Band 47, Heft 7

Gallstone disease is one of the most prevalent gastrointestinal disorders. In addition, the costs associated with the diagnosis and treatment of gallstone disease have been rapidly increasing. The etiology and pathogenesis of gallstone disease remains incompletely understood. Gallstone formation may result from a complex interaction of genetic and environmental factors. This article reviews the prevalence and risk factors associated with gallstone disease. Understanding the pathogenesis of gallstone disease could lead to the development of better therapeutic and preventive strategies for dealing with this disease. Clin Chem Lab Med 2009;47:795–807.

The measurement uncertainty budget for a clinical test-kit – an ELISA technique based on experimental data provided in the instruction manual – is presented. The proper characterization of the calibrators, within-plate homogeneity and the trueness of ELISA test-kits are identified as being major contributors to the combined uncertainty. Clin Chem Lab Med 2009;47:808–10.

Background: Ras homologous (Rho) family GTPases play a pivotal role in the regulation of numerous cellular functions associated with malignant transformation and metastasis. To evaluate the role of these GTPases in colorectal cancer, the mRNA expression levels in matched sets of tumor and non-tumor tissues from surgical specimens were analyzed. The relationship between the mRNA levels in tumor tissues to the clinicopathological features was also assessed. Methods: A total of 68 patients with colorectal carcinoma were recruited and the levels of RhoA and RhoC mRNA transcripts in cancer, paratumoral and normal tissues were characterized by quantitative real-time polymerase chain reaction (QRT-PCR). Their correlation to clinical histopathological parameters was analyzed. Results: The levels of RhoA and RhoC mRNA transcripts in carcinoma tissues were significantly higher than those in the matched paratumor and normal tissues from the same patient (p<0.05). The expression levels of both genes were significantly correlated with metastasis of cancer cells to lymph nodes and liver (p<0.05). The levels of RhoA expression were significantly correlated with the histopathological degree of cancer, while the expression of RhoC was correlated with the extent of local invasion to intestine. Conclusions: This is the first study with QRT-PCR to examine the expressions of RhoA and RhoC genes in colorectal carcinoma of Chinese patients. The significantly up-regulated RhoA and RhoC expressions suggest that they may contribute to the initiation, development, invasion and metastasis of colorectal carcinoma in Chinese patients. Clin Chem Lab Med 2009;47:811–7.

Background: The identification of very low-levels of minority sequences has interesting clinical and diagnostic applications. Among these, non-invasive prenatal diagnosis of genetic diseases on fetal DNA circulating in maternal plasma is an emerging field of application. Methods: A combined approach based on innovative microarray slides coated with a special copolymer was developed for the identification of three polymorphisms located in the causative gene for cystic fibrosis (CF). This technique was applied to the analysis of fetal DNA in maternal plasma from four couples that carried different allelic variants. Results: The use of highly sensitive slides correctly identified fetal paternally inherited alleles without any enrichment strategy. Conclusions: Our results confirm that the high fluorescence signal provided by the optimized substrate may be applied to the identification of any fetal paternally inherited sequence. This helps extend the application of non-invasive prenatal diagnosis to genetic diseases caused by predominant mutations or minor rare molecular defects. Clin Chem Lab Med 2009;47:818–23.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused primarily by defects in the steroid 21-hydroxylase gene ( CYP21A2 ). The CYP21A2 gene is located in the HLA class III region on the short arm of chromosome 6p21.3, along with an inactive pseudogene, CYP21A1P , that is 98% homologous with CYP21A2 in its coding sequence. Most mutations found in the CYP21A2 gene are normally present in the pseudogene, implying that recombination events (microconversion) between these two genes may lead to the transfer of mutations from the pseudogene to the functional gene. Approximately only 5% of all CYP21A2 alleles causing disease harbour rare mutations not originating from the pseudogene. However, detection of these rare and spontaneous mutations has continued to expand worldwide. In this report, we describe the clinical and genetic findings in a Romanian newborn suffering from classic salt wasting form of CAH due to severe 21-hydroxylase deficiency. Clin Chem Lab Med 2009;47:824–5.

Background: Epstein-Barr virus (EBV) is a human herpes virus that persists for life and is associated with several neoplasms. Haptoglobin (Hp), a polymorphic multifunctional plasma protein, exerts several immunomodulatory effects. The present study investigated the influence of the genetic polymorphism of Hp on EBV serology. Methods: Patients without immunoglobulin G (IgG) titers of EBV were compared according to Hp phenotypes (Hp 1-1, Hp 2-1, Hp 2-2; determined by starch gel electrophoresis) in 208 individuals against 918 healthy subjects with unknown EBV status. Results: The Hp 1-1 phenotype was associated with a lower EBV antibody titer (p=0.0005). Conclusions: Our study confirms the involvement of the Hp phenotype in infection with EBV. Phenotypes with a higher Hp concentration (Hp 1-1 and Hp 2-1) are less prone to positive EBV serology. Clin Chem Lab Med 2009;47:826–8.

