Volume 47, Issue 9

Chronic kidney disease (CKD) is defined as the presence of kidney damage or a glomerular filtration rate (GFR) <60 mL/min/1.73 m 2 for three or more months. Measurement of serum creatinine is the most commonly used method to evaluated kidney function, but it must be included in formulas to estimate GFR, adjusting for age, gender and ethnicity, such as the Modification of Diet in Renal Disease (MDRD) study equation. The performance of this equation is acceptable for patients with CKD but appears to under-estimate GFR in populations with unknown kidney status. A new formula has been developed recently. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation appears to perform better than the MDRD equation. Cystatin C has been widely evaluated as a marker for GFR and seems to be more sensitive than creatinine. The aim of this review is to discuss the recommendations for detecting CKD, emphasizing the characteristics and limitations of GFR estimating equations and pitfalls in the evaluation of urinary albumin excretion. Clin Chem Lab Med 2009;47:1023–32.

Background : Leptin plays an important role in energy homeostasis. There is polymorphism on the leptin ( LEP ) gene. Our aim was to compare the tetranucleotide repeat (TTTC) n polymorphism in the 3′-flanking region in the LEP gene on DNA samples from patients with pre-eclampsia (PE), hemolysis, elevated liver enzymes, and low platelet (HELLP) syndrome and healthy pregnant controls. Methods : Blood samples were collected from healthy pregnant women (n=88), patients with PE (n=79) and HELLP (n=77) syndrome. Fluorescent PCR and DNA fragment analysis was performed from the isolated DNA for the detection of (TTTC) repeats. The electrophoretograms were evaluated and patients were assigned to two groups; class I low (<190 bp) or class II high (≥190 bp) PCR fragments. Results : We observed similar distributions of the class I and class II (TTTC) alleles in the groups studied (class I allele: healthy pregnant 58.5%; severe pre-eclamptic 58.3%; HELLP syndrome 52.6%). We detected a higher frequency of the II/II genotype in HELLP syndrome patients (32.4%) compared to healthy controls (22.7%). However, the difference was not statistically significant. Conclusions : In an ethnically homogenous population, the LEP gene (TTTC) microsatellite polymorphism in the 3′-flanking region does not show a significant difference in the allele and genotype distribution in healthy pregnant, pre-eclamptic and HELLP syndrome patients. Furthermore, we recommend a new classification of the class I and class II alleles based on the distribution of the (TTTC) microsatellites. Clin Chem Lab Med 2009;47:1033–7.

Background : Cholesterol ester transfer protein (CETP) plays a pivotal role in the remodelling of triglyceride (TG)-rich and high-density lipoprotein (HDL) particles. Sequence variations in the CETP gene may interfere with coronary atherosclerosis. However, clinical studies of various CETP polymorphisms have shown controversial data in coronary artery outcome. We aimed to investigate whether TaqIB CETP gene polymorphism could predict clinical outcome in a prospective cohort of patients hospitalized for an acute coronary syndrome (ACS). Methods : Two hundred and seventy consecutive Caucasian patients hospitalized for an ACS, and having a significant coronary artery disease in at least one major vessel (stenosis >50%), were prospectively enrolled and followed for 57 months. The mean age was 65.1±12.5 years, and 77% were males. One hundred and thirty-nine patients (51.5%) suffered from unstable angina at inclusion and 131 patients (48.5%) presented with an acute myocardial infarction (MI). The follow-up data were obtained from questionnaires. The major recurrent events recorded were 32 deaths comprising 28 cardiovascular deaths and 49 combined cardiovascular events (28 cardiovascular deaths, 19 non-fatal ACS and 2 non-fatal strokes). CETP genotyping was performed using a restriction fragment length polymorphism based method. Results : A significant relation was found between B2B2 genotype and combined cardiovascular end-point (p<0.02), mainly driven by a link with cardiovascular death (p<0.05). The hazard risk ratio for cardiovascular death associated with B2B2 genotype was 2.2 [95% confidence interval (CI): 1.01–4.94, p<0.05]. In multivariate analyses, no modification except for a significant interaction with statin therapy was observed by inclusion of potential confounders for the association of B2B2 genotype with cardiovascular death. Conclusions : These results suggest that patients homozygous for the B2 allele and not taking statin had a strong increase of recurrent cardiovascular event after an initial acute coronary event. This cardiovascular risk seems to be corrected with statin therapy. Clin Chem Lab Med 2009;47:1039–46.

