P. Hyltoft Petersen

The Nordic reference interval project 2000 : recommended reference intervals for 25 common biochemical properties

Each of 102 Nordic routine clinical biochemistry laboratories collected blood samples from at least 25 healthy reference individuals evenly distributed for gender and age, and analysed 25 of the most commonly requested serum/plasma components from each reference individual. A reference material (control) consisting of a fresh frozen liquid pool of serum with values traceable to reference methods (used as the project “calibrator” for non‐enzymes to correct reference values) was analysed together with other serum pool controls in the same series as the project samples. Analytical data, method data and data describing the reference individuals were submitted to a central database for evaluation and calculation of reference intervals intended for common use in the Nordic countries. In parallel to the main project, measurements of commonly requested haematology properties on EDTA samples were also carried out, mainly by laboratories in Finland and Sweden. Aliquots from reference samples were submitted to storage in a central bio‐bank for future establishment of reference intervals for other properties. The 25 components were, in alphabetical order: alanine transaminase, albumin, alkaline phosphatase, amylase, amylase pancreatic, aspartate transaminase, bilirubins, calcium, carbamide, cholesterol, creatine kinase, creatininium, γ‐glutamyltransferase, glucose, HDL‐cholesterol, iron, iron binding capacity, lactate dehydrogenase, magnesium, phosphate, potassium, protein, sodium, triglyceride and urate.

Analytical goals for the acceptance of common reference intervals for laboratories throughout a geographical area

Analytical goals required for the successful transfer of reference intervals between laboratories within a specified limited geographical area, with a population homogeneous for the quantities, are presented. Diagrams are shown which allow the investigation of the influence of analytical imprecision and bias, both separately and in combination, on the percentage of the population outside each reference limit. Figures to evaluate the effect of population sample size on the size of confidence interval around each reference limit are combined with the diagrams for analytical imprecision and bias. The maximum acceptable percentage of the population outside the limit for the 0.90 confidence interval of each of the means +/- 1.96 s reference limits is 4.6% for a population sample size of 120. Based on this, the maximum acceptable imprecision, with no bias, is 0.6 of the total biological standard deviation (sB) and the maximum acceptable bias, with no imprecision, is 0.25 sB.

Effects of posture on concentrations of blood constituents in healthy adults: practical application of blood specimen collection procedures recommended by the Scandinavian Committee on Reference Values

Different procedures for the collection of blood for the determination of reference values in healthy adults have been subjected to practical testing. The Scandinavian Committee on Reference Values suggested that subjects lying in bed after an overnight sleep and ambulatory individuals, after 15 min of sitting in a chair, were two suitable and different reference populations. In forty subjects we found an increase from lying to sitting position of about 6.5% in the serum concentrations of proteins, enzymes and lipids. The corresponding increases for S-calcium and B-haemoglobin were approximately 3% whereas S-thyroxine, unexpectedly, changed by 11%. There were no significant changes of potassium and sodium values. For some constituents the changes were age and sex-dependent being greater in a group of elderly women as compared to a group of younger men. After 1 h of recumbency the concentrations of the constituents generally returned to those in the initial, lying position.

Optimization of preanalytical conditions and analysis of plasma glucose. 1. Impact of the new WHO and ADA recommendations on diagnosis of diabetes mellitus

