Volume 45, Issue 10

In recent years, biomarkers have been recognized as important tools for diagnosis, risk stratification, and therapeutic decision-making in cardiovascular diseases. Currently, the clinical potential of several natriuretic peptides is under scientific investigation. The well-known counter-regulatory hormones are atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP) and urodilatin, which play an important role in the homeostasis of body fluid volume. ANP and BNP have already been demonstrated to have diagnostic usefulness in a great number of studies, which have progressed from bench to bedside. This article summarizes existing data on ANP and related peptides in cardiovascular and other disorders, and outlines the potential clinical usefulness of these markers. Clin Chem Lab Med 2007;45:1259–67.

Background : Polymorphisms of the receptor for advanced glycation end products ( RAGE ) gene have been associated with diabetes, coronary artery disease (CAD) and inflammatory processes. The –374T>A RAGE gene promoter polymorphism was shown to affect gene transcription. The aim of this study was to evaluate the association of the –374T>A polymorphism with the severity of CAD in patients with or without type 2 diabetes mellitus. Method s: We studied 246 Euro-Brazilians with angiographically defined CAD (stenosis >50%), comprising type 2 diabetic (n=98) and non-diabetic subjects (n=148). Genotyping was performed by PCR-restriction fragment length polymorphism using Tsp 509I restriction enzyme. Results : The AA genotype was associated with a significant decrease in CAD severity estimated by the number of diseased vessels (1.43±0.5 vs. 2.49±1.1; p=0.002) and the Duke score (27.3±10.8 vs. 49.3±20.1; p=0.001) only in the group of CAD subjects with type 2 diabetes mellitus. The protective effect of the AA genotype against severity of CAD was not observed in the non-diabetic group. Conclusion : This result confirms that the –374 AA genotype of the RAGE gene promoter is a protective factor against the severity of CAD lesions in type 2 diabetic patients. Clin Chem Lab Med 2007;45:1268–72.

Background : Presenilin 1 (PS1) and presenilin 2 (PS2) are membranous proteins involved in the pathology of Alzheimer's disease. The development of specific therapies targeted at PS1 or PS2 requires the determination of biochemical properties of presenilins. Hence, in this study we analyzed the hydrophobic and ionic properties of endogenous presenilins. Methods : Lysates of immortalized human B-lymphocytes were used as a source of endogenous presenilins. The presence of 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) detergent in lysates favored preservation of PS1 and PS2 native protein complexes. We compared Kyte-Doolittle hydropathicity profiles and hydrophobic interactions of PS1 and PS2 with phenyl-agarose. We also compared the ionic properties of presenilins using anion-exchange chromatography. Results : The hydropathicity profiles of PS1 and PS2 revealed similarly located hydrophobic regions and more hydrophobic region in the C-terminal fragment of PS2. However, both PS1 and PS2 under physiological conditions showed no interactions with phenyl-agarose. Despite similar predicted isoelectric points, PS1 and PS2 exhibited different ionic behavior during anion-exchange chromatography. Conclusions : The different than expected hydrophobic and ionic behavior of PS1 and PS2 may be caused by interactions with other proteins present in complexes formed by endogenous presenilins. The observed difference in ionic properties of PS1 and PS2 can be further explained assuming that PS1 and PS2 form complexes with different sets of proteins. The composition of such variegated PS1 and PS2 complexes can be explored using a proteomic approach. The difference in PS1 and PS2 ionic behavior can be used for purification of endogenous PS1 from PS2, which has not yet been achieved by any other means. Clin Chem Lab Med 2007;45:1273–6.