Background: Variation in the peroxisome-proliferator-activated receptor γ ( PPARγ ) gene has been reported to alter the risk for adiposity in adults. Methods: We investigated the gender related association between the Pro12Ala variant (rs1801282) in obesity and insulin resistance traits in 794 peri-adolescent children aged 10–12 years of Greek origin from the Gene and Diet Attica Investigation (GENDAI) cohort. Results: Gender stratified analysis suggested that in peri-adolescent boys, Ala carriers exhibited lower measures of skinfold (triceps: 16.9±6.9 vs. 19.4±7.9 mm, p=0.014; subscapular: 9.6±4.5 vs. 11.2±5.4 mm, p=0.016) and lower adiponectin concentrations (3.9±1.3 vs. 4.7±2.4 μg/mL, p=0.05). In peri-adolescent girls, Ala carriers had lower insulin concentrations (7.3±3.7 vs. 8.5±4.4 μU/mL, p=0.026) and lower values of homeostasis model assessment of insulin resistance (HOMA-IR) (1.5±0.8 vs. 1.8±0.96, p=0.019). Linear regression analysis revealed that the presence of the Ala allele in boys was a nominally significant predictor of obesity indices, including skin-folds (triceps: β±SE: –2.3±1.1, p=0.032; subscapular: β±SE: –2.3±1.1, p=0.04) and adiponectin concentrations (β±SE: –0.7±0.4, p=0.05) after adjusting for potential covariates. In girls, the Ala allele was a predictor of insulin concentrations (β±SE: –1.2±0.6, p=0.037) and HOMA-IR (β±SE: –0.24±0.13, p=0.037). Conclusions: Our results suggest that adiposity in children is influenced by the Pro12Ala polymorphism in a gender specific manner. Clin Chem Lab Med 2009;47:1047–50.

Background: A high number of mutations associated with cystic fibrosis have been identified in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, most of which are rare and, therefore, hamper extensive molecular diagnosis. In couples undergoing prenatal diagnosis where no mutation is found in one or both partners, additional analysis of intragenic polymorphisms may allow for the identification of fetal alleles associated with cystic fibrosis. Methods: We developed novel, rapid and accurate assays for CFTR genotype determination using pyrosequencing technology; a simple, automated and reliable technique with low cost. Results: Assays were optimized for the identification of the seven most frequent CFTR mutations ( p.DeltaF508, p.N1303K, p.G542X, c.2183AA>G, c.1717-1G>A, p.W1282X, p.R1162X ) in the Italian population and two common intragenic polymorphisms ( rs213950 and rs1800136 ). Blind validation on 15 known control samples, typed for each sequence variation, allowed correct identification of all 135 genotypes. Conclusions: We demonstrated that this procedure is highly specific for the identification of individual CFTR sequence variations associated with cystic fibrosis, allowing both population screening and prenatal diagnosis. Clin Chem Lab Med 2009;47:1051–4.

