Volume 47, Issue 2

The role of vitamin D in maintaining bone health has been known for decades. Recently, however, the discovery that many tissues expressed the vitamin D receptor and were able to transform the 25-OH vitamin D into its most active metabolite, 1,25-(OH) 2 vitamin D, has led to a very promising future for this “old” molecule. Indeed, observational studies, and more and more interventional studies, are raising the importance of a significant vitamin D supplementation for not-only skeletal benefits. Among them, 25-OH vitamin D has been found to play an important role in prevention of cancers, auto-immune diseases, cardiovascular diseases, diabetes, and infections. Vitamin D deficiency, defined as serum 25-OH vitamin D levels <30 ng/mL, is very common in our population. The cost/benefit ratio and some recently published studies are clearly now in favor of a controlled and efficient vitamin D supplementation in these patients presenting a 25-OH vitamin D level <30 ng/mL. More attention should also be focused on pregnant and lactating women, as well as children and adolescents. Clin Chem Lab Med 2009;47:120–7.

Low-density lipoproteins (LDLs) are susceptible to structural modifications by oxidation, particularly the small dense LDL particles. The formation of lipid peroxidation derivates, such as thiobarbituric reactive substances, conjugated dienes, lipid hydroperoxides, and aldehydes, is associated with changes in apolipoprotein conformation and affects the functional properties of LDLs. Oxidized LDL (oxLDL) formation in the subendothelial space of the arterial wall is a key initiating step in atherosclerosis because it contributes to foam cell generation, endothelial dysfunction, and inflammatory processes. In the last decade, immunoassays were developed using monoclonal antibodies against oxidation-dependent epitopes of LDL which made it possible to directly measure oxLDL in the circulation. Increased circulating oxLDL concentrations have been related to cardiovascular disease in some studies, although not always independently after adjustment of classical lipid markers. The Asklepios Study, investigating 2524 healthy middle-aged subjects, showed that circulating oxLDL is affected by many biological and lifestyle factors, as well as (generalized) subclinical atherosclerosis. Clin Chem Lab Med 2009;47:128–37.

In 2007, the Fourth Joint European Societies' Task Force on cardiovascular disease (CVD) prevention in clinical practice issued an update of the guidelines on how to prevent CVD. These guidelines are endorsed by 10 international societies interested in the subject. Particular attention is given to lifestyle changes, risk factor management, and the use of cardioprotective drugs. In this paper, the content of the guidelines is summarized with emphasis on what is new compared to the previous version and on what could be of particular interest to clinical chemists. For those who would like to read more on this, please refer to the full document (112 pages including 1105 references). Clin Chem Lab Med 2009;47:138–42.

Laboratory diagnostics, a pivotal part of clinical decision making, is no safer than other areas of healthcare, with most errors occurring in the manually intensive preanalytical process. Patient misidentification errors are potentially associated with the worst clinical outcome due to the potential for misdiagnosis and inappropriate therapy. While it is misleadingly assumed that identification errors occur at a low frequency in clinical laboratories, misidentification of general laboratory specimens is around 1% and can produce serious harm to patients, when not promptly detected. This article focuses on this challenging issue, providing an overview on the prevalence and leading causes of identification errors, analyzing the potential adverse consequences, and providing tentative guidelines for detection and prevention based on direct-positive identification, the use of information technology for data entry, automated systems for patient identification and specimen labeling, two or more identifiers during sample collection and delta check technology to identify significant variance of results from historical values. Once misidentification is detected, rejection and recollection is the most suitable approach to manage the specimen. Clin Chem Lab Med 2009;47:143–53.

Background : An E670G polymorphism of the exon 12 of the proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) gene was recently found to be associated with increased plasma low-density lipoprotein cholesterol (LDL-C) levels and severity of coronary atherosclerosis. This case-control study tested for a possible link between this PCSK9 polymorphism and the risk of coronary artery disease (CAD) in an ethnic Chinese population in Taiwan. Methods : The subjects included 202 CAD patients and 614 unrelated controls. Genotypes were determined via polymerase chain reaction, restriction mapping with Mbo II, and gel electrophoresis. Results : Contradictory to the results of a previous report, a significantly lower level of LDL-C was noted in 670G carriers than in non-carriers (2.78±0.82 mmol/L vs. 3.02±0.85 mmol/L; p=0.029) among controls, after adjusting for age, gender, smoking, hypertension, diabetes mellitus, body mass index, and use of lipid-lowering agents. The 670G carrier was identified less frequently in patients with CAD than in controls (9.9% vs. 11.9%), but the difference was not significant in a multivariable logistic regression analysis (odds ratio=0.73; 95% CI=0.24–2.22; p=0.575). The G allele also occurred at similar frequencies in the two groups (5.0% vs. 6.0%; p=0.421). Conclusions : These results indicate that the E670G polymorphism of the PCSK9 gene modulates plasma LDL-C levels, but that it is not a risk variant for CAD in ethnic Chinese in Taiwan. Clin Chem Lab Med 2009;47:154–8.

