Band 46, Heft 6

The description of this familial longitudinal cohort was published in this journal 10 years ago, in 1998. To date, 117 publications on the STANISLAS Cohort (SC) have appeared, corresponding to five main categories of results: familial resemblance and heritability; genetics and gene-environment interactions; mRNA and proteins as gene products; reference values and biological variations of proteins; and finally preventive medicine and prepathological epidemiological data. More than 600 data values on demographic and laboratory data have been collected on each individual taking part out of the 1006 families at the beginning and for all three recruitments. Serum and plasma are stored in liquid nitrogen for all participants for all three recruitments. DNA has been extracted from all participants and mRNA from 357 families. They are stored at −80°C. Owing to the SC study, heritability and many gene-environment interactions have been described. The expression of 166 genes related to cardiovascular diseases was measured in peripheral blood mononuclear cells RNA. Reference values for proteins and vitamins have been established in addition to reference values for the carotid and femoral intima media thickness in adults and children. The data obtained contribute to a better understanding of the relation between the studied polymorphisms (161 polymorphic sites) and health, and predisposition to obesity, high blood pressure and metabolic syndrome. To the best of our knowledge, the SC study is internationally the only longitudinal family cohort of subjects who are presumed to be healthy, which enables the study of the chain DNA-RNA-proteins. Clin Chem Lab Med 2008;46:733–47.

The practice of cardiology is in continual evolution, in parallel with the progress achieved by medical research in understanding the pathophysiology of cardiovascular disease and in developing new therapeutic procedures. Consequently, manufacturers of cardiac biomarkers are pressed with new demands to improve the performance of the existing and the development of novel ones. Several highly sensitive and/or specific assays for myocardial ischemic damage and myocardial function detection have already become commercially available. Moreover, an increasing number of novel risk factors have been added to the classical risk factors of cardiovascular disease. Finally, the recent surge of genetic analysis procedures will likely soon provide the clinical cardiologist with a number of laboratory tests for defining the molecular diagnosis, assessing new risk factors, and better targeting the pharmaceutical approaches in patients with cardiovascular disease. In this review, we first present the general characteristics of a biomarker followed by the analytical and clinical performance of assay methods. Clin Chem Lab Med 2008;46:748–63.

Prevention of medical errors is a major goal of healthcare, though healthcare workers themselves have not yet fully accepted or implemented reliable models of system error, and neither has the public. While there is widespread perception that most medical errors arise from an inappropriate or delayed clinical management, the issue of laboratory errors is receiving a great deal of attention due to their impact on the quality and efficiency of laboratory performances and patient safety. Haemolytic specimens are a frequent occurrence in clinical laboratories, and prevalence can be as high as 3.3% of all of the routine samples, accounting for up to 40%–70% of all unsuitable specimens identified, nearly five times higher than other causes, such as insufficient, incorrect and clotted samples. This article focuses on this challenging issue, providing an overview on prevalence and leading causes of in vivo and in vitro haemolysis, and tentative guidelines on identification and management of haemolytic samples in clinical laboratories. This strategy includes continuous education of healthcare personnel, systematic detection/quantification of haemolysis in any sample, immediate clinicians warning on the probability of in vivo haemolysis, registration of non-conformity, completing of tests unaffected by haemolysis and request of a second specimen for those potentially affected. Clin Chem Lab Med 2008;46:764–72.

Background : The importance of the apolipoprotein A5 ( APOA5 ) gene in determining plasma triglyceride (TG) levels has been demonstrated in transgenic and knockout mice and confirmed by human association studies in different ethnic groups. Methods : We screened for nonsynonymous APOA5 mutations in patients with plasma TG levels >10 mmol/L. The coding sequence and promoter region of the APOA5 gene were sequenced in 95 individuals with severe hypertriglyceridemia (HTG). A large population sample of 3202 individuals was screened by PCR and restriction analysis for presence of detected mutation. Results : In total, three heterozygous carriers of 944C>T (Ala315>Val) were identified in the severe HTG patients, while 22 carriers were identified in the population sample. The rare allele frequency of the Val315 was significantly higher in the HTG sample than in controls (0.016 vs. 0.003, p<0.01, respectively). Most of the control Ala315Val carriers, however, had plasma lipid levels (TGs, total cholesterol and high-density lipoprotein cholesterol) within the usual range detected in the population. Conclusions : APOA5 Ala315>Val does not play any dominant/important role in the genetic determination of plasma TG levels, but the increased frequency in HTG patients compared to controls suggests that it might interact with other gene variants to cause HTG. Clin Chem Lab Med 2008;46:773–7.