Background: The association of serum bilirubin concentrations with kidney function and albuminuria has not been established in the US general population. Methods: We performed a cross-sectional analysis of data from the National Health and Nutrition Examination Survey (NHANES) 2001–2006 and examined the associations of serum total bilirubin concentrations with estimated glomerular filtration rate (eGFR) and albuminuria in a nationally representative sample of 13,184 adults aged 20 years or older. eGFR was estimated using the abbreviated Modification of Diet in Renal Disease equation. Urinary albumin excretion was measured using the albumin/creatinine ratio. Results: An eGFR <60 mL/min/1.73 m 2 and urinary albumin/creatinine ratio ≥30 mg/g were present in 8.1% (n=1072) and 10.6% (n=1402) of participants, respectively. A total of 427 (3.2%) individuals had a serum total bilirubin concentration >22.23 μmol/L. After adjustment for demographics, comorbidities, alcohol consumption, viral hepatitis status and other laboratory measures, serum bilirubin was negatively associated with eGFR, and positively with albuminuria both in the whole cohort and in the subgroup of non-diabetic individuals. In contrast, serum bilirubin was not significantly associated with eGFR or albuminuria in persons with diabetes (n=1253). Conclusions: Increasing serum concentrations of total bilirubin are independently associated with decreasing eGFR and increasing albuminuria in the US adult population. Clin Chem Lab Med 2009;47:1055–62.

Background : Clinical laboratories accredited according to ISO 15189 quality standards are obliged to implement and continuously monitor quality indicators for evaluation of the laboratory's contribution to patient care. Reporting laboratory results to the requesting physician is one important phase of the clinical laboratory testing process. Failure to report results may indicate the ineffectiveness of the laboratory service. We aimed to analyze the proportion and type of laboratory reports for outpatients that were not delivered to the requesting physician. Methods : This retrospective observational study was conducted during an 11-month period from January to December 2007 at our outpatient biochemistry laboratory unit. Data on demographic characteristics, request types and laboratory findings for all uncollected reports were retrieved from the laboratory information system and compared with one random 2-week representative period. Results : During the study period our laboratory issued 22,445 patient reports with more than 150,000 biochemistry analyses. Of these, 464 (2.1%) were uncollected laboratory reports. When compared to the representative period, patients who never collected their laboratory reports were younger (p<0.001) or suffering from some chronic disease. Routine biochemistry tests were the most prevalent (>50%). The majority of routine biochemistry tests were almost equally represented during the study and representative period, while molecular diagnostic tests were several times more frequently uncollected (p<0.001). Reports with electrolytes, metabolites and glucose were the least likely to be uncollected (p<0.001). The total cost for those tests was 30% of the average monthly laboratory budget. Conclusions : A significant amount of the laboratory budget is wasted for tests that never reach the requesting physician. Such misutilization of the laboratory reveals the substantial lack of medical necessity for test requests. Further studies are needed to explore the possible efficiency of the various interventions in reducing the volume of unnecessary and erroneous testing. Clin Chem Lab Med 2009;47:1063–6.

Background: It has been shown that low concentrations of high-density lipoprotein cholesterol (HDL-C) are associated with non-alcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM). HDL2b, a major subfraction of high-density lipoprotein (HDL), is more significantly correlated with coronary heart disease (CHD) compared with total HDL. In this study, we analysed HDL2b in a cohort of Chinese T2DM subjects with or without NAFLD. Methods: A highly sensitive and reliable microfluidic chip method was adopted to measure HDL2b. In total, 48 T2DM patients with NAFLD diagnosed by a B-ultrasound were enrolled from our Beijing Community Pre-Diabetes (BCPD) study cohort. A total of 48 age and gender matched diabetic controls without NAFLD were selected from the same population. Results : Clinical characteristics and serum biochemical analyses demonstrated a significantly increased body mass index (BMI), waist circumference, homeostasis model assessment-insulin resistant index (HOMA-IR), total cholesterol (TC), and triglyceride (TG) concentrations in the NAFLD group. However, the concentrations of HDL2b and its ratio to total HDL in NAFLD patients was decreased, compared with controls (p<0.01). Significantly increased concentrations of high sensitive C-reactive protein (hs-CRP) (p<0.01) were also found. Multifactor logistic regression analysis showed that BMI and TG were the predominant risk factors for fatty liver, while HDL2b was a protective factor. Conclusions : T2DM patients with NAFLD have characteristics including obesity, marked insulin resistance, high TG, high hs-CRP, low HDL2b and a low HDL2b ratio to total HDL. These factors may increase the incidence of atherosclerosis as well as the risk of CHD. Clin Chem Lab Med 2009;47:1067–72.

