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Molecular diagnostics is one of the most rapidly growing areas of laboratory medicine. This rapid growth of clinical molecular tests has outpaced the availability and development of reference methods and reference materials. Such methods and materials are important for the development, validation, and interpretation of diagnostic methods and tests. Yet, there is a lack of harmonization between the numerous international organizations currently either certifying or defining reference materials. The objective of this position paper is to review and clarify the definition, attributes and applications for the use of reference materials in the context of molecular diagnostics. Clin Chem Lab Med 2010;48:1531–5.

When conducting studies to derive reference intervals (RIs), various statistical procedures are commonly applied at each step, from the planning stages to final computation of RIs. Determination of the necessary sample size is an important consideration, and evaluation of at least 400 individuals in each subgroup has been recommended to establish reliable common RIs in multicenter studies. Multiple regression analysis allows identification of the most important factors contributing to variation in test results, while accounting for possible confounding relationships among these factors. Of the various approaches proposed for judging the necessity of partitioning reference values, nested analysis of variance (ANOVA) is the likely method of choice owing to its ability to handle multiple groups and being able to adjust for multiple factors. Box-Cox power transformation often has been used to transform data to a Gaussian distribution for parametric computation of RIs. However, this transformation occasionally fails. Therefore, the non-parametric method based on determination of the 2.5 and 97.5 percentiles following sorting of the data, has been recommended for general use. The performance of the Box-Cox transformation can be improved by introducing an additional parameter representing the origin of transformation. In simulations, the confidence intervals (CIs) of reference limits (RLs) calculated by the parametric method were narrower than those calculated by the non-parametric approach. However, the margin of difference was rather small owing to additional variability in parametrically-determined RLs introduced by estimation of parameters for the Box-Cox transformation. The parametric calculation method may have an advantage over the non-parametric method in allowing identification and exclusion of extreme values during RI computation. Clin Chem Lab Med 2010;48:1537–51.

Scientists of disciplines in clinical laboratory sciences have long recognized the need for a common language for efficient and safe request of investigations, reporting of results, and communication of experience and scientific achievements. Widening the scope, most scientific disciplines, not only clinical laboratory sciences, rely to some extent on various nominal examinations, in addition to measurements. The ‘International vocabulary of metrology – Basic and general concepts and associated terms’ (VIM) is designed for metrology, science of measurement. The aim of the proposed vocabulary is to suggest definitions and explanations of concepts and terms related to nominal properties, i.e., properties that can be compared for identity with other properties of the same kind-of-property, but that have no magnitude. Clin Chem Lab Med 2010;48:1553–66.

The need for harmonizing laboratory results is particularly intense in the field of quantitative protein assays in consideration of the clinical impact of specific protein measurements and their relevance in monitoring disease. We report the efforts made by the Committee on Plasma Proteins of the IFCC Scientific Division to achieve worldwide comparability in plasma protein results. We focus on the production of reference materials and the methods applied throughout their production process. Particularly, the recent characterization of ERM-DA470k/IFCC and ERM-DA472/IFCC has demonstrated that it is possible to reproduce the earlier established procedures and thereby maintain standardization. Plasma protein reference materials have had a substantial impact in improving the harmonization of patient protein results that should translate into better patient care. Clin Chem Lab Med 2010;48:1567–75.

Background: Laboratory testing is an essential tool for diagnosis and management of thyroid diseases. However, the current status of standardization hampers the interchangeability of results. To improve this situation, the Working Group for Standardization of Thyroid Function Tests was established. Methods: Method comparisons were organized for measurement of human thyroid stimulating hormone (TSH), and free and total thyroid hormone in serum from apparently healthy donors. The aim was to assess the status of standardization and the quality of the performance of current routine assays. A second objective was to investigate the effect of mathematical recalibration of the results using their relationship to the overall mean (TSH) or the reference measurement procedure values (other thyroid hormones). Results: The need for standardization was shown to be highest for free thyroid hormone and total triiodothyronine measurements, while the majority of TSH and total thyroxine assays agreed within 10% of the reference. Most assays showed good performance. However, some could benefit from improved precision, consistency of calibration, or within- and between-run stability. Recalibration eliminated assay-specific bias. Thus, the residual spread was due to within-method effects. Not withstanding, sample-related effects remained. Conclusions: These studies confirmed the feasibility of standardization based on method comparison with native sera, but highlighted the need to resolve issues, such as sample-related effects. In view of the fact that in this phase the project worked with samples from individuals with euthyroid status, the next method comparison shall place emphasis on challenging the performance of the assays with clinical samples and expanding the covered measurement range. Clin Chem Lab Med 2010;48:1577–83.