Background: CD36 is a multifunctional membrane receptor and a cell-adhesion molecule that is expressed in platelets, monocytes/macrophages, microvascular endothelial cells, cardiac monocytes and adipocytes. In this study, we investigated whether genetic polymorphisms of the CD36 gene are associated with risk of coronary artery disease (CAD) in a Korean population. Methods: PCR and polyacrylamide gel electrophoresis or PCR-restriction fragment length polymorphism assays were performed to analyze the following CD36 gene polymorphisms: a (TG) repeat in intron 3 and the base substitution 478C>T (Pro90Ser). A total of 219 patients with significant CAD and 236 control subjects were examined with regard to their genotypes, lipid profiles and other risk factors for CAD. Results: The frequency of (TG) 11- or 12-repeat homozygotes was significantly higher in male CAD patients than in control men (28.4% vs. 15.7%, OR=2.13, p=0.018). Homozygosity for the (TG) 11- or 12-repeat allele was also significantly associated with a higher body mass index (BMI) compared to non-carriers in 134 control men after controlling for age, smoking and hypertension, and explains a 13% BMI variation observed in this study (p=0.015, analysis of covariance). For the 478C>T mutation, which has been reported to be associated with CD36 deficiency, there was no difference in the frequency of the 478T allele between CAD patients and control subjects. However, the 478T allele was found to be closely linked with a (TG) 11- or 12-repeat allele of intron 3 in the control subjects (χ 2 =18.88, p<0.001). Conclusions: The (TG) repeat polymorphism in intron 3 of the CD36 gene is associated with a higher BMI and cardiovascular risk for men in a Korean population. Clin Chem Lab Med 2007;45:1277–82.

Background: The aim of the present study was to compare RNALater ® with the usual method of liquid nitrogen snap freezing as a surrogate mRNA preservation method for functional analysis of separated alleles in yeast (FASAY). Methods: A total of 81 patients with transitional cell carcinoma of the bladder underwent fresh tissue biopsies directly transferred into RNALater ® and stored at room temperature or at 4°C for increasing time intervals until RNA processing. From this cohort of patients, 53 paired snap-frozen and RNALater ® preservative-suspended tissues were obtained. Samples immediately frozen in liquid nitrogen were further stored at −80°C. Results: Of the 81 RNALater ® samples, 14 were not processed for FASAY because of RNA degradation. Of the remaining 67 samples, 15 (22%) were FASAY-positive. Identical FASAY results were found for 50 of 53 (94.4%) paired samples and the percentage of red yeast colonies was highly correlated (Cohen's κ<0.82; p<0.00001). A single p53 missense mutation was found in each of the three discordant positive FASAY and was identical in each concordant positive sample (10/53). Storing samples in RNALater ® at room temperature for 3 days and at 4°C for less than 1 month provided high-quality mRNA suitable for FASAY. Conclusions: Our results demonstrate that RNALater ® is a suitable and flexible alternative to snap freezing for FASAY analysis. Clin Chem Lab Med 2007;45:1283–7.

Background : In a paternity dispute case, 17 autosomal short tandem repeats (STR) were examined and signified a possible paternal mismatch at vWA locus and a maternal mismatch at D5S818 locus in the child under investigation. Methods : Seventeen autosomal, 17 Y-chromosome and six X-chromosome repeat loci were used in parentage analysis. The mutated vWA and D5S818 alleles were amplified, cloned and sequenced to analyze the repeat structure. Results : The vWA locus genotype in the mother, questioned child and suspected father were 18/19, 16/18 and 14/18, and were 13/15, 11/12 and 11/14, respectively, for the D5S818 locus. A complete match with the mother at six X-chromosome STR loci and with the father at 17 Y-chromosome STR loci was observed. Nucleotide sequence analysis of the family at vWA alleles indicated the maternal loss of the repeat motif TCTA by two repeat units and a loss of AGAT repeat by one unit in the D5S818 locus leading to an allele mismatch in the child. The probability of maternity and paternity were 0.999999 and 0.999999, respectively. Conclusions : This is the first study of a maternally transmitted microsatellite mutation in the loci D5S818 and vWA in paternity DNA testing. The results convincingly established that the mother and suspected father are the biological parents of the questioned child. Clin Chem Lab Med 2007;45:1288–91.

