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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.

In a cohort of 13 patients, peripheral blood stem cells (PBSC) were harvested by apheresis after mobilization with chemotherapy and rhG-CSF. Nine patients who had excellent mobilization were transplanted with PBSC concentrates from a minimal number of apheresis procedures (mean of 1.5, range = 1-3). During collection, the number of circulating progenitors was on average 50 times higher than those observed at the steady state in the peripheral blood of healthy unstimulated individuals. The mean number of CFU-GM/kg reinfused per patient was 28.1 x 10(4) (range = 18.0-50 x 10(4)). The use of rhG-CSF, at either 1 or 5 micrograms/kg/day, resulted in a significantly greater yield of CFU-GM per mononuclear cells than that observed previously in a comparable group of patients receiving chemotherapy alone. Prompt and durable engraftment occurred after myeloablative chemotherapy. The average duration of absolute neutropenia was 9 days. Transfusion requirements were low with an average of four packed red cell units and two platelet transfusions per patient. The shortest follow-up is 5 months and the longest is 20+ months. The convenience of this new approach to support myeloablative therapy offers new possibilities for the administration of a higher dose-intensity of chemotherapeutic agents. A limited number of apheresis procedures timely harvested will improve the cost effectiveness of transplant programs.

The efficacy of loratadine as prophylactic therapy for seasonal allergic rhinitis was evaluated in a randomized, double-blind, parallel group, placebo-controlled study. One hundred eighteen subjects received either loratadine, 10 mg once daily, or placebo for 6 weeks. Treatment was begun prior to the onset of grass pollen seasonal symptoms of allergic rhinitis. Total symptom-free days occurred more frequently in subjects receiving loratadine. More loratadine than placebo subjects (65% versus 49%) had no symptoms or mild rhinitis at the end of the study. In contrast, the differences between loratadine and placebo in symptom scores did not achieve significance. The incidence of sedation and anticholinergic effects were comparable between the groups. Prophylactic loratadine therapy was effective in suppressing symptoms of seasonal allergic rhinitis and providing patients with symptom-free days throughout the pollen season.

In order to determine calcium intake, 1,450 French-Canadians of both sexes, all age groups and various socio-economic status, were recruited in Montreal and Laval using a stratified randomized selection. One thousand and sixty-five (73%) subjects completed food records: 82.4% for a period of seven days, the others for periods varying from one to six days, providing 6,590 days of food records. Subjects ranged in age from one month to 95 years with a mean age of 35.1 years. No seasonal variation in calcium intake was observed. On average, men consumed more calcium than women. This difference was significant for age groups 13 to 15, 19 to 24 and 25 to 49 (p < 0.05). Womens' diets, however, had a higher calcium density than those of men, except among 10-15-year-olds. Mean calcium intake exceeded the RNI for the Canadian population in all age groups except females aged 10 to 15 years, and both men and women aged 50 years and over. The results of this study suggest that those in the study region most at risk for insufficient calcium intake are adolescent females and older men and women.

The efficacy and safety of an extended-release combination of loratadine plus pseudoephedrine sulfate (SCH 434) was compared with that of a tablet containing chlorpheniramine maleate plus pseudoephedrine sulfate (CTM-D) in 131 patients with symptomatic seasonal allergic rhinitis. Patients were randomly assigned to receive either SCH 434 (loratadine 5 mg and pseudoephedrine sulfate 120 mg) or CTM-D (chlorpheniramine maleate 12 mg and pseudoephedrine sulfate 120 mg) twice daily for 2 weeks. Evaluations were made after 3, 7, and 14 days of treatment. Demographics (age, race, sex, and duration of seasonal allergic rhinitis) and baseline total symptom scores were comparable between groups. Both combination products were effective in relieving the symptoms of allergic rhinitis. Improvement in total symptom scores was 54% on day 3 and 65% on day 14 in the SCH 434 group versus 57% on day 3 and 64% on day 14 in the CTM-D group. Individual symptom scores (nasal discharge, stuffiness, nasal itching, sneezing, and ocular symptoms) responded similarly. A smaller proportion of patients in the SCH 434 group reported side effects, especially dry mouth (7% vs 19%, P = 0.07), fatigue (6% vs 25%, P < 0.01), and sedation (7% vs 22%, P < 0.03). In conclusion, the combination of loratadine plus pseudoephedrine sulfate was equally as effective as a classic antihistamine (chlorpheniramine maleate) plus pseudoephedrine sulfate but had a lower incidence of side effects.

In a double-blind study, 185 patients with seasonal allergic rhinitis were randomly assigned to receive 10 mg of loratadine or placebo once daily for three days. On day 1 of treatment, the onset of relief of symptoms within 30 minutes of drug administration was reported by 13% of the loratadine-treated patients and by 4% of the placebo patients (P less than 0.05). At two hours after drug administration, 65% of the loratadine-treated patients and 48% of the placebo patients reported symptom relief. On day 3, the loratadine-treated patients reported a significantly greater relief of symptoms, and according to both physician and patient evaluations, the treatment response was significantly superior in the loratadine-treated than in the placebo patients. The incidence of sedation was 2% in the loratadine group and 1% in the placebo group.

