 0.38 (0.17–1.10) mg/l, 0.62 (0.20–1.90) mg/l and 0.98 mg/l (0.31–3.13) mg/l respectively. The interindividual variability ranged from 138% to 759% among different age classes. Biological factors associated with CRP concentration variations were examined and accounted for 25% of the CRP variability in men and 40% in women. The main biological factors statistically associated with CRP concentration variations in men were: drugs, leukocyte count, body mass index, tobacco consumption, age, and in women: drugs, leukocyte count, age, body mass index and hemoglobin concentration. These factors were used to define the exclusion and partition criteria when obtaining the reference samples. Medians for reference values ranged from 0.20 to 0.68 mg/l in males and from 0.20 to 0.78 mg/l in women.

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!

Since its establishment, the Center for Preventive Medicine in Vandoeuvre-les-Nancy, France, performed specific studies on healthy humans, and its approach was very useful for defining reference values. Prevention should extend its interest to chronic diseases. The majority of important adult disorders are partially genetically determined. Genetic markers are also useful as exclusion or as partition criteria in the production of reference values. Results are presented that were obtained for apolipoproteins E, B and AIV, frequencies of these polymorphisms in the Lorraine population, and relationships between these polymorphisms and lipid metabolism-related parameters. Health checkup centers, in particular those involved in family screening, are well suited for reassembling many data concerning environmental factors: tobacco consumption, alimentation habits, or alcohol and drug consumption. Simultaneous determination of genetic markers could allow the determination of an individual’s susceptibility or resistance to developing a disease and to prepare a preventive action.

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.

High sensitivity C-reactive protein is a useful marker in clinical practice; however, reference intervals are not available for all age groups. Therefore, the aim of this study was to determine reference intervals for elderly people. The non-parametric reference limits were calculated for the two genders, subdivided into two age classes (50-64 and 65-91 years). In our selected sample population, we did not observe significant gender differences.

The aim of this study was to establish soluble serum transferrin receptor (sTfR) reference limits. sTfR was measured in 885 healthy subjects from 3 to 91 years old (433 men, 409 women), without hematological abnormalities, using an immunonephelometric assay. The sTfR median concentrations in our population decreased gradually from the group aged 3-10 years to the group aged 21-40 years, then there were no changes in the older groups except for the females >60 years of age. The interindividual variability ranged from 12.6% to 30.3% among different age groups, and the analytical variability was 5%. Biological factors and other factors associated with sTfR concentration variation were examined and accounted for 35% of the sTfR variability in men aged 20 years or less, and 18% in those older than 20 years. Also, they accounted for 45% of the variability in women aged 20 years or less and 14% in those older than 20 years. The main factors statistically associated with sTfR concentration in males were ferritin, orosomucoid, hemoglobin, and tobacco in all age groups and only mean corpuscular volume (MCV) in males less than 20 years old. In the females the main factors were age, orosomucoid, and hemoglobin in all age groups, MCV and tobacco in females less than 20 years old, and ferritin and physical activity in females more than 20 years old. These factors were used to define the exclusion and partition criteria for obtaining the reference samples. Medians for reference values were: 1.60 mg/l in the 3-10-year old group (males and females); 1.42 mg/l in males between 11 and 20 years of age, and 1.33 mg/l in females of the same age. In the other age groups, the median of the reference values was 1.16 mg/l, except in females over 60 years old, for whom it was 1.26 mg/l.

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.