Background : Apolipoprotein A5 ( APOA5 ) gene polymorphisms are usually associated with plasma triglyceride levels. We evaluated the relationship of the APOA5 −1131T>C and c.56C>G polymorphisms [single nucleotide polymorphism (SNP)] with serum lipids, dyslipidemia [low high-density lipoprotein (HDL)/high triglyceride] and the risk for metabolic syndrome (MS) in the Turkish Adult Risk Factor study. Methods : We genotyped SNPs using the Taqman allelic discrimination assays in 1564 Turkish adults (51.4% female, mean age 54.1±11.6 years). MS and dyslipidemia were defined using the criteria of the National Cholesterol Education Program. Results : For both SNPs, rare allele carriers had significantly higher fasting triglyceride levels in both genders, except the c.56G allele in men. The −1131C allele was associated with lower HDL cholesterol (HDL-C) levels in women. In relation to dyslipidemia, the c.56C>G and haplotype 1 had significant gender-genotype interactions (p<0.05). Otherwise, both SNPs were significantly associated with dyslipidemia after adjustment for risk factors in women. After similar adjustment, non-carriers of the haplotype 1 (odds ratio=4.1, p=0.003) increased the MS risk in women. However, no significant associations emerged between SNPs and HDL-C, dyslipidemia or MS in a similar analysis in men. Conclusions : Excess risk for low HDL-C, dyslipidemia and MS is associated with the rare alleles of the APOA5 SNPs and non-carriers of common haplotype in women. Clin Chem Lab Med 2008;46:778–84.

Background : The metabolic syndrome is a cluster of cardiovascular risk factors and essential components of metabolic syndrome are hyperglycemia, hypertension, visceral obesity, hypertriglyceridemia and low high-density lipoprotein cholesterol. Oxidative stress plays a critical role in the pathogenesis of metabolic syndrome components and insulin resistance. The aim of this study was to investigate the role of oxidative stress, C-reactive protein and heat shock protein 70 levels in the pathogenesis of this disease. Methods : A total of 36 patients diagnosed with metabolic syndrome and 33 controls were included in the study. Malondialdehyde, carbonyl protein, C-reactive protein and heat shock protein 70 levels and xanthine oxidase and superoxide dismutase activities were measured in the serum of the subjects. Results : Mean serum malondialdehyde, carbonyl protein, C-reactive protein (p<0.01, p<0.05 and p<0.001, respectively) and xanthine oxidase activity were significantly higher (p<0.01) in serum of the patients than the control group. Superoxide dismutase activity and heat shock protein 70 levels were significantly lower (p<0.01 and p<0.05, respectively) in serum of the patients. Conclusions : These results suggest that oxidative stress parameters and components of metabolic syndrome are closely related; therefore, significant alterations may occur in the antioxidant and inflammatory status. However, further studies are required to evaluate the possible molecular mechanisms of heat shock protein 70 levels in metabolic syndrome. Clin Chem Lab Med 2008;46:785–90.

