Volume 46, Issue 12

Cystatin C is a low-molecular-weight protein which has been proposed as a marker of renal function that could replace creatinine. Indeed, the concentration of cystatin C is mainly determined by glomerular filtration and is particularly of interest in clinical settings where the relationship between creatinine production and muscle mass impairs the clinical performance of creatinine. Since the last decade, numerous studies have evaluated its potential use in measuring renal function in various populations. More recently, other potential developments for its clinical use have emerged. This review summarises current knowledge about the physiology of cystatin C and about its use as a renal marker, either alone or in equations developed to estimate the glomerular filtration rate. This paper also reviews recent data about the other applications of cystatin C, particularly in cardiology, oncology and clinical pharmacology. Clin Chem Lab Med 2008;46:1664–86.

Background : Cardiac B-type natriuretic peptides (NPs) are products of a complex neuro-hormonal equilibrium also involving heart and kidneys; they should be carefully handled as heart failure (HF) markers. Meta-analysis of variance (ANOVA) could extrapolate heterogeneity factors (HeFs) in an attempt to reliably evaluate the outcome. Methods : A total of 98 samples from 67 studies were considered – 52 samples from 41 studies evaluating brain natriuretic peptide (BNP), 46 from 24 evaluating N-terminal pro-brain natriuretic peptide (NT-proBNP). Case-control apparently unbiased studies linking NP values to a specific degree of HF clinical severity (HFd), and considering marker sensitivity and specificity, were selected. As an outcome measurement, the natural logarithm of [(true positives/false negatives)/(false positives/true negatives)] was considered. On data negative to the distribution normality and to the publication bias tests, the random-effect heterogeneity test was applied; heterogeneous data – adjusted for the candidate HeFs – were tested using the one-way meta-ANOVA. Afterwards, contrast tests among level means were carried out on the HeFs significant to the one-way meta-ANOVA and not significant to the interaction two-way meta-ANOVA tests. A sensitivity analysis was carried out on the data without the outlier results. Results and conclusions : Reference method (RM), disease prevalence (DP) and HFd resulted in significant BNP HeFs, RM and DP being responsible for, respectively, reference and spectrum biases. No significant NT-proBNP HeFs were found. Clin Chem Lab Med 2008;46:1687–95.

Background : Cyclin D1 (CCND1) is known to regulate function in G1 arrest and therefore may play an important role in carcinogenesis. The aim of this study was to investigate the effect of G870A polymorphism of the CCND1 gene on gastric precancerous condition, on histological chronic gastritis, and on the risk of peptic ulcer diseases. Methods : Restriction fragment length polymorphism analysis was performed for polymorphisms at 870GA in the CCND1 gene in 524 cancer-free subjects, including 111 gastric and 54 duodenal ulcers, and 359 non-ulcer subjects. Gastritis scores of antral gastric mucosa were assessed according to the updated Sydney system in 384 subjects. Results : CCND1 genotype was significantly associated with the severity of intestinal metaplasia by the Kruskal-Wallis test, and this tendency was especially stronger among older subjects of 61 years or older (overall subjects: p=0.035, 61 years∼: p=0.007). We also found that the 870AA genotype held a significant high risk of intestinal metaplasia [ Helicobacter pylori infection adjusted odds ratio (OR)=1.8, 95% confidence interval (CI)=1.03–3.15, p=0.04]. The same genotype was more closely associated with the risk of intestinal metaplasia in older subjects of 61 years or older ( H. pylori infection adjusted OR=3.45, 95% CI=1.48–8.08, p=0.004). A non-significant association was found between CCND1 G870A genotypes and the risk of peptic ulcer diseases as well as histological severity of acute or chronic inflammation. Conclusions : It appears that the G870A polymorphism of CCND1 is associated with gastric premalignant condition especially in older subjects. Clin Chem Lab Med 2008;46:1696–701.