Background: Matrix metalloproteases (MMPs) play an important role in cardiovascular remodeling by degrading the extracellular matrix. The aim of this study was to compare two different methods for MMP-2 and MMP-9 concentration and activity determination. Methods: MMP-2 and -9 levels were measured by immunometric and enzymatic assays to determine total and active levels. The two procedures differ in assay principle and in the extent of cross-reactions with interfering substances present in biological samples. Both human serum and culture medium from an ex vivo human model of intimal hyperplasia were checked. Results: All methods were able to detect MMP-2 and -9 with similar levels of sensitivity, reproducibility and accuracy, and furnished positively related results, although significantly different, in both types of sample. Both systems were able to detect changes in MMP production such as the time-course of MMP-2 and -9 release by cultured saphenous vein associated with intima hyperplasia progression. Conclusions: Our data indicate that different values for MMP concentrations can be obtained using different analytical methods, even if they are intrinsically reliable. This suggests that methodological differences should be taken into account when comparing MMP results from different studies. Clin Chem Lab Med 2007;45:1292–8.

Background : Knowledge about the stability of drugs and metabolites in biological fluids is important information when the analytical results are evaluated and interpreted. This study examines changes in blood-ethanol concentration (BEC) during the storage of specimens for up to 12 months at 4°C. Methods : Venous blood samples were taken from drunk drivers in evacuated glass tubes containing sodium fluoride and potassium oxalate as chemical preservatives. The concentrations of ethanol in blood were determined in duplicate by headspace gas chromatography on arrival at the laboratory and again after storage in a refrigerator at 4°C for up to 12 months. Results : The relationship between the standard deviation (SD) of analysis of ethanol at concentration intervals of 0.2 mg/g (BEC) was defined by the linear regression equation SD=0.00243+0.0104 BEC (r=0.99). At a mean BEC of 1.64 mg/g, the SD was 0.019 mg/g which corresponds to a coefficient of variation of 1.1%. The mean decrease in BEC (±SD) between first and second analysis was 0.105± 0.0686 mg/g (t=19.3, d.f.=158, p<0.001) and the loss of alcohol was positively correlated with the duration (days) of storage (r=0.44, p<0.001), although with large inter-tube variations. A correlation also existed (r=0.23, p<0.01) between the loss of ethanol and the starting BEC. When blood samples (n=49) were opened 17 times to remove aliquots for analysis over 6.5 months, the BEC decreased by 0.217±0.054 mg/g compared to a fall of 0.101±0.076 mg/g in tubes kept unopened. None of the blood samples showed a significant increase in BEC after storage. Conclusions : To be considered analytically significant, the BEC had to decrease by 0.013 (2.6%), 0.028 (1.9%) and 0.045 mg/g (1.8%), respectively at starting concentrations of 0.5, 1.5 and 2.5 mg/g. Clin Chem Lab Med 2007;45:1299–304.

Background : The arginine derivatives asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) interfere with endothelial nitric oxide synthesis. Plasma ADMA and SDMA have been shown to be risk factors for cardiovascular disease and/or kidney function deterioration in a variety of patient populations. Methods : We developed a method to quantitatively measure arginine, ADMA, and SDMA using HPLC-tandem mass spectrometry. 13 C 6 -L-Arginine was used as the internal standard, while the derivatives were separated on a silica column in less than 14 min. Plasma levels of ADMA, SDMA, and arginine were measured in children with stage II or III chronic kidney disease (CKD) and age- and gender-matched siblings. Results : The chromatography exhibited no observable ion suppression in the patient specimens tested. There was no apparent carryover for any of the analytes. The assay was linear over 0.32–2.29, 0.23–4.43, and 1.00–303.89 μmol/L for ADMA, SDMA, and arginine, respectively. Plasma ADMA, SDMA, and arginine (mean±SD) were 1.10±0.35, 2.06±1.11, and 57.93±22.10 μmol/L for children with CKD, and 0.78±0.16, 0.71±0.23, and 65.29±21.30 μmol/L for the healthy siblings. Conclusions : The method exhibited no observable ion suppression in the patient specimens tested and has an acceptably short analytical cycle time. Children with CKD had higher levels of ADMA and SDMA than the healthy siblings. Clin Chem Lab Med 2007;45:1305–12.