Background : The plasma total and low-density lipoprotein-cholesterol (LDL-C) levels that are used as diagnostic criteria for familial hypercholesterolaemia (FH) probands in the general population are too stringent for use in relatives, given the higher prior probability of a first-degree relative being FH (50% vs. 1/500). Our objective was therefore to develop more appropriate LDL-C cutoffs to identify “affected” first-degree relatives found by cascade testing, to test their accuracy and utility in case identification, and to compare them with the published “Make early diagnosis to prevent disease” (MEDPED) cutoffs from the US. Methods : Using a large, anonymised sample of genetically tested first-degree relatives of Netherlands FH probands (mutation carriers/non-carriers, n=825/2469), age- and gender-specific LDL-C diagnostic cutoffs for first-degree relatives were constructed. These were used to test similar data from Denmark (n=160/161) and Norway (n=374/742). Results : Gender-specific LDL-C diagnostic cutoffs were established for six different age groups, which achieved an overall accuracy (measured as Youden's index) of 0.53 in the Netherlands data, and performed significantly better amongst younger (<25 years) compared to older first-degree relatives (0.68 vs. 0.42 Youden's index, p<0.001). Compared with the Netherlands data, age- and gender-adjusted mean LDL-C levels were significantly higher (approximately 0.5 mmol/L) in the Denmark and Norway subjects for both mutation carriers and non-carriers. After adjusting for this difference, the LDL-C cut-offs showed a similar accuracy in identifying mutation carriers from Denmark (81%, range 78%–86%) and Norway (84%, range 82%–86%). Although the MEDPED cutoffs performed significantly worse than these for the Netherlands data (p<0.001), they performed equally well in overall accuracy for the Norwegian and Danish data, although the LDL-C cutoffs had a significantly higher sensitivity but lower specificity for all three countries. Conclusions : The cutoffs developed here are designed to give the greatest overall accuracy when testing relatives of FH patients in the absence of a genetic diagnosis. They have a more balanced specificity and sensitivity than the MEDPED cutoffs that are designed to achieve higher specificity, which is more appropriate for cascade testing purposes. The data suggest that country-specific LDL-C cutoffs may lead to greater accuracy for identifying FH patients, but should be used with caution and only when a genetic diagnosis (DNA) is not available. Clin Chem Lab Med 2008;46:791–803.

Background : The aim of this study was to evaluate the distribution of cardiac troponin I (cTnI) values, measured by the ADVIA TnI-Ultra method (Siemens Medical Solutions Diagnostics SrL) in healthy subjects and to characterize its relation to gender, age, as well as to N-terminal fragment of pro-brain natriuretic peptide (NT-proBNP). Methods : A Caucasian population of 692 healthy subjects (311 males and 381 females) with a mean (SD) age of 45.3 (17.3) years [range 11–89 years; females 46.5 (17.3) years, males 43.8 (17.1) years] was enrolled. The presence of cardiac or systemic acute or chronic diseases was excluded by history and accurate clinical evaluation. Results : A significant difference was found between the cTnI values in men and women (men: median 0.012 μg/L, range from undetectable values to 0.196 μg/L; women: median 0.008 μg/L, range from undetectable values to 0.130 μg/L; p<0.0001 by Mann-Whitney U-test). When a multiple regression analysis was performed, NT-proBNP, gender and age significantly contributed to the regression with cTnI (R=0.444, p<0.0001). Conclusions : Our data indicate that cut-off values, based on the 99th percentile of cTnI distribution in apparently healthy subjects, can significantly vary according to age and gender of the reference population. Clin Chem Lab Med 2008;46:804–8.

Background : Oxidative stress plays an important role in atherosclerosis. Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme that inhibits low-density lipoprotein (LDL) oxidation and may play a protective role against coronary heart disease. The aim of this study was to analyze the relationship between HDL-cholesterol (HDL-C) and PON1 in a Spanish prepubertal population with high plasma HDL-C levels. Methods : The study population included 1266 children between the ages of 6 and 8 years. Serum PON1 activity was measured by the hydrolysis of paraoxon. PON1 192Q/R and PON1 55L/M polymorphisms were analyzed by PCR and restriction analysis. Results : The prevalence of the less common PON1 192R and PON 55M alleles in this population was 30% and 38%, respectively. No significant correlations between serum PON1 activity and lipid profile were observed. Multiple linear regression analysis showed that the PON1 192Q/R polymorphism accounts for 69% of PON1 activity in the children in the study, with the PON1 55L/M polymorphism accounting for an additional 5% of this variation in boys, and for an additional 3% together with HDL-C concentration in girls. Conclusions : PON1 192Q/R polymorphism is the main determinant of PON1 activity in the prepubertal population in this study, accounting for around 70% of serum PON1 activity. HDL-C concentration has a small contribution to serum PON1 activity in girls. Clin Chem Lab Med 2008;46:809–13.