Background: Cardiac troponins are considered the cornestore for risk stratification and diagnosis of patients whit acute coronary syndrome (ACS). Following Clinical Laboratory Standards Institute (CLSI) guidelines, we assessed the analytical performances of the Pathfast ® (Mitsubishi, Japan) cTnI method. Methods: We evaluated different sample types. Control materials and lithium heparin plasma pools were used to determine: limit of blank (LoB), limit of detection (LoD), imprecision and linearity. The effects of potential endogenous interfering substances and the possibility of falsely increased cardiac troponin I (cTnI) concentrations attributable to the presence of heterophilic antibodies (HA), rheumatoid factor (RF) and human anti-mouse antibodies (HAMA) in high concentrations were evaluated. The 99th percentile limit of the cTnI value distribution was determined from 320 Caucasian reference individuals. Results: No significant differences were found when cTnI concentrations of 40 lithium-heparin plasma samples were compared with the matched values of K 2 -EDTA plasma, whole blood and serum samples. The LoB and the LoD of the cTnI method were 0.0048 and 0.0066 μg/L, respectively. cTnI mean values from 0.66 to 6.0 μg/L showed a total CV% from 6.0 to 6.4. cTnI at a concentration of 0.02 μg/L was associated with a total CV of 9.6%. The method gave a linear response for cTnI concentrations within the measurement range. In six of 12 samples containing HA, a positive interference was demonstrated. The 99th percentile limit of the cTnI distribution in the reference population was 0.013 μg/L. Conclusions: The data indicate that the cTnI Pathfast method may be suitable for helping clinicians in the management of patients with ACS. Clin Chem Lab Med 2009;47:829–33.

Background: Measurement of C-peptide under standardized conditions provides a sensitive and well-established assessment of β-cell function. We describe the analytical and clinical validation of an automated, microparticle-based chemiluminescent immunoassay method. The assay is designed to measure C-peptide in human serum, plasma and urine. Methods: Assay performance characteristics such as precision and recovery were measured according to protocols established by the Clinical Laboratory Standards Institute (CLSI). A reference range study was conducted. Analytical sensitivity and specificity, interfering substances, recovery, and linearity studies were performed. Method comparison, against the ADVIA Centaur C-Peptide assay (Siemens), was evaluated with clinical specimens from patients with abnormal insulin secretion. Results: The detection limit for this assay was 0.01 ng/mL. Functional sensitivity (inter-assay imprecision ≤20%) was 0.015 ng/mL at a coefficient of variation (%CV) of 11.2%. Total imprecision was below 6.5% CV. The assay was linear upon dilution. Comparison with the ADVIA Centaur C-Peptide assay yielded a correlation coefficient (r) of 0.99. Conclusions: The ARCHITECT C-Peptide assay measures C-peptide rapidly, accurately, and precisely in human serum, plasma and urine. It provides useful improvements for β-cell function testing and for evaluating the clinical status of a patient in combination with other diabetes markers. Clin Chem Lab Med 2009;47:834–41.

Background: Symmetric polyarthritis associated with hepatitis C virus (HCV) infection frequently displays a clinical picture like rheumatoid arthritis (RA). Antibodies to cyclic citrullinated peptide (CCP) have high specificity for the diagnosis of RA. This study examined the frequency and clinical significance of anti-CCP antibodies in patients with chronic HCV infection, with and without manifestations of joint involvement, compared to RA patients. Methods: Serum anti-CCP antibodies and rheumatoid factor (RF) were evaluated in 30 patients with RA and 47 patients with chronic HCV infection. Of those with HCV infection, 20 patients had chronic HCV infection associated with articular involvement and 27 patients had chronic HCV infection without any articular involvement. Results: Anti-CCP antibody level was positive in 70% of RA patients, 8.5% of HCV-infected patients, and in 20% of HCV patients with articular manifestations. RF was positive in 76% of RA patients and in 60% of HCV patients with articular involvement. Cryoglobulins were found in 29% of HCV-infected patients and in 16% of RA patients. Cryoglobulins were more frequent among HCV patients with articular affection (35%) compared to HCV patients without articular affection (26%). Conclusions: Although anti-CCP antibodies remain a useful diagnostic tool for RA, their interpretation in HCV-infected patients with arthritis should be applied with caution. The possibility that those patients could be prone to develop RA cannot be ruled out. Those patients need careful clinical and radiological follow-up. Further large-scale studies are warranted. Clin Chem Lab Med 2009;47:842–7.