Background : Mutations in the ABCD1 gene lead to X-linked adrenoleukodystrophy, a neurodegenerative disorder. Hundreds of hereditary mutations of the gene have been reported in patients with X-linked adrenoleukodystrophy, but there have been no reports of de novo mutations. Methods : The coding region of ABCD1 cDNA of two patients was amplified and sequenced. To confirm the mutations in the ABCD1 gene of the patients and screen for mutations in their family members, the genomic DNA was analyzed by direct sequencing and denaturing high performance liquid chromatography. Results : Two missense mutations (C631Y and G512S) were identified in the probands, but the mutations were not found in their parents. Tests for paternity identification excluded the possibility of misparentage. Conclusions : The mutations identified in the two male patients were de novo mutations. Mutation analysis of parents of the proband may be helpful for pregnancy planning and evaluation of the recurrence risk to siblings of the proband. Clin Chem Lab Med 2008;46:1702–6.

Background : Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease most often due to steroid 21-hydroxylase deficiency (21OHD). The incidence of the CYP21A2 gene mutations in 21OHD has been extensively studied in recent years. The p.Q318X mutation presents an ethnic-specific distribution with a higher prevalence (40%) in Tunisia. Methods : A total of 20 Moroccan patients were studied, using PCR amplification and sequencing, to determine the mutation spectrum and to evaluate whether the incidence of the p.Q318X mutation is similar in Morocco and in Tunisia. Results : Results revealed that 15 patients were with the salt wasting (SW) form and five with the simple virilizing (SV) form of the disease. All patients were homozygous or compound heterozygous for severe mutations of the CYP21A2 gene. The IVS2-13A/C>G was the most common mutation (47% of chromosomes) and the p.I172N (11%) was associated with the SV form. The p.Q318X was the second most frequent mutation (19.4%) with a regional distribution: the mutation was especially detected (75%) in patients from the midland of Morocco (Fez). We found a novel p.L353R mutation associated with the p.V281L mutation on the same chromosome in one patient at homozygous state. Conclusions : Genotyping for the four common mutations (IVS2-13A/C>G, p.Q318X, large lesions of the CYP21A2 gene and p.I172N) should allow identifying the diseased alleles and providing genetic counseling in 94% of CAH Moroccan cases. The regional distribution of mutations should help in screening studies. Clin Chem Lab Med 2008;46:1707–13.

Background : The –1131T>C polymorphism in the apolipoprotein gene A5 ( APOA5 ) was found to be associated with increased levels of plasma triglyceride and decreased levels of high-density lipoprotein cholesterol (HDL-C), which are characteristic dyslipidemic components of metabolic syndrome. This study aimed to identify a link between this polymorphism and the risk of metabolic syndrome. Methods : The sample population comprised 615 unrelated subjects, 18.7% of whom had metabolic syndrome. Genotypes were determined via polymerase chain reaction, restriction mapping with Mse I, and gel electrophoresis. Results : A significantly higher level of triglycerides and a lower level of HDL-C were noted in carriers of the –1131C allele than in the non-carriers (p<0.001 and p=0.044, respectively). The frequency of the –1131C allele in the metabolic syndrome-affected subjects was significantly higher than that of the group of unaffected subjects (37.4% vs. 27.7%, p=0.004). Even after adjusting for age, gender, smoking, regular exercise, and waist-to-hip ratio, the APOA5 –1131C allele carriers remained significantly associated with an increased risk of metabolic syndrome (OR=1.77, 95% CI, 1.13–2.77; p=0.012). Conclusions : These results indicate that the association of APOA5 –1131T>C polymorphism with dyslipidemia can also contribute to an increased susceptibility to metabolic syndrome in the Chinese, as a result of its effect on triglyceride metabolism. Clin Chem Lab Med 2008;46:1714–9.

Background : Recent studies suggest that the common variant in the glutathione S-transferase ( GST ) M1 ( GSTM1 ) and T1 ( GSTT1 ) gene is associated with the risk of smoking-related coronary artery disease (CAD). Intra-ethnic as well as inter-ethnic differences are known to impact the frequencies of GST gene polymorphisms, thus influencing its interactive effect with tobacco smoking on CAD risk. The aim of the present study was to evaluate the interaction of the genetic polymorphisms of GSTM1 and GSTT1 with cigarette smoking and the risk of CAD in a Chinese population. Methods : We conducted a study with 277 CAD patients and 277 controls matched by age and sex to examine the prevalence of GSTM1 and GSTT1 polymorphism in CAD. Results : We found that homozygous deletion of GSTM1 had a frequency of 32.1% among patients with CAD and 21.3% among those without CAD (p=0.004). The frequency of the GSTT1 null genotype was 27.8% among the patients with CAD and 19.1% among CAD-free subjects (p=0.016). Patients who smoked having both the wild-type genotypes of GSTM1 and GSTT1 were protected from developing coronary heart disease (p<0.001). Moreover, smokers with combined GSTM1 null GSTT1 null genotypes had a significantly higher number of stenosed vessels than those with the positive genotype (p=0.02). Conclusions : Our results suggest that GST polymorphisms may be a susceptibility factor to smoking-related CAD in the Chinese population. Clin Chem Lab Med 2008;46:1720–5.