Background : Uterine leiomyomas are common, benign, smooth muscle tumors representing a significant public health problem. The aim of this study was to investigate CYP17A1 , CYP19 , and androgen ( AR ) polymorphisms, their relative risks for uterine leiomyomas and possible associations with clinical parameters. Methods : Uterine leiomyoma tissues and blood samples were obtained from 87 patients, as were peripheral blood samples from 68 control women. Clinical data were recorded in both groups. The CYP17A1 (rs743572) polymorphism was analyzed by PCR-RFLP, and the CYP19 [TTTA] n repeat and AR [CAG] n repeat were analyzed using PCR-based GeneScan analysis. AR loss of heterozygosity (LOH) and microsatellite instability were also evaluated, while samples exhibiting LOH were analyzed for X inactivation. Results : Clinical parameters related to disease development did not differ between cases and controls. CYP17A1 *A2/*A2 genotype was prevalent in non-white women. CYP17A1 , CYP19 , and AR genotypes and alleles did not differ between groups. However, alleles presenting [TTTA] 7 repeats in intron 4 of CYP19 were more frequent in the control group (p=0.0550). Shorter and longer [CAG] n repeat alleles of AR were exclusive to the leiomyoma group. The LOH assay showed allele losses at AR locus in four informative tumors and X chromosome inactivation analysis revealed that these tumors retained the active allele. Conclusions : The overall lack of association between uterine leiomyomas with polymorphisms involved in steroidogenesis or steroid metabolism is consistent with the hypothesis that these polymorphisms do not substantially contribute to the development of these tumors. Clin Chem Lab Med 2008;46:814–23.

Background : This report describes clinical, biochemical and molecular findings regarding two Italian monozygotic twins carrying a novel multiple endocrine neoplasia type 1 ( MEN1 ) mutation inherited from their mother. Methods : Clinical, biochemical and genetic evaluations of the above-mentioned family members were performed. Results : All three members were heterozygous for a deletion involving the first nucleotide at codon 98 in exon 2 of the MEN1 gene, which results in early termination of the protein. The clinical phenotypes were as follows: one out of the two twins suffered from insulinoma and hyperparathyroidism, while the second one was asymptomatic. Furthermore, the mother suffered from hyperparathyroidism, as well as from hypergastrinemia for several years before the daughter was diagnosed of MEN-1. Conclusions : We describe a family with a new heterozygous mutation (g.292delC) in the MEN1 gene not described previously. The mutation leads to a truncated protein without activity, explaining the clinical picture of this family. Clin Chem Lab Med 2008;46:824–6.

Background : Structural hemoglobin variants can affect the accuracy of hemoglobin A1c (HbA1c) testing and represent the most common pitfall in the determination of HbA1c. We here describe the characterization of an α chain variant in diabetic patients as the cause of an abnormal presentation of the HbA1c fraction on the HLC-723 G7 analyzer. Methods : HbA1c analysis was performed using various HPLC-based HbA1c analyzers and by immunoassay. α-Globin mutation analysis was performed by GAP-PCR and DNA sequencing. Results : The peak partially overlapping HbA1c in the chromatogram represents the glycated fraction of the silent α chain variant Hb Riccarton [α51(CE9)Gly→Ser]. This aberrant peak is uniquely identified by the HLC-723 instrument, as it is not observed on other HPLC-based HbA1c analyzers. Occasionally, the HLC-723 may fail to properly integrate both glycated Hb fractions, resulting in a falsely low HbA1c result. The variant was confirmed in samples from other diabetic patients with identical chromatographic patterns. Conclusions : The silent α chain variant Hb Riccarton [α51(CE9)Gly→Ser] leads to an abnormal chromatographic presentation on the HLC-723 analyzer with a risk of erroneous HbA1c determination. Manual validation of chromatograms to detect abnormalities caused by Hb variants is important to prevent incorrectly produced HbA1c results from being reported. Clin Chem Lab Med 2008;46:827–30.