The new diagnostic criteria for type 2 diabetes from the American Diabetes Association (ADA) and World Health Organization (WHO) recommend measurements on plasma and a lowering of the glucose threshold for diabetes by 0.8mmol/L. This narrows the distance between the upper end of the reference limit and the discriminatory level to a degree where analytical quality becomes critical. The quality demands for the preanalytical and analytical phase and their consequences on diagnostic performance have to be established in the new technical system, measuring in plasma rather than in capillary whole blood. Because of the instability of glucosein blood samples it is necessary to clarify the influence of different preanalytical and analytical factors on the number of false-positive and false-negative classifications. Thus the aim of the present study was to find optimal conditions for sampling, additives, storage, transport and analysis of plasma glucose combining feasibility with an analytical bias close to zero and a within-imprecision around 1%. We have documented the analytical performance of the method itself and its traceability to an international standard. The preanalytical conditions, such as influence of antiglycolytic agent NaF, conditions for plasma separation, storage temperature and storage time before and after plasma separation were investigated. In conclusion, we recommend that blood should be drawn in tubes containing heparin and NaF and kept on ice water for not more than 1h until centrifugation at minimum 1000 g for 10min. The plasma is then stable for at least 48h at room temperature.The new diagnostic criteria for type 2 diabetes from the American Diabetes Association (ADA) and World Health Organization (WHO) recommend measurements on plasma and a lowering of the glucose threshold for diabetes by 0.8 mmol/L. This narrows the distance between the upper end of the reference limit and the discriminatory level to a degree where analytical quality becomes critical. The quality demands for the preanalytical and analytical phase and their consequences on diagnostic performance have to be established in the new technical system, measuring in plasma rather than in capillary whole blood. Because of the instability of glucose in blood samples it is necessary to clarify the influence of different preanalytical and analytical factors on the number of false-positive and false-negative classifications. Thus the aim of the present study was to find optimal conditions for sampling, additives, storage, transport and analysis of plasma glucose combining feasibility with an analytical bias close to zero and a within-imprecision around 1%. We have documented the analytical performance of the method itself and its traceability to an international standard. The preanalytical conditions, such as influence of antiglycolytic agent NaF, conditions for plasma separation, storage temperature and storage time before and after plasma separation were investigated. In conclusion, we recommend that blood should be drawn in tubes containing heparin and NaF and kept on ice water for not more than 1 h until centrifugation at minimum 1000 x g for 10 min. The plasma is then stable for at least 48 h at room temperature.

Descriptive analytical data and consequences for calculation of common reference intervals in the Nordic Reference Interval Project 2000.

In the Nordic Reference Interval Project (NORIP), data from 102 Nordic clinical chemical laboratories were obtained. Each laboratory reported analytical data on up to 25 of the most commonly used clinical biochemical properties, including results from each of a minimum of 25 reference individuals. A reference material consisting of a liquid frozen pool of serum with values traceable to reference methods (used as the project “calibrator” for non‐enzymes to correct reference values) was measured together with other serum pool controls in each laboratory in the same analytical series as the project samples. The data on the controls were used to evaluate the analytical quality of the routine methods. For reference interval calculations, only such reference values on enzymes were accepted that were obtained by applying the International Federation of Clinical Chemistry (IFCC) compatible methods (37°C), while “calibrator”‐corrected reference values were used in the cases of non‐enzymes. For each property, gender‐ and age‐specific reference intervals were estimated, based on simple non‐parametric calculations and using objective criteria to perform partitioning into subgroups. It is concluded that the same reference intervals are applicable in all five Nordic countries. The following descriptive data for the considered properties are presented in the tables: number of measurement values from each country and measurement system, certified/indicative target values for controls, differences between methods and measurement systems together with coefficients of variation, effects of control correction on the measurement values, differences between subgroups as determined by age, gender, country and material, and comparison of the new reference intervals with those presented in standard textbooks. The 25 components involved in this project were (listed in alphabetical order): Alanine transaminase, albumin, alkaline phosphatase, amylase, amylase pancreatic type, aspartate transaminase, bilirubin, calcium, carbamide, cholesterol, creatine kinase, creatininium, γ‐glutamyltransferase, glucose, HDL‐cholesterol, iron, iron‐binding capacity, lactate dehydrogenase, magnesium, phosphate, potassium, protein, sodium, triglyceride and urate.

Screening for haemochromatosis: prevalence among Danish blood donors

Abstract. Hereditary haemochromatosis is an autosomal recessive disease that is genetically expressed by excessive accumulation of iron in the tissues, resulting in cirrhosis, diabetes mellitus, cardiomyopathy and hypogonadism. As the disease may be diagnosed before the appearance of symptoms, and prevented by repeated phlebotomies, there are strong implications for adoption of a screening procedure. Determinations of transferrin saturation (TS) and serum ferritin concentration (SF) were used to screen 4302 blood donors, who were selected for follow‐up studies if they fulfilled any of the following three criteria: (i) TS ≥ 0.7; (ii) TS ≥ 0.5 together with SF ≥ 150 μg l−1; (iii) SF ≥ 300 μg l−1. A total of 58 subjects who fulfilled at least one of these criteria were reinvestigated, after which 18 individuals still fulfilled at least one criterion. Fifteen subjects having SF ≥ 300 μg l−1 were offered liver biopsy and thirteen of these accepted. In one individual, no stainable iron was detected, and two subjects did not fulfil the previously established diagnostic criteria for the diagnosis of hereditary haemochromatosis. Ten subjects who had a high TS and liver iron grade 2–4 according to Bassett were classified accordingly as homozygotes. On the basis of these results, the prevalence of haemochromatosis in Denmark was estimated to be 0.0037–0.0046.