Background : Evidence is accumulating that mitochondrial (Mt) oxidative stress plays a role in the pathogenesis of preeclampsia. The current study analyzes the stress levels, energy status and associated enzymatic alteration in placental mitochondria of preeclamptic (n=30) and normotensive (n=35) subjects. Methods : Total Mt stress was measured using dichlorofluorescin (DCFH) oxidant analysis, malondialdehyde (MDA) concentrations, protein carbonyl (PC) concentrations and measurement of nitrite (NO 2 − ) and nitrate (NO 3 − ). Activity of antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx) and the glutathione redox ratio (GRR) were measured. The ATP/ADP (adenosine triphosphate/adenosine diphosphate) concentrations and respiratory chain enzyme activities were also analyzed. The expression of heat shock protein 70 (HSP70) was measured in mitochondria. Results : The DCFH oxidants, MDA, PC concentrations, and concentrations of NO 2 − and NO 3 − were significantly higher in the preeclamptic group (p<0.01) compared with the control group. The activities of SOD, GPx, GRR [glutathione (GSH)/glutathione disulfate (GSSG)] (p<0.01, p<0.001), ATP/ADP and respiratory chain enzyme activities were reduced significantly (p<0.001) in preeclamptic conditions. The placental mitochondrial HSP70 (mtHSP70) showed significant over expression in the preeclamptic group (p<0.001) compared with the control group. Conclusions : These results provide the first line of evidence for accumulated Mt stress demonstrated by increased stress markers, decreased antioxidants and enhanced mtHSP70. The study illustrates the probable protective mechanism of mtHSP70 against the generated stress. This is primarily to combat the enzymatic and free radical mediated damage produced in preeclampsia. Clin Chem Lab Med 2009;47:1073–80.

Background: Bedside tests for C-reactive protein (CRP) have been studied in pediatric patients, but not in neonates. Methods: This study compared the results of two rapid bedside tests for CRP (Quick-Read CRP, Orion Diagnostic, Espoo, Finland and NycoCard CRP-Single Test, Axis-Shield, Oslo, Norway) with those of our central laboratory method (CRP-Lab) in newborn infants. CRP concentrations were determined using 72 samples obtained from 43 infants with suspected sepsis occurring between 1 and 28 days of life. Results: Considering positive CRP concentrations to be ≥10 mg/L, both bedside tests had good specificity (Quick-Read 80.5%, NycoCard 83.3%) and sensitivity (Quick-Read 97.2%, NycoCard 94.4%) when compared with our CRP-Lab. The agreement of measurement with central laboratory values was high for both the bedside tests, without statistically significant differences between the methods. The Quick-Read and NycoCard methods did not show any statistically significant systematic proportional bias when compared with the central laboratory values. The accuracy of the results of both bedside tests is somewhat decreased when CRP concentrations are >100 mg/L. Conclusions: This study shows that both the Quick-Read and the NycoCard test can be used for serial determinations of CRP concentrations in newborn infants. They require small volumes of blood and provide reliable results in <5 min. Clin Chem Lab Med 2009;47:1081–4.