Background : In a prospective study, we measured plasma markers of myocardial damage induced by radiofrequency catheter ablation (RFA) with the protein biochip microarray system. Methods : A total of 32 consecutive patients undergoing RFA for atrioventricular nodal re-entry tachycardia (AVNRT), right atrial flutter (AFL) and atrial fibrillation (AF) were included in the study. Cardiac troponin I (cTnI), creatine kinase isoenzyme MB (CK-MB), heart-type fatty acid binding protein (hFABP) and glycogen phosphorylase BB (GPBB) were measured using biochip array technology at baseline and 24 h after the procedure. Results : Values for all markers increased 24 h after RFA (cTnI: 0.92±0.49 μg/L vs. 0.33±0.06 μg/L, p<0.001; CK-MB: 3.79±2.04 μg/L vs. 1.85±0.55 μg/L, p<0.001; hFABP: 2.82±0.95 μg/L vs. 2.00±0.95 μg/L, p<0.001; GPBB: 9.07±5.83 μg/L vs. 4.70±2.50 μg/L, p<0.001). The correlations between plasma marker levels and RFA time were cTnI: r=0.63, p<0.01; CK-MB: r=0.75, p<0.01; hFABP: r=0.55, p<0.05, GPBB: r=0.51, p<0.05; the correlation between RFA time and number of RF applications was significant (r=0.81, p<0.001). Patients with RFA due to AF or flutter had elevated cTnI, CK-MB and hFABP levels compared to patients with AVNRT (cTnI: 1.14± 0.49 μg/L vs. 0.59±0.25 μg/L, p<0.05; CK-MB: 4.46± 2.07 μg/L vs. 2.81±1.54 μg/L, p<0.05; hFABP: 3.21± 0.98 μg/L vs. 2.25±0.54 μg/L, p<0.01). Conclusions : Myocardial injury induced by RFA can be detected by cTnI, CK-MB, hFABP and GPBB. Plasma cTnI, CK-MB and hFABP levels significantly increased in patients with AFL and AF compared to patients with AVNRT. The increase of cTnI, CK-MB and GPBB levels correlates with the total duration of RFA. Clin Chem Lab Med 2008;46:1726–8.

Background : Hepatitis C virus (HCV) is one of the most common viral infections worldwide causing major human chronic pathology. Viral RNA can be detected 1–3 weeks after infection and in cases where HCV RNA is still detectable after 6 months, the patient is considered to be chronically infected. Early detection is crucial in preventing loss of treatment opportunities. Methods : Here, we present an in-house real time PCR TaqMan ® probe based screening method for the detection and quantification of the six recognized HCV genotypes and evaluate its efficiency with the use of quality control for molecular diagnostics HCV quantification panels. Results and conclusions : The quantification method presented performed well with all quality control panels and is therefore an attractive approach for the clinical setting. Clin Chem Lab Med 2008;46:1729–31.