The importance of imprecision

A recent editorial by Barnett’ stated: ‘There is no evidence that patients or physicians are harmed by our present techniques as long as the techniques meet existing standards’. While this may have some elements of truth, it is important to recognize that there appears to be evidence that many of our existing standards have either deteriorated with time (or not continued to improve) perhaps due to either complacency or the possibly inferior performance of newer analytical system^.^^^ There are, however, other plausible reasons for this. In evaluations of reagent kit sets and instruments, desirable standards of performance are used rarely as criteria for a c ~ e p t a b i l i t y ; ~ . ~ empirical comments such as ‘the analytical performance is acceptable’, ‘it gave acceptable precision’ and ‘precision was excellent’ are often used,6 which imply, without objectivity or good documentation, that the analytical performance achieved is satisfactory. Moreover, expansion of the use of instruments designed to be operated by relatively unskilled staff has occurred, even though many studies have shown that the standards of specimen collection technique and analytical performance are generally inferior to those obtained by skilled staff in laboratories. In addition, many have stated that the imprecision of analytical techniques is no longer a problem and that available resources should be directed to lowering inaccuracy and improving betweenlaboratory transferability of results. We fully support improvements in analytical methodology which reduce inaccuracy and non-specificity. However, analytical imprecision cannot be neglected. We believe that the current situation should be a matter of concern and that further improvement in analytical imprecision is warranted and of both clinical and laboratory importance.

Reference individuals, blood collection, treatment of samples and descriptive data from the questionnaire in the Nordic Reference Interval Project 2000

The rules for recruitment of reference individuals, inclusion and preparation of individuals, blood collection, treatment of samples (and control materials) and analysis at the 102 medical laboratories attending the Nordic Reference Interval Project (NORIP) are given as well as the rules for central exclusion of reference individuals. The individuals (18–91‐year‐olds) should be evenly distributed on age and gender groups. The 3002 reference individuals who contributed at least one reference value to the finally suggested reference intervals were characterized using the information in the questionnaire. Gender, age and country are the main entries in the tables. Other variables in the cross‐tables or figure are height, weight, body mass index, ethnic origin, heredity for diabetes, chronic disease, oestrogens or oral contraceptives, other medication, hard physical activity, previous blood donations, smoking habits, use of alcohol, hours since last meal and time of blood collection (hour, day of week, month, year). The Danes had the highest alcohol consumption and the Icelanders had the highest body mass index. The information in this article may interest potential users of the Nordic Reference Interval Project bio‐bank and database (NOBIDA) in which serum, Li‐heparin plasma and EDTA buffy coat from the mentioned individuals are stored below −80°C.

Can capillary whole blood glucose and venous plasma glucose measurements be used interchangeably in diagnosis of diabetes mellitus

According to new proposals from the American Diabetes Association (ADA) and WHO, venous peripheral plasma is the preferred system for measuring glucose for diagnosing diabetes mellitus. Owing to the instability of glucose in plasma after blood sampling, strict well-defined and standardized preanalytical conditions are essential to ensure that glucose concentration measured in plasma reflects real blood glucose in the patient. This is in contrast to the capillary whole blood measurements, which are easy to perform and well established. We investigated whether it is possible to perform analysis on capillary whole blood but express the results as plasma glucose values and hence obtain comparable results and the same predictive values for diagnosis in the individual patient? The conclusion of our investigations is that these two systems are not interchangeable and that conversion should not be done for diagnostic purposes where plasma determinations are recommended.

The stability of blood, plasma and serum constituents during simulated transport

The aim of the study was to simulate physical conditions occurring during mail transport and to study how these conditions influence the stability of material for clinical chemistry analysis. Periods of transport at constant temperatures did not result in big changes. We found that all serum and plasma constituents tested should be considered stable for 4 days at -20 degrees C. Except for the coagulation factors studied all other serum and plasma constituents were stable also at 4 degrees C and 20 degrees C. Blood cells were studied only at 4 degrees C at which temperature they were stable for 4 days except for thrombocytes. The condition that caused the largest effect was changing of temperature. Temperature gradient from ambient to 50 degrees C influenced the concentrations of all cells in blood, of coagulation factors in plasma and of enzymes in serum. The constituents were remarkably stable during shaking. The results agreed with those of a field study on the effect of mail transport by Berg et al. [3].