Background : Non-invasive biochemical markers are needed to support the diagnosis of ulcerative colitis (UC), an incurable disease of unknown pathology. Midkine is an angiogenic cytokine, chemotactic towards neutrophils and macrophages, and a T-regulatory cell suppressor. Methods : Serum midkine was measured immunoenzymatically in 93 UC patients and 108 healthy subjects, and evaluated with respect to disease status, endoscopic, inflammatory and angiogenic activity. The diagnostic value of midkine was compared to C-reactive protein (CRP) using receiver operating characteristics (ROC) analysis. Results : Midkine was higher (p<0.0001) in inactive (199 ng/L) and active UC (351 ng/L) compared with controls (93 ng/L), and reflected disease activity (r=0.427, p<0.001). Midkine was correlated with CRP, erythrocyte sedimentation rate (ESR), leukocytes, platelets, interleukin-6, paraoxonase-1, albumin, transferrin, iron, hemoglobin, and hematocrit. Midkine correlated with angiogenic factors: vascular endothelial growth factor-A and platelet-derived growth factor-BB. As a marker of UC, midkine showed a diagnostic accuracy of 85%, sensitivity of 72%, specificity of 82%, whereas CRP showed 83%, 65% and 91%, respectively. As a marker of active UC, midkine showed a diagnostic accuracy of 87%, sensitivity of 84%, specificity of 75%, whereas CRP showed 75%, 63% and 83%, respectively. Combined assessment of midkine and CRP improved sensitivity but substantially decreased specificity. Conclusions : UC is associated with increased circulating midkine, which corresponds with clinical, endoscopic, inflammatory and angiogenic activity, and anemia. Performance of midkine as a marker of UC or active UC was comparable to that of CRP. Clin Chem Lab Med 2009;47:1085–90.

Background: Hyperthyroidism in Graves' disease (GD) is often associated with the production of autoantibodies (TRAb) to the thyrotropin receptor (TSHR). Current manual second generation TRAb assays demonstrate high clinical sensitivity, but are labor-intensive and time consuming. Until recently, technical difficulties prevented the availability of an automatic TRAb assay. Methods: Development of a fast and fully automated TRAb assay on the Elecsys ® /cobas ® e electrochemiluminescence immunoassay platform. Results: A labeled thyroid-stimulating human monoclonal TSHR autoantibody (M22) was used in an automated M22-binding inhibition assay. TRAb are detected by their ability to competitively inhibit M22-binding to solubilized porcine TSHR (pTSHR). High clinical sensitivity could be maintained by assembling multiple TSHR binding sites within a soluble oligomeric immunocomplex for improved TRAb binding. Requirement of sufficient on board stability of the delicate TSHR structure in solution for several days was met by pre-complexation of the pTSHR with a capture antibody to its C-terminus in combination with the use of structure-stabilizing chemical chaperones. Total imprecision coefficient of variation (CV) at 1.71 (approximate cut-off) was found to be 11.4%. TRAb results were available within 30 min. Conclusions: Availability of a fast and automatic TRAb assay offers an attractive alternative to the manual TRAb assays for the differential diagnosis of hyperthyroidism. Clin Chem Lab Med 2009;47:1091–5.

Background: Fatty acid synthase (FASN) is an enzyme synthesized by the liver and plays an important role in lipogenesis. The present study aimed to assess whether serum FASN concentrations are altered in patients with chronic liver disease, and to investigate whether its measurement may be a useful tool in the clinical evaluation of this derangement. Methods: We investigated 93 patients with chronic liver disease (14 minimal change disease, 79 steatohepatitis) and 100 control subjects. Serum FASN concentrations were measured using ELISA. Results: Patients had a significant increase in serum FASN concentration (p<0.001), which was specifically associated with the hepatic Knodell sub-index III of portal inflammation (p=0.019). In addition, serum FASN concentrations were significantly correlated with the circulating levels of the monocyte chemoattractant protein-1 (MCP-1) (Spearman ρ =0.375; p<0.001) and type III procollagen-N-peptide (P-III-P) (Spearman ρ =0.297; p<0.001). Conclusions: Serum FASN concentrations are increased in patients with chronic liver impairment, and are associated with specific histological alterations and biochemical markers of portal inflammation. These data suggest that FASN measurement may contribute significantly to the evaluation of these patients. Clin Chem Lab Med 2009;47:1097–9.