Average per capita consumption of eggs, milk, and meat; total caloric intake; and protein and fat consumption in 29 countries from 1964 through 1966 was related to the average age-adjusted mortality rates from cancer of the pancreas in these same countries for the period 1978 through 1979. A direct and significant correlation between mortality rates from cancer of the pancreas and per capita consumption of eggs, milk (p less than 0.001), and meat (p less than 0.01 for males and p less than 0.05 for females) was found. The total caloric intake was directly correlated with mortality rates from pancreatic cancer (p less than 0.01). This correlation was stronger for calories derived from animal sources of food (p less than 0.001) for both sexes, while consumption of vegetable calories correlated with decreased rates of mortality from pancreatic cancer. The average per capita intake of both total and animal fat was also directly correlated with mortality from cancer of the pancreas (p less than 0.001). This suggests that animal sources of calories, protein, and fat may play an important role in the etiology of pancreatic cancer.

The average per capita consumption of certain foods in 29 countries during 1964-66 was related to the average mortality rates for cancers of the oesophagus, stomach and pancreas in these same countries for the period 1978-79. No significant correlation was found between consumption of nutrients from different food groups and mortality rates for oesophageal cancer, with the exception of meat intake in females. Consumption of eggs, milk and particularly meat was inversely related to mortality from stomach cancer in males and females. Caloric and fat intakes from animal sources were also significantly and negatively related to mortality from stomach cancer in both sexes, while caloric intake from vegetable sources was positively associated, particularly in females. The absence of correlation between vegetable fat intake and mortality from stomach cancer strongly suggests carbohydrates as a major caloric source related to stomach cancer. Mortality from pancreatic cancer was strongly and positively related to consumption of eggs, milk and meat in both sexes. Caloric and fat intakes from animal sources had a strong positive correlation with mortality from pancreatic cancer in males and females, while caloric intake from vegetable sources showed a strong negative correlation in both sexes. The weak and not significant correlation of vegetable fat with mortality from pancreatic cancer mortality strongly suggests carbohydrates as a major caloric source related to pancreatic cancer. No significant correlation was found between age-adjusted mortality rates from stomach or pancreatic cancer or a given per capita caloric intake from animal or vegetable source. For both cancers, age-adjusted mortality rates were about twice those of females.

Alkaline phosphatase (EC 3.1.3.1) bound to trophoblastic cells in rat placenta is activated by Mg2+ and inhibited by Zn2+ in the same way as is found with partially purified soluble alkaline phosphatase in the same tissue (PetitClerc, C., Delisle, M., Martel, M., Fecteau, C. & Brière, N. (1975) Can. J. Biochem. 53, 1089-1100). In studies done with tissue sections (6-10 micron), it is shown that alkaline phosphatase activity and labelling of active sites by orthophosphate are lost during incubation with ethanolamine at pH 9.0. Addition of Mg2+ causes total recovery of catalytic activity and active sites labelling. Zn2+ displaces and replaces at the Mg2+ binding sites. The affinity for both ions is similar, and dissociation of Zn2+ from the enzyme is a very slow process, even in the presence of Mg2+. The Zn2+-alkaline phosphatase and Mg2+-alkaline phosphatase, which only differ by the ion bound to an apparent modulator site, have the same catalytic activity at pH less than 7.0, but the Zn2+ species has little activity at alkaline pH. Phosphorylation of the enzyme by orthophosphate indicates that with both enzyme species phosphoryl intermediate does not accumulate at alkaline pH. These results suggest that with orthophosphate, the phosphorylation step is rate determining for both enzymes, and that Zn2+ affects this step to a much greater extent. It is proposed that Zn2+ and Mg2+ regulate alkaline phosphatase in rat placenta. The concentration of both ions in maternal serum and placenta suggest that such a mechanism could exist in vivo.

Generally, the parietal layer of Bowman's capsule in the mammal kidney consists of a squamous epithelium resting upon a basement lamina. However, tall cylindrical cells resembling those of the proximal tubules were observed on the outer wall of Bowman's capsule in the mouse and rat kidneys. These cells were provided with an apical brush border and were positive for alkaline phosphatase and gamma-glutamyl transpeptidase suggesting phosphate and amino acid transport at the capsular level.

Wolfe has described different cancer risks associated with a classification of four patterns of the breast parenchyma on mammography, but there is however little information available on the ability of radiologists to agree on the classification of the different patterns. We have assessed inter-rater agreement on the assignment of films to one of the four mammographic patterns described by Wolfe. One hundred xeromammograms were selected, copied and distributed to 10 radiologists who were experts in mammography. Films were classified according to the presence or absence of several radiological signs, according to diagnosis and recommendation, and according to mammographic pattern. Agreement was assessed after correction for agreement expected by chance, using the Kappa statistic. In general, high levels of agreement were found for the classification of mammographic pattern. Agreement on the classification of mammographic pattern was substantially greater than agreement for any other feature of mammographic interpretation, including diagnosis and recommendation.