Background : Disturbances in the function of renal proximal tubules increase the activity of several enzymes in urine. Among them is fructose-1,6-bisphosphatase (FBP-1), the key enzyme of gluconeogenesis normally present in the renal convoluted, and to smaller degree, proximal renal tubular cells cytosol. FBP-1 activity in urine and serum was used for evaluation of the degree of graft ischemia during human kidney transplantation. The aim of our present research was to determine FBP-1 activity in urine as an indicator of damage to renal proximal tubules in children with idiopathic nephrotic syndrome (INS). Methods : We evaluated the excretion of FBP-1 into urine of 21 children (10 girls and 11 boys) with INS, aged from 10 to 15 years and 30 healthy children (14 girls and 16 boys), aged from 2 to 15 years. FBP-1 activity was determined by the Latzko and Gibbs method. Creatinine (mg%) in urine and blood serum was measured by the Jaffe method in Larsen modification. Protein in blood serum was determined by the biuret method (g/L), and albumin (mg%) by the Young method. Proteinuria in the urine collected over 24 h was measured with the Exton turbidimetric method by Tomaszewski with modification and expressed in mg/kg body weight/24 h. Results : In the urine of 30 healthy children, FBP-1 activity was in the range from 0–1.74 μmol FPB/h/mmol of creatinine. In 43% of the healthy children, FBP-1 activity in urine was not detectable. In the period of intensive proteinuria during the INS in children, FBP-1 activity and protein concentrations in urine were significantly higher than in the control group (p<0.0008 and p<0.0001, respectively). In the urine of children with active INS, we observed a very weak negative linear correlation between protein concentration and FBP-1 activity (r=−0.5018, p=0.067). After treatment with Encorton (prednisone), FBP-1 activity and protein concentration in urine dropped to values of the control group. Conclusions : “The overload” of proximal renal tubules by proteins in children with INS releases FBP-1 into urine. FBP-1 activity in urine may therefore be considered as a marker of damage to the proximal renal tubules in children with INS. Clin Chem Lab Med 2008;46:831–5.

Background : Oxidative stress is associated with scleroderma (systemic sclerosis) and is supposed to favor disease progression by complex effects on the vascular endothelium and on fibroblasts. Methods : Plasma oxidative process marker, thiobarbituric acid-reactive substances, and several markers of antioxidant defense capacity (plasma total antioxidant activity, serum albumin, uric acid and glutathione, superoxide dismutase and catalase) were evaluated by spectrophotometric methods using blood samples collected from 23 scleroderma patients and 21 healthy controls. Results : In scleroderma patients, thiobarbituric acid-reactive substances levels (mmol/L plasma) were significantly elevated (29.3±5.8) compared with healthy controls (16.6±3.1, p<0.001). Total antioxidant activity (mmol Trolox/L) was significantly lower in scleroderma patients than in controls (1.29±0.13 vs. 1.55±0.23, p<0.001), as well as the antioxidant gap (mmol Trolox/L) (0.57±0.18 vs. 0.92±0.22, p<0.001). Superoxide dismutase activity (IU/g hemoglobin) was markedly decreased in patients as compared with controls (395±184 vs. 659±211, p<0.001). Conclusions : Lower plasma total antioxidant activity and plasma antioxidant gap in scleroderma patients show that plasma antioxidant defense is deficient in scleroderma patients. As previous studies on this issue are controversial, the decreased erythrocyte superoxide dismutase activity found in the patients in this study needs further investigation. Clin Chem Lab Med 2008;46:836–41.

Background : Blood contamination is commonly observed in ventricular cerebrospinal fluid (CSF) samples from patients with extraventricular drainage systems. Because the introduction of blood may interfere with the white blood cell count as a useful marker for the diagnosis of an infection, correction for blood content would be desirable. Methods : In a retrospective study, we analysed the use of correction formulas in 724 blood-contaminated ventricular CSF samples. Results : Using a standard correction method the white blood cell count was not normalised in most CSF samples, with pleocytosis indicating an inflammatory stimulus set by the blood itself or by the foreign body. When correcting white blood cell counts in the CSF of culture-positive patients, some samples were normalised or overcorrected. In addition, correction of the CSF white blood cell count did not increase sensitivity and specificity for the detection of culture-positive CSF samples. Conclusions : Correction is not necessary when using the white blood cell count as a parameter to predict CSF infection in ventricular CSF samples. Clin Chem Lab Med 2008;46:842–8.