Background : Infections with hepatitis B virus (HBV) may lead to a distinct clinical outcome which is partially related to host genetic variability. Our aim was to investigate the relationships between the polymorphisms of the E-selectin gene and disease progression in a HBV-infected Chinese Han population, and also to determine the plasma soluble E-selectin (sE-selectin) levels in this population. Methods : Genomic DNA was extracted from the peripheral blood of 367 HBV carriers and 281 healthy controls. Two polymorphisms ( Pst I for A561C and Hph I for G98T ) of the E-selectin gene were analyzed by polymerase chain reaction-restriction fragment length polymorphism. Circulating sE-selectin levels were measured by specific enzyme-linked immunosorbent assay (ELISA). Results : The frequency of the C allele (AC or CC) of the A561C polymorphism was significantly higher in patients with liver cirrhosis (LC) compared to controls (p=0.002). There was no difference in allele distribution of the G98T polymorphism. But in patients with LC, classified according to the Child-Pugh classification, the frequency of the T carrier (GT and TT) was significantly different between Child-Pugh class A and class B plus C (p=0.009). Levels of plasma soluble E-selectin (sE-selectin) were significantly increased in HBV carriers with chronic hepatitis (CH) and LC (mean±SD 68.94±34.09 and 43.39±18.00 ng/mL) compared to controls (13.96±7.50 ng/mL) (p<0.01). In the LC subgroup, levels of sE-selectin were significantly decreased from Child-Pugh class A to class C (p<0.05). In each group, individuals with the C allele showed higher sE-selectin levels than those with the A allele (p<0.05). Conclusions : This is the first report describing the association between E-selectin polymorphisms and HBV-related chronic liver diseases. Our data suggest that the A561C polymorphism of the E-selectin gene may be associated with disease progression in patients with chronic HBV infection and control the expression of plasma soluble levels, while the G98T polymorphism may be related to fibrotic severity in the Chinese population. Clin Chem Lab Med 2009;47:159–64.

Background : Serum human chorionic gonadotropin (hCG) and estradiol levels are higher in women with hyperemesis gravidarum (HG) than in unaffected pregnant controls. We postulated that higher levels of these hormones may be associated with clinically more severe HG. The aim of this study was to evaluate the effect of maternal hCG and estradiol levels on the severity of HG. Methods : A prospective study was performed on 167 first trimester women hospitalized for HG. Venous blood was taken for hCG and estradiol levels. Scattergrams were plotted for hCG or estradiol levels vs. gestational age. A curve of best fit was drawn. Women were categorized into two groups according to their position above or below the curve. Prolonged hospital stay (≥4 days) was used as a marker for HG severity. Multivariable logistic regression analysis was used to control for differences in characteristics, laboratory results on admission, and treatment received. Results : After adjustment, high hCG level adjusted odds ratio (AOR) (2.2, 95% CI 1.0–4.9, p=0.04) and hyponatremia AOR (2.8, 95% CI 1.2–6.6, p=0.02) were independently associated with prolonged hospital stay. With bivariate analysis, high estradiol level was not associated with prolonged stay. Conclusions : High hCG but not estradiol is associated with more severe HG. Clin Chem Lab Med 2009;47:165–71.