Background : Measurement of serum free light chains (FLCs) has recently become available for the diagnosis and monitoring of patients with plasma cell dyscrasias. The aim of this study was to investigate the performance of the serum FLC assay as a tumour marker by comparing FLC concentrations with serum protein electrophoresis (PE) results in the diagnosis of multiple myeloma (MM). In addition, we also evaluated the prognostic value of the baseline serum FLC ratio in patients with MM. Methods : We measured FLC concentrations and calculated the kappa/lambda (κ/λ) FLC ratios for three groups (control, polyclonal gammopathy and MM). Results : The FLC ratio at a cut-off threshold of 2.0 showed higher sensitivity and specificity compared with serum electrophoresis for the diagnosis of MM. We used the median FLC ratio of >57.5 and <0.04 for κ and λ secretors, respectively, for assessing survival. Survival was 30 months in patients with the κ/λ ratio of >57.5 and <0.04 compared to 47 months in patients with the ratio <57.5 and >0.04, indicating that more abnormal serum FLC ratios are associated with poorer survival (p<0.011). Conclusions : Despite the limitations of the assay, the results of our study indicate that the FLC assay in combination with serum PE has an increased sensitivity in the diagnosis of MM. Also, baseline measurement of the κ/λ ratio provides prognostic information in these same patients. Clin Chem Lab Med 2009;47:1101–7.

Background: Detection of plasma cell dyscrasias (PCD) requires screening of serum and urine for monoclonal proteins. Several studies have demonstrated increased sensitivity and specificity when measurement of serum free light chain (SFLC) is part of the screening protocol. In addition, omission of immunofixation (IFE) in the standard work-up that includes SFLC assay has been proposed. This study attempts to define the role of the SFLC assay in a screening strategy limited to serum only. It compares outcomes to a serum-only screening strategy that omits serum IFE. Methods: Serum from 691 patients was analysed for the presence of monoclonal protein using standard serum IFE, serum protein electrophoresis (SPE) and measurement of SFLC. Data were analysed retrospectively. Results: Specificity and sensitivity of abnormal SFLC-ratios for the detection of monoclonal protein using IFE were 96% and 41%, respectively. Eighteen patients with negative monospecific and Bence Jones IFE, but abnormal SFLC-ratios were identified. In most cases, this could be attributed to kidney and inflammatory disease or haematological disorders. In four cases, this resulted in further diagnostic investigation and light chain disease was later detected in two cases. Light chain disease was confirmed in one case but not confirmed in the other patient. In 14 patients, Bence Jones IFE was negative, although the concentrations of SFLC suggested the presence of monoclonal Bence Jones protein at concentrations detectable by IFE. Thus, either the anti-serum failed at detection, there was polymerisation of the free light chains or the SFLC assay overestimated protein concentrations. Simulating a work-up that included IFE only if abnormalities were detected by SPE or the SFLC assay would have resulted in 26% fewer IFEs being performed, but three patients with monoclonal proteins by IFE would have been missed. Conclusions: Abnormal SFLC concentrations are neither sensitive nor specific for the detection of monoclonal proteins by IFE. Not all PCD are accompanied by excessive production of SFLC, and several other conditions, such as renal disease are associated with increased SFLC concentrations. An abnormal SFLC-ratio is a specific marker for PCD, and occurs primarily in patients with haematological disease. If renal and inflammatory diseases are excluded, this should prompt further diagnostic investigation. Screening of serum without performing an IFE as a standard procedure leads to a reduction of sensitivity when compared to screening of serum that includes IFE. Clin Chem Lab Med 2009;47:1109–15.