Background : The soluble transferrin receptor (sTfR) is a parameter of erythropoietic activity and iron deficiency. Increased levels have also been described in hematological malignancies, especially in chronic lymphocytic leukemia (CLL). Methods : We investigated the value of sTfR in the assessment of tumor mass in 61 previously untreated CLL patients. Both hemolysis and iron deficiency were excluded. sTfR was measured nephelometrically (normal 0.81–1.75 mg/L). Results : All Binet A patients had normal sTfR values (1.36±0.22 mg/L). In Binet B patients, the sTfR was increased (3.08±1.70 mg/L, p<0.0001) compared to Binet A patients. Binet B patients with normal sTfR had a small tumor load and no abdominal involvement. A further increase of sTfR in Binet C (3.75± 2.32 mg/L) was not significant compared to Binet B patients. sTfR values decreased or even normalized after successful treatment, whereas relapse or disease progression was associated with another increase of sTfR. Conclusions : The sTfR concentration directly reflects the tumor burden in CLL. Therefore, sTfR may be of clinical value in monitoring disease activity, response to treatment and disease progression. Clin Chem Lab Med 2007;45:1313–8.

Background : Most clinical laboratories use calculated (C) low-density lipoprotein cholesterol (LDL-C) for measurement. Some studies have questioned the linearity of CLDL-C in the clinically useful low range. Moreover, it is generally believed that calculation leads to poor precision such that variation in CLDL-C is greater than the 4% guideline since the calculation is dependent on three primary variables. Actually, the degree of variability of a calculated value will be small if the variability of each primary value is small as compared to its contribution to the calculated value. When LDL-C is low, high-density lipoprotein cholesterol (HDL-C), that has poorer precision, becomes more important in defining the precision of CLDL-C. New homogeneous (direct) HDL-C (dHDL) methods show better precision than the older heterogeneous methods. We hypothesized that a direct homogeneous HDL-C method would substantially improve the low range precision of LDL-C as compared to older heterogeneous HDL-C methods. Methods : We compared CLDL-C to a standardized electrophoretic method that shows very high precision. We also compared the precision of CLDL-C calculated using a homogeneous dHDL and a heterogeneous indirect method. Results : We found good linearity for CLDL-C down to 500 mg/L (×0.002586). The main source of CLDL-C variation was HDL-C. Precision was within guidelines when the dHDL method was used. Using our automated methods for lipoprotein lipids, assuming our reference method is accurate, the formula that calculated CLDL-C (mg/dL) using triglyceride (mg/dL) (×0.001129) ×0.2 suggested by some gave more accurate results than the formula using triglyceride (mg/dL) ×0.16 suggested by others. Conclusions : Given the potential for CLDL-C to meet the precision guidelines, until direct LDL-C methods can be refined, CLDL-C should continue to be the primary test used for assessing LDL-C clinically. Standardized testing for CLDL-C for manufacturers should be available so that the formula used for each instrument can provide well-defined accuracy. Clin Chem Lab Med 2007;45:1319–25.

Background: Various studies have described decreased serum angiotensin-converting enzyme (ACE) activity in patients with pneumonia. The aim of the present study was to evaluate the role of ACE in pneumonia by comparing ACE insertion/deletion (I/D) genotype corrected serum ACE activity and to establish whether the severity of the disease correlates with lower ACE activity. Methods: This was a prospective hospital-based observational study including 134 patients with pneumonia. Serum ACE activity was determined at admission, on days 2, 3, 5 and 10 of hospital stay, and at recovery. Based on ACE genotype and reference values, corresponding Z-scores were calculated. Disease severity, quantified by the acute physiology score (APS), and clinical outcome were compared between tertile groups of the Z-scores. Results: A significant decrease in serum ACE activity during an episode of pneumonia with return to control range during recovery was observed for all three genotypes (II, ID and DD). The calculated Z-scores showed a negative correlation with APS scores (p=0.050). No significant association between decreased serum ACE activity and clinical outcome was observed. Conclusions: Serum ACE activity is significantly decreased during the acute phase of pneumonia. Despite correction for ACE I/D genotype, decreased ACE activity did not show a prognostic value. Further studies are needed to examine the mechanisms behind and diagnostic value of decreased ACE activity in community-acquired pneumonia. Clin Chem Lab Med 2007;45:1326–31.