Carbohydrate-deficient transferrin (CDT) is a descriptive term used for a temporary change in the transferrin glycosylation profile caused by alcohol, and used as a biomarker of chronic high alcohol consumption. The use of an array of methods for measurement of CDT in various absolute or relative amounts, and sometimes covering different transferrin glycoforms, has complicated the comparability of results and caused confusion among medical staff. This situation prompted initiation of an IFCC Working Group on CDT standardization. This second publication of the WG-CDT covers the establishment of a network of reference laboratories running a high-performance liquid chromatography (HPLC) candidate reference measurement procedure, and evaluation of candidate secondary reference materials. The network laboratories demonstrated good and reproducible performance and thus can be used to assign target values for calibrators and controls. A candidate secondary reference material based on native human serum lyophilized with a cryo-/lyoprotectant to prevent protein denaturation was found to be commutable and stable during storage. A proposed strategy for calibration of different CDT methods is also presented. In an external quality assurance study involving 66 laboratories and covering the current routine CDT assays (HPLC, capillary electrophoresis and immunoassay), recalculation of observed results based on the nominal values for the candidate calibrator reduced the overall coefficient of variation from 18.9% to 5.5%. The logistics for distribution of reference materials and review of results were found to be functional, indicating that a full reference system for CDT may soon be available. Clin Chem Lab Med 2010;48:1585–92.

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.

In this study, the first steps in the development of a secondary reference measurement procedure (RMP) ‘higher metrological order measurement procedure’ to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serum-based secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 μg/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and inter-comparison studies. Clin Chem Lab Med 2010;48:1603–10.

Background: In 2004, a working group on the standardization of hemoglobin A 2 (HbA 2 ) was created within the IFCC, with the aim of developing a reference system for this analyte. One goal was to prepare a certified reference material in collaboration with the Institute for Reference Materials and Measurements (IRMM). This paper describes the properties of a first batch of this candidate study material. Methods: Eighty millilitre of fresh whole blood, collected from a healthy blood donor, was treated by removing plasma, white blood cells and platelets. Red cells were hemolyzed to prepare 100 vials of lyophilized material (approximately 155 mg per vial). After reconstitution, the HbA 2 content was measured with a total of seven HPLC methods, three electrophoretic techniques, and two capillary electrophoresis (CE) methods. Homogeneity was tested in a subset of five vials. Stability during storage at +4°C and –20°C was tested monthly over a period of 1 year. The commutability of this material was assessed by analysing the study material together with a set of 54 fresh blood samples, with a subgroup of the above mentioned methods, only by one routine HPLC (Bio-Rad Variant II, dual kit) and by a CE method (Beckman PA800, Analis kit), respectively. Results: The chromatographic and electrophoretic patterns obtained by all the HPLC, electrophoretic and CE techniques did not show any difference between those obtained using the first study material and those obtained with fresh blood samples. The lot was found to be homogeneous on the basis of the content of lyophilized powder per vial. The HbA 2 concentration in the lyophilized material remained stable at +4°C and –20°C, even after 1 year of storage. After reconstitution, the HbA 2 concentration did not change for more than 2 weeks in the refrigerator at +4°C. The normalized residual of the study material, measuring the degree of its commutability was 0.9, similar to that obtained on other home prepared and some commercial controls. Conclusions: Ideally, fresh whole blood is the best reference material in the meterological traceability chain for HbA 2 analysis. However, for a number of reasons the preparation of large batches of fresh whole blood to be used as secondary reference material for HbA 2 is not practical. In our work, we have proven that lyophilization does not appear to cause any matrix effect or inhomogeneity in the study material, which also confirmed to be commutable for the Bio-Rad Variant II (dual kit) and Beckman PA800 (Analis kit) methods. We conclude that a material similarly prepared as the current study material and value assigned with the candidate reference measurement procedure still under development will be suitable to calibrate various routine methods for HbA 2 . This will result in improvement of the inter-method variability for this important biochemical marker. Clin Chem Lab Med 2010;48:1611–8.