Background: The purpose of this study was to determine the positive predictive value (PPV) of positive serum human epidermal growth factor receptor-2 (HER-2) for monitoring women with breast cancer following diagnosis and treatment in a routine clinical setting. Methods: Serum HER-2 was measured in 1348 patients with breast cancer: 837 during routine oncology clinic visits and 511 following new diagnosis. All patients with positive serum HER-2, 1/5 of negative patients from the oncology clinic, and all the newly diagnosed were followed; a total of 862 patients. Serum HER-2 was measured using the Bayer ADVIA Centaur assay. Tissue HER-2 was determined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). IHC +3 or IHC +2 and FISH>2.0 were positive. Patients were considered to have positive serum HER-2 when at least two values were >15 ng/mL. Recurrence, progression and regression were diagnosed according to usual clinical practice. Serum HER-2 concentrations did not contribute to diagnostic decision-making or selection of treatment. Results: From January 2004 to January 2009, 149 patients were found to have positive serum HER-2. Of these, 35 were tissue HER-2 positive at surgery, 69 tissue-negative and 45 were not determined. Fifty-five of 149 that were serum HER-2 positive (37%, 95% CI: 29–45) had metastases. Among the 35 tissue-positive patients, 25 had recurrence in the form of metastases and there was good correlation between recurrence/progression and increase in serum HER-2 (p<0.0003). There was also a high correlation between effect of treatment and decline in serum HER-2 (p<0.0003). Of the 69 tissue-negative patients, 29 had recurrence in the form of metastases, and there was good correlation with serum HER-2 levels (p<0.000004). In this routine application of serum HER-2, the PPV for metastases recurrence detection in both tissue-positive and tissue-negative was 54 of 104 (52%, 95% CI: 42%–62%), in tissue-positive 25 of 35 (71%, 95% CI: 54%–85%), in tissue-negative 29 of 69 (42%, 95% CI: 30%–54%). The lead time of increases in serum HER-2 before recurrence could be determined in ten tissue-positive patients was 3–24 months (mean 11.3 months), when compared to standard clinical imaging methods. Conclusions: Serum HER-2 is a useful marker for the detection of recurrence of breast cancer and for monitoring the effect of treatment, especially in tissue HER-2 positive patients. Clin Chem Lab Med 2009;47:1117–23.

Background: Low concentrations of selenium (Se) in humans have been associated with risk of cancer. Selenoprotein mRNAs can potentially be regulated by Se status. Methods: Se status, GPx1 Pro198Leu and Sep15 1125G/A genetic polymorphism and human (h)GPx1 , hGPx3 , hSep15 and hSeP1 transcript levels in peripheral leukocytes of 33 males with bladder cancer and 47 healthy male controls were analysed. Results: All the subjects expressed detectable selenoprotein mRNA concentrations in leukocytes. Significantly lower expression of hGPx1 , hGPx3, hSep15 and hSeP1 in leukocytes of bladder cancer patients compared to controls was observed. hGPx1 , hGPx3 and hSep15 expression was significantly lower in non-smokers in the control group compared with smokers in the control group. A positive relationship between expression of all studied genes was also observed in non-smoking controls. Expression of hGPx3 and hSep15 gradually increased with tumour grade in patients with cancer. We did not find any association between selenoprotein mRNA levels, Se status and selenoprotein genetic polymorphism. Conclusions: This study showed significant down-regulation of hGPx1 , hGPx 3, hSep15 and hSeP1 mRNA levels in leukocytes of patients with bladder cancer compared to controls. Selenoprotein transcript levels in circulating leukocytes of patients with bladder cancer and controls revealed no potential impact of Se status on selenoprotein expression. Clin Chem Lab Med 2009;47:1125–32.