Background : There is frequent discussion on the susceptibility of direct high-density lipoprotein cholesterol (HDL-C) methods to matrix effects. In Vitro Diagnostics manufacturers recognize this issue and regularly improve their HDL-C reagent formulations in subsequent generations. Methods : The 3rd generation direct HDL-C assay from Roche was investigated for matrix effects in comparison to the former generation, a Beckman direct HDL-C method and a conventional phosphotungstate (PTA)/Mg 2+ precipitation method. In addition, 235 heparin plasma samples were measured freshly and after a freeze-thaw cycle with the Roche 2nd and 3rd generation direct HDL-C. Biases, outliers, and intraclass correlation coefficients (ICCs) were calculated for both experiments. Multivariate analysis was used to investigate interference by matrix components. Results : In fresh samples, Roche 2nd and 3rd generation HDL-C methods averaged +0.15 mmol/L (95% CI: 0.13–0.16) and +0.08 mmol/L (95% CI: 0.07–0.09) higher compared to frozen samples. In frozen aliquots, ICCs for Roche 2nd and 3rd generation and Beckman direct HDL-C as compared to PTA/MgCl 2 were 0.963, 0.966, and 0.924, respectively. Predictors of outliers (defined as having an absolute difference >0.21 mmol/L) in comparisons of direct methods to the PTA/MgCl 2 precipitation method were high triglyceride and low albumin levels. Conclusions : The 3rd generation direct HDL-C from Roche has become insensitive to most matrix effects, bringing along more accurate results in hypoalbuminemic and hypertriglyceridemic samples. Surprisingly, Roche direct assays produced significantly higher HDL-C levels in fresh samples compared to frozen plasma samples. If confirmed by others, the latter finding has implications for patient management and necessitates further reagent optimization. Clin Chem Lab Med 2009;47:172–6.

Background : The aim of this study was to evaluate the diagnostic value of different detection methods for cytomegalovirus (CMV) infection after renal transplantation and also to establish a system to monitor therapy for CMV infection. Methods : We retrospectively studied 1516 renal transplant recipients from June 1994 to December 2006. All patients were screened for CMV-DNA. A total of 1402 patients had received CMV-IgG/IgM detection since June 1996 and 660 had received CMV antigen detection since June 2000. Results : A total of 664 (43.8%) recipients developed CMV infection. The sensitivity, specificity and Youden index of the three methods, respectively, were 18.84%, 100% and 0.1884 for ELISA, 91.86%, 82.98% and 0.7484 for PCR, and 88.06%, 96.95% and 0.8501 for the CMV-pp65 antigenemia test. The sensitivity and specificity of the two combined detection methods (CMV-DNA and CMV-pp65) for post-operation CMV infection were 93.49% and 99.06%; the two detection methods had significant dependability (p<0.05) in diagnosis of CMV infection and in evaluation of therapeutic effect of antiviral drugs. Conclusions : Only ELISA can be used as a screening index in order to distinguish whether the donors or recipients are infected with CMV or not. CMV-pp65 antigenemia can help guide clinical therapy for CMV infection. CMV-pp65 and CMV-PCR combined together provide a more effective method to monitor CMV infection and predict its outcome. Clin Chem Lab Med 2009;47:177–81.

Background : Human milk and occasional serum samples contain high concentrations of unsaturated haptocorrin, which influence accurate measurement of cobalamins. Methods : Cobalamins in serum samples spiked with increasing amounts of unsaturated haptocorrin were measured employing the Centaur, Cobas and Architect analysers. Cobinamide-coated EAH sepharose was employed for pretreatment of the samples. Human milk samples were collected from 24 healthy mothers. Haptocorrin was measured by ELISA. Results : The measured concentration of cobalamins either increased (Centaur analyser) or decreased (Architect, Cobas analysers) significantly for haptocorrin >10 nM, and was 220%, 52% or 45% of the expected values in a serum sample containing 50 nM haptocorrin. Following pretreatment with cobinamide-sepharose, the expected cobalamin concentration was obtained (Centaur). The milk samples contained 4.5–180 nM haptocorrin. In samples containing >10 nM haptocorrin (n=19), the median concentration of cobalamins decreased from 1.3 nM to 0.67 nM after pretreatment with cobinamide-sepharose. Conclusions : Haptocorrin in concentrations above 10 nM influences measurement of cobalamins giving rise to falsely elevated or decreased results. Removal of unsaturated haptocorrin by pretreatment with cobinamide-sepharose solves the problem. Clin Chem Lab Med 2009;47:182–7.

Background : Most human metabolomics studies have shown that spectral outputs of 1 H nuclear magnetic resonance fingerprinting are strongly influenced by inter- and intra-individual variations; however, few studies have been performed to evaluate the inter- and intra-individual variations in urinary endogenous metabolites. Methods : We recruited 30 male college students to evaluate the factors affecting intra- and inter-individual variations in urinary endogenous metabolites. Statistical analysis for variations in urinary metabolites was performed after eliminating outliers found in principal component analysis (PCA) plots. Results : Inter-individual variations were relatively low for 2-oxoglutarate, succinate, citrate, dimethylglycine, and taurine, but high for trimethylaminoxide (TMAO), hippurate, and lactate. Intra-individual variations for 2-oxoglutarate, citrate, dimethylglycine, and taurine were relatively low, but high for TMAO and hippurate. The factors affecting inter-individual variation of lactate were age, body mass index, beverages, and alcohol, whereas the factors affecting intra-individual variation of lactate were age and fish. Kim Chi intake affected the inter-individual variation of succinate, citrate, TMAO, and hippurate; however, it did not affect the intra-individual variation of endogenous metabolites. Conclusions : Our results showed that inter- and intra-individual variations in urinary endogenous metabolites were very large, and significant factors affecting inter- and intra-individual variation were diverse, even after eliminating outliers in PCA analysis. Clin Chem Lab Med 2009;47:188–94.