The IFCC Working Group for the Standardisation of Cystatin C (WG-SCC), in collaboration with the Institute for Reference Materials and Measurements (IRMM), announces the availability of the new certified reference material ERM-DA471/IFCC. The material was characterised using a pure protein primary reference preparation (PRP) as calibrant. The PRP was prepared from recombinant cystatin C, and its concentration measured using dry mass determination. The characterisation of ERM-DA471/IFCC was performed by particle enhanced immuno-nephelometry, particle enhanced immuno-turbidimetry, and enzyme amplified single radial immuno-diffusion. The certified cystatin C mass concentration in ERM-DA471/IFCC, if reconstituted according to the specified procedure, is 5.48 mg/L, the expanded uncertainty ( k =2) being 0.15 mg/L. Clin Chem Lab Med 2010;48:1619–21.

Background: The objective of this study was to determine if there is an association between serum hepatic markers and the metabolic syndrome in postmenopausal women. Methods: This study involved 1229 postmenopausal women aged 44–85 years, who visited the Center for Health Promotion for a health check-up. We excluded subjects from the analysis if they had a daily alcohol consumption of more than 1.5 drinks (alcohol consumption ≥20 g/day) or had chronic viral hepatitis. We also excluded subjects who had abnormal hepatic function, as defined by serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >100 IU/L, serum γ-glutamyltransferase (GGT) >100 IU/L, or serum total bilirubin concentrations >2 mg/dL. Results: Serum ALT and GGT concentrations increased in proportion to the number of elements of the metabolic syndrome (p<0.01). However, total bilirubin concentrations decreased (p=0.01). After adjusting for age, body mass index, and the presence of fatty liver in the patients with metabolic syndrome, the odds ratios (95% confidence interval) were 1.38 (0.89–2.15) for log (ALT), 1.69 (1.30–2.20) for log (GGT), and 0.53 (0.33–0.86) for log (total bilirubin). Conclusions: We found that an increase in GGT and a decrease in total bilirubin was associated with metabolic syndrome in postmenopausal women. Hepatic enzymes could be proposed as simple clinical metabolic markers that identify the metabolic syndrome. Clin Chem Lab Med 2010;48:1623–8.

Background: Homeostasis model assessment (HOMA) is a surrogate index widely used to study the role of insulin sensitivity or resistance in associated disease states. However, reported values for the definition of insulin resistance (the top 25% of the distribution in non-diabetic subjects) vary widely. This study evaluates the effect of HOMA computational methods [original HOMA model formula (HOMA1) and online calculator computer model (HOMA2)] on the associations and cut-off limits for insulin resistance. Methods: Anthropometric measurements, fasting adiponectin, leptin, leptin receptor, insulin, glucose, high-sensitivity C-reactive protein and a lipid profile were measured in type 2 diabetic patients (n=226) and their normoglycemic first degree relatives (n=319). HOMA1 and HOMA2 were used to estimate insulin resistance, β-cell function and insulin sensitivity. Subjects were classified as metabolic syndrome positive or negative (International Diabetes Federation criteria). Bland-Altmann analysis was used to evaluate agreement between the computational methods. Univariate and multivariate logistic regression analyses were used to relate the HOMA computational methods with metabolic variables and metabolic syndrome status. Results: The two computational methods had different cut-offs for the definition of insulin resistance: HOMA1 formula ≥2.5; HOMA2 calculator ≥1.4. Correlations of the two HOMA computational methods with anthropometric and metabolic variables showed some degree of variation. The odds ratios of the associations with the metabolic syndrome for the HOMA1 formula and HOMA2 calculator computational methods were 2.04 and 1.43, respectively. Receiver operating characteristic analysis for diagnosis of the metabolic syndrome showed that areas under the receiver operating characteristic curves for the HOMA1 formula and HOMA2 calculator computational methods were 0.741 and 0.680, respectively. Conclusions: The HOMA computational method is a significant determinant of the associations and classification of insulin resistance. Clin Chem Lab Med 2010;48:1629–34.