Background : Several drugs may affect the diagnosis of brain death by depressing the electroencephalographic signal. Serum levels of these drugs must be below their respective therapeutic ranges. Methods : A high performance liquid chromatography-based fast and simple method was developed for determination of thiopentone, pentobarbitone, phenobarbitone, methohexital and propofol in serum and validated according to international recommendations. Results : Separation of extracted analytes was performed on a reversed phase column [Agilent Zorbax SB C18, 5 μm, 4.6×250 mm; mobile phase 50% 50 mM NaH 2 PO 4 pH 4.6 mixed with 35% (v/v) acetonitrile and 15% (v/v) methanol]. Calibration curves were linear throughout the selected ranges (μg/mL, thiopentone 0.25–50, pentobarbitone 0.25–25, phenobarbitone 2.5–50, methohexital 0.125–2.50, propofol 0.25–5.0). The standard deviations for the regression line, recovery, imprecision and accuracy results were all highly satisfactory. The lower limits of quantification for propofol, thiopentone and pentobarbitone were set at 0.25 μg/mL, for phenobarbitone 2.5 μg/mL, and for methohexital 0.125 μg/mL, which are below the lowest pharmacologically relevant serum concentrations. Intra- and inter-day coefficients of variation were less than 10% throughout as determined with six replicates. Conclusions : The method presented is suitable for drug monitoring to help enhance the reliability of the diagnosis of brain death. Clin Chem Lab Med 2008;46:1732–8.

Background : Accurate assessment of cobalamin profiles in human serum, cells, and tissues may have clinical diagnostic value. However, non-alkyl forms of cobalamin undergo β-axial ligand exchange reactions during extraction, which leads to inaccurate profiles having little or no diagnostic value. Methods : Experiments were designed to: 1) assess β-axial ligand exchange chemistry during the extraction and isolation of cobalamins from cultured bovine aortic endothelial cells, human foreskin fibroblasts, and human hepatoma HepG2 cells, and 2) to establish extraction conditions that would provide a more accurate assessment of endogenous forms containing both exchangeable and non-exchangeable β-axial ligands. Results : The cobalamin profile of cells grown in the presence of [ 57 Co]-cyanocobalamin as a source of vitamin B 12 shows that the following derivatives are present: [ 57 Co]-aquacobalamin, [ 57 Co]-glutathionylcobalamin, [ 57 Co]-sulfitocobalamin, [ 57 Co]-cyanocobalamin, [ 57 Co]-adenosylcobalamin, [ 57 Co]-methylcobalamin, as well as other yet unidentified corrinoids. When the extraction is performed in the presence of excess cold aquacobalamin acting as a scavenger cobalamin (i.e., “cold trapping”), the recovery of both [ 57 Co]-glutathionylcobalamin and [ 57 Co]-sulfitocobalamin decreases to low but consistent levels. In contrast, the [ 57 Co]-nitrocobalamin observed in extracts prepared without excess aquacobalamin is undetectable in extracts prepared with cold trapping. Conclusions : This demonstrates that β-ligand exchange occurs with non-covalently bound β-ligands. The exception to this observation is cyanocobalamin with a non-covalent but non-exchangeable CN – group. It is now possible to obtain accurate profiles of cellular cobalamins. Clin Chem Lab Med 2008;46:1739–46.

Background : Posaconazole is a new broad-spectrum triazole antifungal drug that is used in prophylaxis and therapy of opportunistic fungal infections in immunocompromised patients. Up to now, it is available as an oral suspension only. Due to variable systemic availability known from other azoles, such as itraconazole, it is important to measure blood levels, especially in patients undergoing abdominal surgery which may influence the intestinal resorption. Methods : A sensitive and selective high-performance liquid chromatography method for the precise determination of posaconazole and the internal standard clotrimazole in human plasma was developed and validated. Samples were extracted using solid-phase extraction and separated on a reversed-phase C8 column (150×4.6 mm, 5 μm) using phosphate buffer (pH 6.7, 0.04 M):acetonitrile:methanol (43:49:8, v/v/v) as mobile phase. UV detection was performed at 260 nm. Results : This method showed that a lower limit of quantification was 50 ng/mL and the limit of detection 3 ng/mL. Linearity was tested in the range from 50 to 5000 ng/mL (r 2 =0.9998). Mean recovery was 86%. Conclusions : The method proved to be a useful tool for therapeutic drug monitoring. It is specific, precise and showed excellent reproducibility as well as a favourable accuracy. Clin Chem Lab Med 2008;46:1747–51.

