Handling of hemolyzed serum samples in clinical chemistry laboratories: the Nordic hemolysis project.

Background Some clinical chemistry measurement methods are vulnerable to interference if hemolyzed serum samples are used. The aims of this study were: (1) to obtain updated information about how hemolysis affects clinical chemistry test results on different instrument platforms used in Nordic laboratories, and (2) to obtain data on how test results from hemolyzed samples are reported in Nordic laboratories. Methods Four identical samples containing different degrees of hemolysis were prepared and distributed to 145 laboratories in the Nordic countries. The laboratories were asked to measure the concentration of cell-free hemoglobin (Hb), together with 15 clinical chemistry analytes. In addition, the laboratories completed a questionnaire about how hemolyzed samples are handled and reported. Results Automated detection of hemolysis in all routine patient samples was used by 63% of laboratories, and 88% had written procedures on how to handle hemolyzed samples. The different instrument platforms measured comparable mean Hb concentrations in the four samples. For most analytes, hemolysis caused a homogenous degree of interference regardless of the instrument platform used, except for alkaline phosphatase (ALP), bilirubin (total) and creatine kinase (CK). The recommended cut-off points for rejection of a result varied substantially between the manufacturers. The laboratories differed in how they reported test results, even when they used the same type of instrument. Conclusions Most of the analytes were homogeneously affected by hemolysis, regardless of the instrument used. There is large variation, however, between the laboratories on how they report test results from hemolyzed samples, even when they use the same type of instrument.

Pediatric reference intervals for general clinical chemistry components - merging of studies from Denmark and Sweden.

BACKGROUND: Reference intervals are crucial tools aiding clinicians when making medical decisions. However, for children such values often are lacking or incomplete. The present study combines data from separate pediatric reference interval studies of Denmark and Sweden in order to increase sample size and to include also pre-school children who were lacking in the Danish study. METHODS: Results from two separate studies including 1988 healthy children and adolescents aged 6 months to 18 years of age were merged and recalculated. Eighteen general clinical chemistry components were measured on Abbott and Roche platforms. To facilitate commutability, the NFKK Reference Serum X was used. RESULTS: Age- and gender-specific pediatric reference intervals were defined by calculating 2.5 and 97.5 percentiles. CONCLUSION: The data generated are primarily applicable to a Nordic population, but could be used by any laboratory if validated for the local patient population.

Analytical Bias Exceeding Desirable Quality Goal in 4 out of 5 Common Immunoassays: Results of a Native Single Serum Sample External Quality Assessment Program for Cobalamin, Folate, Ferritin, Thyroid-Stimulating Hormone, and Free T4 Analyses.

BACKGROUND: We undertook this study to evaluate method differences for 5 components analyzed by immunoassays, to explore whether the use of method-dependent reference intervals may compensate for method differences, and to investigate commutability of external quality assessment (EQA) materials. METHODS: Twenty fresh native single serum samples, a fresh native serum pool, Nordic Federation of Clinical Chemistry Reference Serum X (serum X) (serum pool), and 2 EQA materials were sent to 38 laboratories for measurement of cobalamin, folate, ferritin, free T4, and thyroid-stimulating hormone (TSH) by 5 different measurement procedures [Roche Cobas (n = 15), Roche Modular (n = 4), Abbott Architect (n = 8), Beckman Coulter Unicel (n = 2), and Siemens ADVIA Centaur (n = 9)]. The target value for each component was calculated based on the mean of method means or measured by a reference measurement procedure (free T4). Quality specifications were based on biological variation. Local reference intervals were reported from all laboratories. RESULTS: Method differences that exceeded acceptable bias were found for all components except folate. Free T4 differences from the uncommonly used reference measurement procedure were large. Reference intervals differed between measurement procedures but also within 1 measurement procedure. The serum X material was commutable for all components and measurement procedures, whereas the EQA materials were noncommutable in 13 of 50 occasions (5 components, 5 methods, 2 EQA materials). CONCLUSIONS: The bias between the measurement procedures was unacceptably large in 4/5 tested components. Traceability to reference materials as claimed by the manufacturers did not lead to acceptable harmonization. Adjustment of reference intervals in accordance with method differences and use of commutable EQA samples are not implemented commonly.

Recommended Nordic paediatric reference intervals for 21 common biochemical properties.