Background : Preanalytical variables, such as sample collection, handling, transport, and storage, may affect patient results. The number of errors in the preanalytical phase may decrease by following standardized procedures. Methods : A retrospective analysis (2001–2005) of results obtained through the Spanish Society of Clinical Chemistry and Molecular Pathology Quality Assessment Program for the Preanalytical Phase has been carried out to summarize data regarding the main factors affecting the preanalytical phase quality. In such a program, participants are asked to register rejections and causes for rejection of routine or stat samples usually and locally collected at their laboratories. Results : Results discussed refer to 105 laboratories. Of the 4,715,132 tubes expected to be received during the data collection period among participating laboratories and according to determinations included by clinicians in the request form, 32,977 (0.699%) offered a cause for rejection. Whole blood-EDTA samples and serum samples accounted for 75.6% of all samples collected among laboratories, although they only corresponded to 55.8% of all registered rejections. In total, 81% of rejections arose as a result of the following reasons: “specimen not received” (37.5%), “hemolysis” (29.3%), and “clotted sample” (14.4%). Moreover, plasma-citrate-erythrocyte sedimentation rate exhibited the highest percentage of rejection (1.473%), whereas the lowest rate corresponded to whole blood-EDTA (0.381%). Conclusions : Overall percentage of rejection is similar to previously published data. As some of the included variables have turned out to be irrelevant, the program has been simplified from the year 2006 onwards. Clin Chem Lab Med 2008;46:849–54.

Background : Established general risk score models for intensive care patients incorporate several clinical and laboratory data. However, the collection, documentation and classification of clinical data are time-consuming, incur labor-related costs, and are dependent on the experience of the examiner. Therefore, in the present study a general score for medical intensive care patients based solely on routine laboratory parameters is presented. Methods : Parameter selection was performed using stepwise logistic regression analysis. The maximum likelihood estimate of variable influence on mortality provided a relative weighting for each variable. The new score was compared to two established risk models (Acute Physiology And Chronic Health Evaluation II, APACHE II; and Simplified Acute Physiology Score II, SAPS II). Results : The study included 528 medical intensive care patients with a mean age of 65.4±0.7 years. The in-hospital mortality was 16.5% (87/528). Multiple logistic regression analysis revealed eight parameters with significant prognostic power: alanine aminotransferase, cholesterol, creatinine, leukocytes, sodium, thrombocytes, urea, and age. These parameters were used to build a new laboratory score called Critical Risk Evaluation by Early Keys (CREEK). The area under the receiver operating characteristics curve was 0.857 (0.814–0.900). Pearson correlation analysis showed significant correlation between CREEK and APACHE II (r=0.550) and SAPS II (r=0.516; p<0.001; n=387). The areas under curve of the APACHE II and the SAPS II were 0.869 and 0.874, respectively. Conclusions: We show that a general risk score for medical intensive care patients on admission based solely on routine laboratory parameters is feasible. The quality of risk estimation using CREEK is comparable to established risk models. Furthermore, this new score is based on quality controlled low-cost laboratory parameters that are routinely measured on admission to the intensive care unit. Therefore, no additional costs are involved. Clin Chem Lab Med 2008;46:855–62.

Background : Consolidation of analyzers is an emerging issue in clinical chemistry. We evaluated the analytical performance of the Cobas 6000 analyzer (Roche Diagnostics), which is considered a candidate for replacement of current Hitachi 917 analyzers and for consolidation of chemistry and immunochemistry. Methods : The precision, accuracy, linearity and correlation with current field methods were evaluated according to Clinical and Laboratory Standards Institute protocols EP5, EP9 and EP10. A total of 31 routine chemistry assays and 18 immunoassays were studied. Accuracy and linearity were verified for 24 chemistry parameters using value-assigned trueness controls from the Dutch External Quality Assessment Scheme organizers. In addition, traceability to methods endorsed by the Joint Committee of Traceability in Laboratory Medicine was examined. Results : All analytes met allowable precision criteria, apart from the low level for sodium and folate. Total coefficients of variation ranged between 0.6% and 4.4% for routine chemistry and between 0.8% and 5.8% for immunochemistry, apart from folate (12% at the low end). The correlation coefficients for comparison to current field methods were >0.975, except for magnesium and for six out of 18 immunochemistries. Recovery experiments indicated high recovery for most of the 24 routine chemistry assays. Conclusions : Considering the excellent precision data and the result equivalence for most assays, it can be concluded that Cobas 6000 accommodates robust chemistry and immunochemistry, and has good potential for workstation consolidation in medium-sized laboratories. Clin Chem Lab Med 2008;46:863–71.