Background: Peroxynitrite was hypothesized to be involved in the pathogenesis of multiple sclerosis (MS) through its various neurotoxic effects. Uric acid (UA) was shown to be a strong peroxynitrite scavenger. Methods: We analyzed cerebrospinal fluid (CSF) and serum UA concentrations in 30 MS patients and 20 controls with non-inflammatory neurological diseases (NIND) and correlated these findings with demographic and clinical characteristics of MS patients. Disease activity was assessed by brain magnetic resonance imaging (MRI) and the CSF/serum albumin quotient as an indicator of the state of blood-brain-barrier (BBB). Results: Serum UA concentrations were found to be significantly lower in MS patients compared with controls (p=0.019). CSF UA concentrations were lower in MS patients as compared to controls, as well as in patients with active MS (clinical and/or MRI activity) in comparison to patients with inactive MS or controls, but these differences were not statistically significant. Significant correlation was found between CSF and serum UA concentrations (p=0.016) in MS patients, but not in controls; and between CSF UA concentrations and the CSF/serum albumin quotient in MS patients (p=0.043), but not in controls. Conclusions: Our results support the significance of UA in the pathogenesis of MS. Decreased serum UA concentrations in MS patients might be due to both intrinsically reduced antioxidant capacity and increased UA consumption in MS. CSF UA concentrations may not be a reliable marker of disease activity in MS since its concentration is dependent on leakage of UA molecules from serum through the damaged BBB and the balance between consumption/production within the central nervous system (CNS). Clin Chem Lab Med 2009;47:848–53.

Background: The analytical performance of the clinical chemistry module c 501 (cobas ® 6000 analyzer series) was evaluated for therapeutic drug monitoring and drugs of abuse testing using a spectrum of representative assays. Particular attention was paid to potential interactions between reagents using a simulated routine workload. Methods: Within-run and total imprecision were assessed using a selection of representative reagents. Deviation from a consensus mean was tested using samples from a proficiency testing scheme. Method comparison using routine samples was carried out against the MODULAR ANALYTICS SWA and COBAS INTEGRA ® 800 analysis systems. Results: Total coefficients of variation (CV) ranged from 1.9% to 7.8% for individual drugs, and from 3.2% to 8.6% for drugs of abuse testing. Results from proficiency test samples were between 81% and 125% of the consensus mean for therapeutic drugs. Method comparisons (Passing-Bablok regression) showed overall good comparability to MODULAR ANALYTICS SWA and COBAS INTEGRA ® 800 systems, with slopes from 0.93 to 1.17 and correlation coefficients r>0.98. Imprecision in a simulated routine run was tested using a total of 42 methods (10 therapeutic drug monitoring, 9 drugs of abuse testing, 3 enzymes, 12 substrates, 8 specific protein assays). Imprecision in the reference batch run ranged from 0.7% to 5.0% CV for therapeutic drug monitoring assays, except for digoxin (DIG) (7.3%), and from 0.9% to 7.7% for drugs of abuse testing. The CVs of general clinical chemistry and specific protein tests were within the expected limits of 2% and 4%. CV changes in the simulated routine run were within the expected limits for most assays. Negative ΔCVs (≥2%) for DIG, digitoxin (DIGIT), cannabinoids (THC), and phencyclidine (PCP) may indicate improved performance when running these assays in a simulated routine operation. A positive ΔCV (≥3%) was found for amphetamines (AMPHs). Conclusions: In conclusion, the cobas ® c 501 module seems to be well-suited for routine use as consolidated workstation. Except for a potential interaction with AMPH, as indicated by the positive ΔCV, no significant interferences from different reagents could be observed during this study. Clin Chem Lab Med 2009;47:854–9.

