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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!

The concept of reference values is widely accepted, but their application has been quite lax over the years. This is due primarily to the difficulty of properly selecting and documenting samples from a reference population. In the absence of a clear description of reference individuals, reference values lose their meaning, are ambiguous at best, and are often confused with decision limits. The clinical medicine perspective of reference values is to rule out diseases and to define health, while that of preventive medicine is to appreciate the state of health. Defining reference limits and normality in this context is a great challenge. Advances in the fields of genomics and proteomics and the rapid pace of technological advances help highlight the biological diversity among individuals. However, there is a great need for reference values that are representative of healthy humans and presented in a manner that they can be utilized by all laboratories. In addition, as secure information technology becomes available, the goal of using an individual as their own reference during a lifetime is now possible, provided that consistency of databases is ensured.

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 select a homogeneous 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 make a good choice of the interquartile 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 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.