Background : Most errors in laboratory medicine occur in the preanalytical phase and are the result of human mistakes. This study investigated information search procedures, test request management and test tube labelling in primary healthcare compared to the same procedures amongst clinical laboratory staff. Methods : A questionnaire was completed by 317 venous blood sampling staff in 70 primary healthcare centres and in two clinical laboratories (response rate = 94%). Results : Correct procedures were not always followed. Only 60% of the primary healthcare staff reported that they always sought information in the updated, online laboratory manual. Only 12% reported that they always labelled the test tubes prior to drawing blood samples. No major differences between primary healthcare centres and clinical laboratories were found, except for test tube labelling, whereby the laboratory staff reported better practices. Re-education and access to documented routines were not clearly associated with better practices. Conclusions : The preanalytical procedure in the surveyed primary healthcare centres was associated with a risk of errors which could affect patient safety. To improve patient safety in laboratory testing, all healthcare providers should survey their preanalytical procedures and improve the total testing process with a systems perspective. Clin Chem Lab Med 2009;47:195–201.

Background : The changes in androgen levels after the menopause and the effects of hormone replacement therapy (HRT) itself and the mode of HRT have not been established. The objectives of this study were to document the effect of age on androgen levels in a normal population of postmenopausal women and to investigate the effect of oral or transdermal HRT on androgen, oestradiol and sex hormone binding globulin (SHBG) values. Methods : A cross-sectional study was conducted on 182 postmenopausal females aged 45–100 years, randomly recruited from the community. Serum levels of dehydroepiandrosterone sulphate (DHEAS), androstenedione, testosterone, free androgen index (FAI) and SHBG in a reference group of women, subdivided by age, menopausal status, HRT replacement and mode of HRT replacement. Results : Age was a significant factor affecting androstenedione (<55, ≥55 years) and DHEAS (<65, ≥65 years). HRT status was found to influence values for androstenedione, SHBG and FAI. Neither HRT nor age significantly influenced testosterone. Conclusions : The observations suggest the need to provide age-specific reference intervals for androstenedione and DHEAS but not for testosterone and SHBG. The significant effect of HRT treatment would also suggest a need for treatment associated reference intervals for androstenedione, SHBG and FAI. Suggested reference intervals for each of these partitioned groups are presented. Clin Chem Lab Med 2009;47:202–7.

Background : Correlates of preheparin serum lipoprotein lipase (LPL) mass and its associations with the likelihood of metabolic syndrome (MS) and coronary heart disease (CHD) were investigated. Methods : A cross-sectional study was carried out in a population sample (n=352, median age 55). MS was defined according to modified Adult Treatment Panel III criteria. Results : Age-adjusted geometric mean preheparin LPL concentrations were 58.6±1.04 ng/mL in men and 66.9±1.03 ng/mL in women (p<0.004). A positive interaction with both the LPL X447 allele (p<0.034) and age-adjusted smoking status (p=0.026 in men and p=0.11 women) was observed. LPL mass was significantly correlated in both genders with high-density lipoprotein (HDL)-cholesterol and inversely with triacylglycerol levels and HOMA index. In multiple linear regression analysis, LPL mass was significantly associated with genotype, gender, age, adiponectin, smoking status and HDL-cholesterol, and in women with C-reactive protein after adjustment for body mass index, triacylglycerol and insulin. Significantly low sex- and age-adjusted serum LPL mass was observed in cases of MS, hypertension and CHD. Logistic regression analysis after adjustment for age, sex, adiponectin and S447X polymorphism demonstrated that LPL mass was inversely associated with CHD in men and both genders (p=0.02), with hypertension confined to women (p=0.04) and with MS likelihood in both genders combined and women [odds ratio 1.51 (95% CI 1.14–2.00) for halving the likelihood]. Conclusions : LPL X447 genotype, female gender and smoking habit interact in increasing preheparin serum LPL mass in Turkish adults. Serum LPL mass is inversely associated with MS and CHD, independent of confounders, and probably reflects insulin sensitivity. Clin Chem Lab Med 2009;47:208–15.