Background: We recently hypothesized that serum γ-glutamyltransferase (GGT), within its reference range, predicts many diseases as a biomarker for background exposure to various xenobiotics. Even though normal serum GGT was associated with xenobiotics having very long half-lives (heavy metals, dioxin, or organochlorine pesticides), it was unknown whether GGT was associated with xenobiotics with short half-lives, including polycyclic aromatic hydrocarbons (PAHs), well known carcinogens. Methods: Among 1256 adult participants in the National Health and Nutrition Examination Survey (NHANES) 2003–2004, urinary metabolites of PAH (monohydroxy-PAH), and serum GGT were measured. We selected the 10 monohydroxy-PAHs (OH-PAHs) for which at least 90% of participants had concentrations greater than the limit of detection. Results: Among the 10 OH-PAHs, eight had significant positive correlations with serum GGT. These correlations were similarly observed in men and women, and in individuals under 60 years of age. Unlike serum GGT, alanine aminotransferase, another liver enzyme, was not associated with OH-PAHs. Conclusions: Taken together with the previous epidemiological evidence, the associations of serum GGT with OH-PAHs reinforce the concept that serum GGT is a marker for various environmental pollutants encountered at background levels in the general population. Clin Chem Lab Med 2009;47:860–2.

Background: C-reactive protein (CRP) has emerged as an important predictor for cardiovascular disease (CVD). To facilitate clinical and public health interventions, CRP thresholds have been defined as follows: low-risk (<1.0 mg/L), average-risk (1.0–3.0 mg/L), and high-risk (>3.0 mg/L). However, these cut-off thresholds are based on distributions in Western populations, and do not distinguish between men and women. Methods: We examined CRP distribution, gender difference, and determinants of CRP concentrations ranging from 0.02 mg/L to 10.0 mg/L, in 4923 Korean adults (2248 men; 2675 women) who received health checkups at Gangnam Severance Hospital from March 2006 to May 2007. Results: The distribution of CRP was highly skewed toward lower concentrations. CRP was higher in men than women, and the cut-off thresholds for the high-risk tertile of CRP concentrations corresponded to 1.01 mg/L in men and 0.62 mg/L in women, based on the current study population. Age, male gender, cigarette smoking, physical activity, body mass index, total cholesterol, high density lipoprotein cholesterol, γ-glutamyltransferase, and uric acid were independently associated with CRP concentrations. Conclusions: CRP distribution and gender difference in Korean adults were found to be different from previous Western studies, although similar risk factors influence CRP concentrations. Our results suggest that ethnicity and gender specific cut-off thresholds for CRP concentrations should be taken into consideration in CVD risk assessment. Clin Chem Lab Med 2009;47:863–9.

Background: There is accumulating evidence from the epidemiological field of telomere biology that telomere length plays an important role in the pathophysiology of cardiovascular disease. The RNA-dependent DNA polymerase, telomerase, is essential in regulating telomere length by acting as a reverse transcriptase. However, the relationship between telomerase activity and telomere length in cardiovascular disease is unclear. This is due, in part, to the paucity of information on the utility of a quantitative and routine assay for the determination of telomerase activity in circulating blood leukocytes. Methods: We used a validated, high-sensitive real-time quantitative telomeric repeat amplification protocol (RQ-TRAP) to determine telomerase activity in circulating blood leukocytes. Results: The present investigation demonstrated direct and reliable detection of telomerase activity of circulating blood leukocytes. Conclusion: The present investigation suggests the feasibility of using RQ-TRAP assay in routine screening of telomerase activity in blood specimens typically collected in a clinical/epidemiological setting. Clin Chem Lab Med 2009;47:870–3.

Background : Identification of neoplastic cells in cerebrospinal fluid (CSF) by cytological analysis is the key diagnostic feature of leptomeningeal metastasis (LM). Because of the lack of sensitivity of this test, considerable efforts have been made to identify alternative diagnostic markers. Data from the literature suggest that measurement of tumor markers (TM) in CSF may be helpful for improving the diagnosis. Methods : We analyzed the concentrations of the TM carcinoembryonic antigen (CEA), CA15.3, CA125 and CA19.9 in both CSF and serum from 18 patients with neoplastic meningitis diagnosed by CSF cytology. We also performed these same measurements in 50 patients affected by other neurological diseases (OND) in order to evaluate putative intrathecal synthesis. In addition, CSF and serum concentrations of the proangiogenic factor VEGF (vascular endothelial growth factor) were evaluated. Results : All LM patients showed intrathecal synthesis for at least one TM. In one patient, a negative CSF cytology after treatment paralleled normalization of tumor marker synthesis. None of the OND patients displayed intrathecal TM synthesis. The VEGF Index (CSF/serum VEGF relative to CSF/serum albumin ratios) was significantly higher in LM patients compared with the control group. However, significant overlap between LM patients and values seen in those with OND was observed. Conclusions : Evaluation of intrathecal TM synthesis is a specific, sensitive, reliable, and reproducible diagnostic tool, and is useful to support diagnosis of carcinomatous meningitis. Clin Chem Lab Med 2009;47:874–9.