Paediatric reference intervals based on samples from healthy children are difficult to establish and consequently data are often from hospitalized children. Furthermore, biases may present in published data due to differences in the analytical methods employed. Blood samples from 1429 healthy Danish children were collected for establishing reference intervals for 21 common biochemical properties (Alanine transaminase, Albumin, Alkaline phosphatase, Aspartate transaminase, Bilirubin, Calcium, Cholesterol, Creatinine, Creatine kinase, HDL-Cholesterol, Iron, Lactate dehydrogenase, LDL- Cholesterol, Magnesium, Phosphate, Potassium, Protein, Sodium, Transferrin, Triglycerides and Urate). Samples were analyzed on a Roche-Modular-P/ISE-system. The NORIP reference material (NFKK Reference Serum X) was included in all the analytical runs. Reference values were recalculated according to the target values of X for the properties and statistical calculations carried out as performed in the NORIP study. Thus commutable (regarding analytical method) reference intervals for 20 properties were established and for LDL-Cholesterol reference intervals were reported for the specific analytical method employed. The data were compared to previous studies and to those obtained from the youngest age group in the NORIP study. Marked age differences were observed for most of the properties. Several properties also showed gender-related differences, mainly at the onset of puberty. Data are presented as suggested intervals for combined age groups, but can be accessed via the NORIP home page if more detailed division according to age or gender is desired.

Heterophilic antibody interference in commercial immunoassays; a screening study using paired native and pre-blocked sera.

BACKGROUND: Heterophilic antibodies are still an important source of interference in immunoassays. We have conducted a screening study for interference in a panel of commercially available assays using two sera known to contain high titer Fc-reactive heterophilic antibodies. METHODS: The sera were distributed to laboratories participating in the Nordic External Quality Assessment cooperation (EQANord). Duplicate samples pre-blocked with aggregated murine monoclonal MAK33 were also supplied. Discrepancies (>50%) between the results for native and blocked samples were used to classify the tested assays as susceptible to interference. A total of 170 different assay kits covering 91 analytes were tested. RESULTS: We found that 21 assays, covering 19 different analytes, were susceptible to interference from the heterophilic antibodies in the two sera. Many of these are clinically and commercially important assays. Some of the false results were grossly elevated and could have been detrimental to patient care in a clinical setting. CONCLUSIONS: Heterophilic antibodies with Fc-reactivity remain a threat. A more widespread use of antibody fragments and aggregated immunoglobulin could potentially improve the heterophilic antibody resistance of assays intended for clinical use.

[Updated reference intervals for clinical chemical components]. / Oppdaterte referanseintervaller for klinisk-kjemiske komponenter.

BACKGROUND: More or less outdated reference intervals are still in common use with many clinical biochemical components. In order to update them, a Nordic cooperative project (the Nordic reference interval project, NORIP) was organised. MATERIAL AND METHODS: 102 Nordic laboratories participated; 3036 persons aged 18 years or above were included. 25 components were measured in serum and/or plasma. RESULTS: Common reference intervals for all Nordic countries were calculated for each of the 25 components, with age and sex-specific intervals when statistical criteria suggested this to be appropriate. INTERPRETATION: The national clinical biochemical societies of Norway, Sweden, Finland and Denmark have all recommended that the suggested reference intervals should be brought into common use. In Norway, new reference intervals for seven enzymes were implemented in May 2003, the remaining are recommended implemented in spring 2004. Except for four of the enzymes, there will be no simultaneous change in the measuring methods used, hence medical decision limits are essentially not affected.

[New common reference intervals for clinical chemistry in the Nordic countries. A better basis for clinical assessment and cooperation]. / Nya gemensamma nordiska referensintervall inom klinisk kemi. Bättre bas för klinisk bedömning och samarbete.

A project engaging the five Nordic countries has presented common reference intervals for the most frequently used biochemical and haematological analytes. The results are based on samples from up to 3000 healthy adult reference persons and are statistically calculated to include 95% of the reference population's values. Reference samples were analyzed together with commutable control materials traceable to reference methods (IMEP-17). Enzymes were analyzed using methods traceable to IFCC reference methods. The Swedish Society of Clinical Chemistry now recommends its member laboratories to introduce the new reference intervals and new enzyme methods during 2004.

Effect of ambient temperature on analytical performance of self-monitoring blood glucose systems.

BACKGROUND: The analytical quality of self-monitoring of blood glucose (SMBG) can be affected by environmental conditions such as temperature. The objective of this study was to determine the influence of (1) a shift in the ambient temperature immediately before measurement and (2) taking measurements in the lower and upper part of the operating temperature range. METHODS: Nine different SMBG systems on the Norwegian market were tested with heparinized venous blood (4.8 and 19.0 mmol/L). To test the shift in ambient temperature effect, the glucometer and strips were equilibrated for 1 h at 5°C or 1 h at 30°C before the meter and strips were moved to room temperature, and measurements were performed after 0, 5, 10, 15, and 30 min. To test the lower and upper temperature range, measurements were performed at 10°C and at 39°C after 1 h for temperature equilibration of the glucometer and strips. All these measurements were compared with measurements performed simultaneously on a meter and strips kept at room temperature the whole time. RESULTS: Six of nine SMBG systems overestimated and/or underestimated the results by more than 5% after moving meters and strips from 5°C or 30°C to room temperature immediately before the measurements. Two systems underestimated the results at 10°C. One system overestimated and another underestimated the results by more than 5% at 39°C. CONCLUSIONS: The effect on analytical performance was most pronounced after a rapid shift in the ambient temperature. Therefore patients need to wait at least 15 min for temperature equilibration of affected meters and strips before measuring blood glucose.