Background: The aim of this study was to investigate the effect of hypothyroidism on lipid peroxidation and the antioxidant profile, as well as to evaluate the interaction between thyroid hormones and biomarkers of oxidative stress in patients with overt hypothyroidism. We also evaluated the influence of cholesterol concentrations on biomarkers of oxidative stress in these same patients. Methods: Total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), and vitamin E were measured in 20 subjects with overt hypothyroidism (OH) and 20 controls. Results: TC, LDL cholesterol, triglycerides, TBARS, SOD, CAT, and vitamin E were significantly higher in the OH group. Significant correlation was observed for TSH and SOD, CAT, vitamin E and TBARS. Correlation was observed for triiodothyronine (T3) and SOD, CAT, vitamin E and TBARS. Significant correlation was also observed for free thyroxine and vitamin E and TBARS. However, correlation between T3 and CAT remained significant after controlling for TC concentrations. Conclusions: Overt hypothyroidism is associated with an increase in oxidative stress, and hypercholesterolemia has a stronger influence on development of oxidative stress in hypothyroid conditions compared with thyroid hormones. Clin Chem Lab Med 2010;48:1635–9.

Background: Micro-structural changes associated with ultra high molecular weight polyethylene particle (UHMWPE) induced osteolysis, the most frequent cause of aseptic loosening, have been intensively investigated in the mammalian calvarian model by histomorphometry and micro-computed tomography. However, little is known regarding the serological changes that occur during this process. Methods: Serological parameters for bone metabolism [calcium, phosphate, osteocalcin (OCN), deoxypyridinoline (DPD)/creatinine, alkaline phosphatase, osteoprotegerin and receptor activator of nuclear factor-κB] were analyzed in this animal model for particle induced osteolysis. Ten C57BL/6 mice were divided at random into sham operated and UHM-WPE implanted groups. Blood and urine samples were collected prior to and at 14 days after surgery. Results: Implantation of UHMWPE lead to a significant decrease in bone volume (p=0.027). Both groups (sham/UHMWPE) showed a significant increase in calcium (p=0.004/p=0.027) and phosphate (p=0.001/p=0.001), without correlation to particle implantation. Significantly higher concentrations of DPD/creatinine (p=0.034) and OCN (p=0.022) were found after implantation of UHM-WPE. In addition, parameters could not be correlated to particle induced osteolysis. Conclusions: DPD can be regarded as a valuable parameter for detecting UHMWPE induced osteolysis in the calvarian model. Further studies of serum parameters should focus on the clinical relevance in aseptic prosthetic loosening. Clin Chem Lab Med 2010;48:1641–6.

Background: The goal of this study was to develop and to validate an improved isotope-dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of methylmalonic acid (MMA) in urine. Methods: A previously described sample preparation protocol requires two solvent extraction steps, including evaporation. The first extraction is to extract the analyte from the sample, and second occurs following derivatization of the extract. In the method described here, the second evaporation step was substituted by on-line solid phase extraction employing column-switching and a permanent co-polymer based extraction cartridge. A standard validation protocol was applied to investigate the performance of the method. Results: The method was found to be linear in the clinically relevant range of concentrations (6–100 μmol/L). Total coefficients of variation were below 10% and inaccuracy was <10% for quality control samples at three concentrations. Conclusions: By omitting one evaporation step, the semi-automated method described in this article enables for more convenient work-flow in the quantification of urinary MMA compared to the previous protocol. This is of relevance for MMA measurement in the routine clinical laboratory setting. Validation demonstrated acceptable analytical performance. Clin Chem Lab Med 2010;48:1647–50.