Background : The 24-h urine sample is considered as the most reliable material for testing many but not necessarily all constituents in urine. However, its collection is tedious for both patients and research participants. The aim of this study was to compare concentrations of essential elements calcium (Ca), magnesium (Mg), sodium (Na), potassium (K), and zinc (Zn) in 24-h and spot urine samples. Methods : Urine samples were collected from 143 generally healthy women, aged 30–79 years. Fasting spot urine was collected immediately after the end of the 24-h collection, therefore being of the same content as the first morning urine which ended the 24-h collection. Elements were analyzed by flame atomic absorption/emission spectrometry and expressed as mg/g and/or mmol/mol of creatinine (Cr). Spearman rank order correlations between 24-h and spot urine were carried out for each element. Ratios of elements in 24-h to spot urine samples were calculated to estimate the element-proportion of spot in the 24-h sample. Results : All coefficients of correlation between 24-h and spot urine of measured elements and Cr were significant (p<0.05): Zn (0.637), Mg (0.623), Ca (0.603), Na (0.452), K (0.396), and Cr (0.217). Ratios of 24-h to spot urine samples for each element (except K) were similar and close to 2, indicating uniform proportion of elements from spot urine sample in the 24-h sample. In addition, a high correlation between various pairs of elements was obtained in both 24-h and spot urine; the highest being between Na/Ca (0.435) and (0.578), respectively. This is in accordance with theoretical presumptions and previous findings regarding those relationships. Conclusions : Although replacing burdensome 24-h urine collection with spot urine sampling might not provide the solution in all cases, our results show that for the elements analyzed, spot urine could be a reliable alternative. Clin Chem Lab Med 2009;47:216–21.

Background : The VU University Medical Center (VUmc) was the first hospital in the Netherlands to introduce the Delfia Xpress for the analysis of free β-human chorionic gonadotrophin (β-hCG) and pregnancy associated plasma protein-A (PAPP-A) in the first trimester screening program for Down syndrome. Since then, others have implemented this system. In this study, we tested the equality of measurements for free β-hCG and PAPP-A between Delfia Xpress systems and one AutoDelfia system. Methods : A total of 40 serum samples were aliquoted and stored at –20°C. Samples were analyzed by six Delfia Xpress systems and one AutoDelfia system over a time period of 2 years. Results : The relationships between free β-hCG and PAPP-A were excellent for the different Delfia Xpress systems (r>0.99, p<0.0001). For PAPP-A, the agreement between the main system at VUmc and five other systems was linear with slopes between 0.99 and 1.06. Similarly, agreement for free β-hCG was linear with slopes between 0.99 and 1.09. Likewise, agreement for PAPP-A and free β-hCG was excellent for the AutoDelfia vs. the main Delfia Xpress at the VUmc (r>0.99, p<0.0001). For both PAPP-A and free β-hCG, the relationships were linear with slopes of 1.08 and 1.07. Conclusions : We demonstrate an excellent agreement for the analysis of PAPP-A and free β-hCG between Delfia Xpress systems and one AutoDelfia system. Clin Chem Lab Med 2009;47:222–6.

Background : Guidelines on preferred cardiac marker strategies for investigation of patients with acute coronary syndromes (ACS) are available from the laboratory medicine and cardiology communities. Therefore, implementation of these guidelines into daily clinical practice should be a joint effort of laboratory specialists and clinicians. This was investigated in this survey. Methods : A pilot study was performed sponsored by the European Federation of Clinical Chemistry and Laboratory Medicine. A link to an online questionnaire was e-mailed to 990 laboratories from eight European countries in May 2006. The requested information included tests performed, clinical protocol development, and reference limits. Results : We obtained a total of 220 responses. Out of these, 208 responses (95%) were from hospitals that provide 24-h admission of patients. The suggested turn-around-time (<60 min) was apparently met by >88% for cardiac troponin T/I and for CK-MB mass. The majority of the laboratories derive their decision limits from kit inserts provided by the manufacturers. The results revealed a worrying fact that external quality assessments are not used in all testing. Conclusions : Our survey demonstrated that cardiac troponin is the preferred biomarker for the diagnosis of ACS. Half of the participants had written protocols, mostly as a result of collaboration between laboratorians and clinicians. Clin Chem Lab Med 2009;47:227–34.