The time-trend and the relation between smoking and circulating selenium concentrations in Norway.

OBJECTIVES: The objectives of this study were to investigate biomarkers of selenium status in relation to smoking habits and to analyze the time-trend of selenium in serum (S-Se) in Norway during the time period 1995-2006. METHODS: The impact of smoking habits was investigated in a population recruited to a cross-sectional study of blue-collar workers in the southern part of the country (n=98). The time-trend was studied in all subjects who delivered blood samples for the determination of S-Se to a large commercial clinical chemistry laboratory in Norway. RESULTS: Smokers had 0.14 and 0.20 micromol/L lower concentrations of selenium in whole blood (B-Se) and serum, respectively, than non-smokers. The amount of smoking, as assessed by the serum cotinine concentration, was negatively associated with the B-Se concentration (Pearson's r=-0.43). The 1/3 of the blue-collar workers with the lowest concentrations of B-Se or S-Se had lower activity of glutathione peroxidase in serum (S-GSHpx) than the remaining subjects. Snuff users had about the same levels of B-Se and S-Se as the non-smokers, although they had about the same amount of nicotine metabolites in urine and serum as the smokers. A decreasing trend of S-Se was observed during the observation period from 1995 to 2006. The mean concentration was 1.26 micromol/L in 1995, while the lowest mean concentration was measured in 2003 (1.01 micromol/L). CONCLUSION: Smoking, but not snuffing, is associated with lower concentrations of B-Se and S-Se. The reduction of B-Se is negatively associated with the nicotine biomarker cotinine in serum. A substantial proportion of blue-collar workers had not maximized the activity of S-GSHpx. Selenium status may have become poorer since 1995.

Comparison of high-pressure liquid chromatography (HPLC) and Griess reagent-spectroscopic methods for the measurement of nitrate in serum from healthy individuals in the Nordic countries.

OBJECTIVES: Bioavailability of NO can be estimated by measuring the concentration of nitrate (NO(3)) in serum. However, the methods used for the measurement NO(3) in plasma or serum show a great degree of variation. Therefore, we compared two analytical methods for the measurement of NO(3) in serum. DESIGN AND METHODS: The concentration of NO(3) in 600 serum samples collected from healthy individuals was determined by the HPLC and by the Griess reagent-spectroscopic method. RESULTS: The concentration of NO(3) in the samples was 29.4+/-16.1 micromol/L and 26.2+/-14.0 micromol/L (mean+/-SD) measured by HPLC and Griess reagent-spectroscopic method respectively (p<0.0001). We detected a significant correlation between the two methods (R=0.81, p<0.0001). CONCLUSIONS: A significant correlation between the two methods may suggest that either method can be used for the measurement of NO(3) in serum, however the Griess reagent-spectroscopic method measures lower concentrations of NO(3) than the HPLC method.

Proposal for guidelines to establish common biological reference intervals in large geographical areas for biochemical quantities measured frequently in serum and plasma.

A suggestion for a standard procedure to establish biological reference intervals for biochemical quantities by a multicenter approach is presented. This procedure was developed for and used in the Nordic Reference Interval Project 2000 (NORIP). This project established biological reference intervals for 25 frequently requested biochemical quantities through cooperation of 102 Nordic laboratories. Each laboratory performed collection of reference samples and measurement using their routine methods. The bias of each routine method was eliminated by use of common reference materials measured in each of the participating laboratories.

Partitioning of nongaussian-distributed biochemical reference data into subgroups.

BACKGROUND: The aim of this study was to develop new methods for partitioning biochemical reference data, covering in particular nongaussian distributions. METHODS: We recently proposed partitioning criteria for gaussian distributions. These criteria relate to proportions of the subgroups outside each of the reference limits of the combined distribution (proportion criteria) and to distances between the subgroup distributions as correlates of these proportions (distance criteria). However, distance criteria do not seem to be ideal for nongaussian distributions because a generally valid relationship between proportions and distances cannot be established for these. RESULTS: Proportion criteria appear preferable to distance criteria for two additional reasons: (a) The prevalences of the subgroup populations may have a considerable effect on stratification, but these are hard to account for by using distance criteria. Two methods to handle prevalences are described, the root method and the multiplication method. (b) Tied reference values, another complication of the partitioning problem, could also be hard to take care of using distance criteria. Some solutions to the problems caused by tied reference values are suggested. CONCLUSIONS: Partitioning of biochemical reference data should preferably be based on proportion criteria; this is particularly true for nongaussian distributions. Both of the described complications of the partitioning problem, the prevalences of the subgroups and tied reference values, are hard to deal with using distance criteria, but the proposed methods make it possible to account for them when proportion criteria are applied.