Background: Increased cell-free DNA (cf-DNA) and the integrity of cf-DNA in plasma of patients with cancer has been described. We investigated the clinical utility of cf-DNA in the detection and monitoring of progression of leukemia. Methods: Plasma samples from 60 patients with acute leukemia were analyzed in comparison to plasma from 30 healthy controls. Plasma DNA was determined by quantitative PCR (qPCR) by amplifying the β-actin gene ( ACTB ). The DNA integrity index was calculated as the ratio of qPCR results ( ACTB 384/106). Paired diagnostic/complete remission (CR)/relapse samples from eight of 60 patients were analyzed, and the minimum residual disease (MRD) situations were monitored. Results: DNA concentrations (median: 8.80 ng/mL, p=0.004) and DNA integrity (median: 0.51, p<0.001) in cancer patients were significantly higher. Receiver operating characteristic (ROC) curve analysis showed that the area under the ROC curve of DNA and DNA integrity were 0.79 and 0.88, respectively. DNA integrity at CR had a distinct reduction and then an increase at relapse. DNA integrity in CR cases was higher than that observed in healthy controls. Conclusions: Our preliminary data suggest that plasma DNA integrity is increased in acute leukemia and may be a potential biomarker for monitoring MRD. However, more work is needed. Clin Chem Lab Med 2010;48:1651–6.

Background: Heat shock protein 70 (Hsp70) has been shown to act as a chaperone and be associated with a variety of tumors. We investigated HSP70-1 G+190C, HSP70-2 A+1267G, and HSP70-hom T+2437C polymorphisms to assess whether genetic variation in HSP70 plays a role in the occurrence and development of lung cancer. Methods: A case-control study was conducted using 159 patients with lung cancer and 202 control subjects. Genomic DNA was typed for HSP70 polymorphisms using polymerase chain reactions with restriction fragment length polymorphism (PCR-RFLP). Unconditional logistic regression was used to estimate the relative risks of lung cancer. Results: There were significant differences in genotype and allele distributions between patients and controls for the HSP70-1 G+190C polymorphisms with and without adjustment for age, gender, smoking history, drinking history and family history of cancer (p<0.05). No significant differences were found in the polymorphisms of HSP70-2 A+1267G and HSP70-hom T+2437C. The haplotype analysis showed that the G/A/C and C/G/T haplotypes were associated with a significantly increased risk of lung cancer compared to the G/G/T haplotype. After adjustments for other risk factors, such as age, gender, drinking history and family history of cancer, the interactions between the HSP70-1 and HSP70-hom genotypes and smoking were confirmed [I(AB), 2.56 and 5.12, respectively]. Conclusions: HSP70-1 G+190C may be a functional polymorphism and affect susceptibility to lung cancer, and homozygous C/C genotype may enhance the risk of lung cancer. In addition, smoking along with HSP70-1 G+190C and HSP70-hom T+2437C, may increase the risk of lung cancer. Clin Chem Lab Med 2010;48:1657–63.

Background: Colorectal cancer (CRC) is one of the most frequent causes of cancer related deaths worldwide. Recently, the use of cell-free DNA as diagnostic tools to identify cancer has been investigated. The aim of this work was to assess whether circulating DNA could be considered a useful marker for detection of early stage CRC and polyps. Methods: A total of 118 patients with CRC were included in the study along with 49 patients with colorectal polyps and 26 control subjects. Cell-free DNA was quantified using a real-time TaqMan-polymerase chain reaction assay. Non-parametric tests (Mann-Whitney test and Spearman correlation) were utilized for statistical analysis. Results: Serum DNA concentrations were significantly higher in CRC patients and patients with polyps (median value 105.0 ng/mL and 40.0 ng/mL) compared with controls (median value 14.0 ng/mL; p<0.05). Although carcinoembryonic antigen was above the cut-off in only 13/66 (19.7%) patients with early stage CRC, serum free DNA showed values above the threshold identified using receiver operator characteristic (ROC) curve analysis in 53/66 (80.3%) patients. Conclusions: Our data confirm that serum DNA concentrations are significantly increased in CRC patients with early stage disease and in patients with polyps. This marker might be useful for identifying high-risk individuals. Clin Chem Lab Med 2010;48:1665–8.