Background: The invasion and metastases of gastric cancer (GC) depends on the activities of matrix metalloproteinases and tissue inhibitors of metalloproteinases. It was suggested that the concentration of plasma matrix metalloproteinase-9 (MMP-9) is better than the concentration of serum MMP-9 for prediction of evolution of GC. The aim of the present study was to compare the clinical usefulness of plasma and serum tissue inhibitor of metalloproteinases-1 (TIMP-1) in the diagnosis and prognosis of GC. Methods: Plasma and serum concentrations of TIMP-1, MMP-9 and carcinoembryonic antigen (CEA) were assayed in 73 patients with GC and 61 healthy controls. The diagnostic criteria and prognostic value for the measurands were defined. Results: Plasma and serum TIMP-1, MMP-9 and CEA were significantly higher in GC patients compared with healthy controls. The area under the ROC curve (AUC) (0.961), diagnostic sensitivity (89%) and accuracy (91%) of plasma TIMP-1 were higher than those for MMP-9 and CEA. An increased pre-treatment concentration of plasma TIMP-1 was a significant independent prognostic factor for the survival of patients with GC. Conclusions: These findings suggest that the plasma TIMP-1 is a better biomarker than the serum TIMP-1 and might be useful for the diagnosis of GC and prognosis of patient survival. Clin Chem Lab Med 2009;47:1133–9.

Background : The potential for faster detection of human herpes viruses using PCR compared to other methods is undisputed. However, because of fear of contamination, the clinical implication of nucleic amplification methods in routine laboratories is not widespread. Herpes viruses cause a wide spectrum of diseases and can cause morbidity and mortality in immune-compromised patients. Using real-time PCR, most of the problems associated with PCR (contamination, cumbersome detection, and rather expensive tests) are solved, and a rapid, economical, and – most importantly – closed system is at hand. Methods : We evaluated work procedures in our laboratory that enable the routine diagnosis of viral infections with high accuracy and rapid turn-around time. In parallel, inherent problems usually associated with PCR testing, especially cross-contamination could be suppressed to a minimum. The start of the work flow process begins with an automated nucleic acid extraction procedure that yields high quality DNA. A common – internally and externally controlled – PCR program for all six viruses allows rapid sample turn around. Results : In all, 7500 analyses for human herpes virus infection were performed in the last 5 years. Results for various different specimens were produced within 24 h. Contamination occurred rarely and could be ameliorated easily. The use of internal controls identified rare PCR-inhibited samples. The detection limits for our assays are markedly below the clinically relevant range. Conclusions : Our workflow allowed rapid, cost-efficient, and labor saving routine diagnostic detection of viral infections. Clin Chem Lab Med 2009;47:1141–5.

Background : Antibodies in adults living in malaria endemic areas that target specific parasite antigens are implicated in protective immunity to infection and disease. This study aimed to identify, isolate and characterise targets of protective immunity in malaria. A Plasmodium falciparum antigen termed UB05 (Genbank Accession Number DQ235690: PlasmoDB PF10_ 0372) that had been isolated by immunoscreening with semi-immune sera was studied. Methods : Polymerase chain reaction, sequencing and bioinformatics were used to analyse the UB05 gene. A specific mouse anti-UB05 antibody was used in parasite reinvasion growth/inhibition assays and in immunoflourescence to localise the antigen. In a cross-sectional study, enzyme linked immunosorbent assay was used to study immunoglobulin G (IgG) responses to the antigen. Results : The gene revealed significant homologies with gene sequences from Plasmodia and other apicomplexan parasites and had two alleles in the wild P. falciparum isolates. The antigen is expressed by schizonts and segmented merozoites. Mouse antibodies against it marginally inhibit in vitro invasion of erythrocytes by P. falciparum . The IgG responses to UB05 were found to be significantly lower (p<0.05) in the sera of children (2–5 years) compared with adults (>18 years), with or without parasitaemia. However, parasitaemia correlated inversely (r=0.7– 0.75) with serum anti-UB05 IgG concentrations. Furthermore, anti-UB05 IgG concentrations were lower in the sera of febrile patients (body temperature >37.5°C) than their non-febrile counterparts regardless of parasitaemia status. Conclusions : These results are compatible with a role for UB05 in the development of immunity and as a marker of protective immunity to malaria. Clin Chem Lab Med 2009;47:1147–58.