Background : % Microcytic and % hypochromic red cells show an inverse trend in β thalassemia trait and in iron deficiency anemia (IDA). The aim of this study was to assess the discriminant value of % microcytic/% hypochromic ratio, as can be obtained from the Advia 2120 (Siemens) analyzer, in the differential diagnosis of microcytic anemia, compared to the conventional England and Fraser, Green and King, Mentzer, Ricerca, Shine and Lal and Srivastava indices. Methods : Receiver operating characteristic (ROC) curves, sensitivity, specificity and Youden index of indices were calculated for a set of 148 IDA patients and 170 β thalassemia carriers. Results : The Green and King index showed the best area under the curve (AUC, 0.962), with a Youden index of 80.9%; the % microcytic/% hypochromic ratio ranked second (AUC 0.943), with a Youden index of 76.3% and a sensitivity of 99.2%. Conclusions : This index, with high sensitivity for β thalassemia screening, can be a useful tool in the differential diagnosis of microcytic anemia, so samples can be chosen for HbA 2 analysis, to confirm the presumptive diagnosis of the disease. As specificity is 77.1%, we assume it will be 22.9% for false positives, but these samples will have HbA 2 values within the reference range, for correct diagnosis. Clin Chem Lab Med 2008;46:1752–8.

Background : Carbohydrate-deficient transferrin (CDT) in serum is a biomarker of heavy alcohol consumption. In Italy, CDT testing is primarily used for matters of road safety by the commissions that reissue drivers' licenses after alcohol-related offences. The purpose of this study was to examine how CDT determinations are carried out by Italian laboratories. Methods : Public (hospital and university) laboratories, the companies producing CDT assays, and the organizers of two external quality assurance (EQA) programs were approached and telephone interviews were conducted. The study was carried out between October, 2006 and January, 2007, and considered the situation as of 31 December, 2006. Results : In 2006, 142 Italian hospital and university laboratories performed CDT measurements and there were 67 license commissions using different protocols for the evaluation of alcohol abuse. Compared with 2005, the number of laboratories that assayed CDT had doubled in 2006. Several different CDT methods were in routine use and there were large differences in the ways results were expressed and in the cut-off limits applied, even for identical methods and instrumentations. Only approximately one-third of the laboratories participated in an EQA program for CDT. Conclusions : Despite that CDT testing is used almost exclusively for medico-legal purposes in Italy, many different methods, ways of expressing test results, and cut-off limits were routinely applied. This observation points at the urgent need for standardization of CDT measurement. Clin Chem Lab Med 2008;46:1759–62.

Background : In recent years a number of point of care testing (POCT) glucometers were introduced on the market. We investigated the analytical variability (lot-to-lot variation, calibration error, inter-instrument and inter-operator variability) of glucose POCT systems in a university hospital environment and compared these results with the analytical needs required for tight glucose monitoring. Methods : The reference hexokinase method was compared to different POCT systems based on glucose oxidase (blood gas instruments) or glucose dehydrogenase (handheld glucometers). Based upon daily internal quality control data, total errors were calculated for the various glucose methods and the analytical variability of the glucometers was estimated. Results : The total error of the glucometers exceeded by far the desirable analytical specifications (based on a biological variability model). Lot-to-lot variation, inter-instrument variation and inter-operator variability contributed approximately equally to total variance. As in a hospital environment, distribution of hematocrit values is broad, converting blood glucose into plasma values using a fixed factor further increases variance. The percentage of outliers exceeded the ISO 15197 criteria in a broad glucose concentration range. Conclusions : Total analytical variation of handheld glucometers is larger than expected. Clinicians should be aware that the variability of glucose measurements obtained by blood gas instruments is lower than results obtained with handheld glucometers on capillary blood. Clin Chem Lab Med 2008;46:1763–8.

Background : Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis. LDL(–) is present in blood plasma of healthy subjects and at higher concentrations in diseases with high cardiovascular risk, such as familial hypercholesterolemia or diabetes. Methods : We developed and validated a sandwich ELISA for LDL(–) in human plasma using two monoclonal antibodies against LDL(–) that do not bind to native LDL, extensively copper-oxidized LDL or malondialdehyde-modified LDL. The characteristics of assay performance, such as limits of detection and quantification, accuracy, inter- and intra-assay precision were evaluated. The linearity, interferences and stability tests were also performed. Results : The calibration range of the assay is 0.625–20.0 mU/L at 1:2000 sample dilution. ELISA validation showed intra- and inter-assay precision and recovery within the required limits for immunoassays. The limits of detection and quantification were 0.423 mU/L and 0.517 mU/L LDL(–), respectively. The intra- and inter-assay coefficient of variation ranged from 9.5% to 11.5% and from 11.3% to 18.9%, respectively. Recovery of LDL(–) ranged from 92.8% to 105.1%. Conclusions : This ELISA represents a very practical tool for measuring LDL(–) in human blood for widespread research and clinical sample use. Clin Chem Lab Med 2008;46:1769–75.