Fibrinogen is considered to be a strong predictor and independent factor of cardiovascular diseases. The data presented here describe the baseline measurements of fibrinogen in 1008 apparently healthy subjects, aged 4-60 years and their relationship to age, sex, body weight, smoking, alcohol, and use of oral contraceptives. Pearson's correlations and a linear multiple regression model were used. Plasma fibrinogen was measured kinetically in a photometer, the Behring Chromotimer, using the CTS-fibrinogen method. There were neither statistical difference between girls and boys aged 4-20 years nor correlation with variables related to cardiovascular diseases. In adults, we found an increase of plasma fibrinogen concentration with age and no statistical difference between men and women, except in subjects aged 40-50 years. There was a positive correlation between fibrinogen and ponderal index. In women aged 20-30, 30-40, 40-50 and 50-60 years, the mean fibrinogen concentrations increased of 0.009, 0.021, 0.010 and 0.015 g/l for one percent of overweight, in each subgroup respectively. In women aged 20-30 years using oral contraceptives, the mean fibrinogen concentration was 0.19 g/l higher than in women not using oral contraceptives. The smoking effect was observed only in 30-40 year-old men. Each cigarette smoked per day increases of the mean fibrinogen by 0.35 g/l after standardization for ponderal index and alcohol consumption. Alcohol consumption was negatively correlated to plasma fibrinogen in subjects 30-40 years old. In women, 1 g of alcohol per day induces a 0.008 g/l decrease in the mean fibrinogen while in men the decrease is 0.004 g/l.(ABSTRACT TRUNCATED AT 250 WORDS)1008 healthy subjects (510 men and 498 women) aged 4-60 years were studied. 109 women, aged 20-30 years and 40-50 years, were taking oral contraceptives. Plasma fibrinogen was measured kinetically in a photometer, the Behring Chromotimer. Day-to-day controls were tested using CTS-control plasma (Behring). The Student's t-test showed that there was no statistical difference between the sexes except in subjects aged 40-50 years (mean +or- standard deviation [SD] for men = 2.99 +or- 0.59 gm/l [n = 125] vs. 2.81 +or- 0.50 gm/l for women [n = 90], p = 0.017). Fibrinogen was higher in children and adolescents than in adults aged 20-30 years. There were statistically significant differences between children aged 4-14 years and adults aged 20-30 years. 54 boys had a mean +or- SD = 3.07 +or- 0.57 gm/l compared with 109 men with 2.65 +or- 0.52 gm/l (p 0.001). From age 30, plasma fibrinogen increased steadily in both women and men. In adults, a significant effect of smoking appeared only in men aged 30-40 years. Each cigarette smoked per day increased the mean fibrinogen by 0.35 gm/l. In men aged 20-50 years the fibrinogen varied from 2.785 gm/l +or- 0.564 (mean +or- SD) in nonsmokers (n = 163) to 2.974 +or- 0.615 (mean +or- SD) in men (n = 119) smoking more than 10 cigarettes/day. Alcohol consumption was negatively correlated to plasma fibrinogen in subjects aged 30-40. In women 1 gm of alcohol/day induced a 0.008 gm/l decrease of mean fibrinogen. In men the decrease was 0.004 gm/l of alcohol/day. Regression analysis showed that the fibrinogen concentration increased by 0.1, 0.21, 0.1, and 0.15 gm/l/10% overweight for women aged 20-30, 30-40, 40-50, and 50-60 years, respectively. In women aged 20-30 taking oral contraceptives the mean fibrinogen concentration was significantly (0.19 gm/l) higher, weight for weight, than in the women not taking them.

Laboratory-test results for a population of immigrants living in north-east France revealed differences between the concentration of the blood constituents of immigrants and native French people. By immigrants we mean persons now living in France but coming from other geographical locations such as Italy, the Iberian Peninsula, northern or central Europe, northern Africa, or the Near or Middle East. Multiple regression analysis confirmed the need to establish reference limits in immigrants for many blood constituents because of a significant shift of the curve relative to that for the French population. We have determined appropriate reference limits, for some ten blood constituents in each group of immigrants.

We have studied factors affecting biological variation in total plasma alkaline phosphatase in a population of 32 329 apparently healthy subjects four years old or older. Quantification of the bone and liver isoenzymes after thermal denaturation made it possible to specify the contributions of each isoenzyme to variations in the total activities. The main factors that modify plasma alkaline phosphatase activity are age, sex, hormonal state (puberty or menopause), and morphometric parameters (height, body weight, or degree of overweight). The bone isoenzyme is mainly responsible for the variations associated with age, sex, and puberty and to some extent with the menopause. Activity of the liver isoenzyme was also altered at the menopause and by certain drugs, such as oral contraceptives and blood-lipid-lowering agents. These data allow us to propose reference limits for total plasma, bone, and liver alkaline phosphatases according to age and sex.