Background : The serum insulin-like growth factor I (IGF-I) level is accepted to diagnose the growth hormone (GH) status. Here, we evaluated the DRG IGF-I 600 ELISA, DSL IGF-I ELISA, IDS OCTEIA IGF-I, Mediagnost IGF-I-ELISA, and the Siemens Immulite 2500 IGF-I in comparison to the former Nichols Advantage IGF-I assay. Methods : Imprecision was determined by use of a serum pool and commercial control materials. Accuracy was evaluated by means of a method comparison to Nichols in 173 serum samples of GH deficient patients. Results : The Siemens and the IDS IGF-I assays showed the lowest imprecision with coefficients of variation up to 3.6% and 6.9%, respectively. Both correlated best to Nichols (Siemens: y=0.667X+8.8 μg/L, r=0.950; IDS: y=0.527X+4.6 μg/L, r=0.927) with the lowest dispersion of residuals from a linear equation. The DSL assay had the highest comparability to Nichols (y=1.000X+35.5 μg/L, r=0.864), but with a considerable scattering. Conclusions : To yield IGF-I determination comparable to the former Nichols IGF-I, either the Siemens or the IDS assay should be applied, and the results should be converted by a linear method transformation. Where a conversion factor is not desired, the DSL assay should be selected. Clin Chem Lab Med 2008;46:1776–83.

Background : Logarithmic transformation is recommended in method comparison or commutability studies when the standard deviation of the measurement results is heteroscedastic. We show that in the case of a considerable constant difference in the relationship between the x - and y -data, logarithmic transformation introduces non-linearity. Methods : We used a simulated bivariate dataset [n=50; no systematic differences between the x - and y -data; x -data without error and y -data with concentration-dependent random, normally distributed error (CV=7%)], from which we generated two new sets of data: one by i) multiplying the y -data by 1.1, and the second by ii) adding a constant value of 15 to the y -data. Results : The runs test (p<0.001) confirms that logarithmic transformation of the second dataset introduces non-linearity. Consequently, applying a linear regression model to the transformed data would result in erroneous decisions about commutability and in erroneously high estimates of the limits of agreement in method comparison studies. Conclusions : We recommend applying a linearity test after logarithmic transformation of bivariate data and, if necessary, to calculate the prediction intervals of a non-linear regression function. Clin Chem Lab Med 2008;46:1784–5.

Background : Hyperhomocysteinemia is a risk factor for cardiovascular, cerebrovascular diseases and is also associated with congenital birth defects, pregnancy complications and cancer. Many homocysteine assays have been developed since the 1960s and most companies have commercially available kits for homocysteine determination. In this paper, we report the performance characteristics of the new Architect homocysteine assay and the correlation with HPLC and other routine analytical methods. Methods : Recovery, limit of detection (LOD), total imprecision and interferences were evaluated; furthermore, the method was compared with HPLC and with two routine methods. Results : Recovery was higher than 95%, LOD was lower than 1.0 μmol/L, the range for total imprecision was 1.7%–3.8%. No significant degree of interference was observed up to 5 g/L hemoglobin, 20 mg/dL (342.5 μmol/L) bilirubin and 60 g/L lipids. For the comparison study, this new method shows a lightly positive bias with HPLC (mean±SD: 0.03±1.7 μmol/L) with R 2 =0.96, a positive bias with the immunonephelometric method (mean±SD: 1.7±1.2 μmol/L) with R 2 =0.98, and a negative bias with the enzymatic method (mean±SD: –1.8±2.2 μmol/L) with R 2 =0.94. Conclusions : This new method from Abbott can be included among the methods suitable for homocysteine routine analysis. Clin Chem Lab Med 2008;46:1786–8.