Introduction : Pleural effusions are often classified into transudates and exudates based on Light's criteria. In this study, the diagnostic properties of Light's criteria were compared to those of several other analytes for the classification of pleural fluids into transudative and exudative. Methods : A total of 471 patients with pleural effusions were evaluated. In pleural effusions and simultaneously drawn blood samples, lactate dehydrogenase (LDH), total protein, albumin, cholesterol, amylase, glucose, pH and the cell number were measured. Retrospectively, the clinical records were used to establish a clinical diagnosis. The diagnostic properties of the biochemical tests were calculated using the clinical diagnoses as gold standard. Results : By clinical diagnosis, 108 patients had transudative and 300 patients had exudative pleural effusions. In addition to pleural LDH activity (accuracy 89%, sensitivity 86%, specificity 97%) and fluid to serum LDH ratio (accuracy 89%, sensitivity 91%, specificity 85%), pleural cholesterol concentration readily identified exudates (accuracy 82%, sensitivity 76%, specificity 98%). Combination of these three parameters achieved a higher overall accuracy (accuracy 95%, sensitivity 93%, specificity 100%) than the Light's criteria (accuracy 93%, sensitivity 100%, specificity 73%). Combination of effusion cholesterol concentration and effusion LDH activity had the highest discriminatory potential (accuracy 98%, sensitivity 98%, specificity 95%). Conclusions : Including effusion cholesterol, concentration in the routine biochemical work-up of pleural fluid allows for correct classification of more pleural effusions than achieved by use of Light's criteria. Combination of cholesterol and LDH had the highest discriminatory potential and the added advantage that no patient plasma is needed for correct classification. Clin Chem Lab Med 2007;45:1332–8.

Background: Although high-sensitivity C-reactive protein (hs-CRP) has emerged as a cardiovascular marker, questions arise regarding the relative information provided by other inflammatory molecules. Therefore, as a first step, we examined interrelationships between serum hs-CRP concentrations and inflammatory, adhesion and growth factors in healthy adults. Methods: Circulating concentrations of hs-CRP, haptoglobin, orosomucoid, interleukin-6 (IL-6), IL-8, IL-18, tumor necrosis factor-α (TNF-α), TNF-receptor II (TNF-RII), E-, P-, and L-selectins, intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1, endothelial growth factor (EGF), vascular EGF (VEGF), insulin-like growth factor-1 (IGF-1) and IGF-binding protein (IGFBP-3) were measured in 154 men and 161 women of the Stanislas cohort. Leukocyte and platelet counts were also determined. Results: Correlations were significant between hs-CRP concentrations and leukocyte and platelet counts, as well as haptoglobin, orosomucoid, IL-6, and ICAM-1 concentrations (p≤0.001). Correlation coefficients for ICAM-1 were higher in men than in women (p≤0.05). When stratifying subjects according to hs-CRP levels, the group with high hs-CRP levels had significantly higher haptoglobin and orosomucoid concentrations than the others, in addition to higher leukocyte counts and IL-6 concentrations in women, and platelet counts and ICAM-1 concentrations in men. Conclusions: Further studies are warranted to explain the association pattern for hs-CRP. Partition of these factors according to their association with hs-CRP concentration opens a new perspective for choice of the best factors in terms of cardiovascular risk in relation to hs-CRP, while non-associated markers could be used to give additional information. Clin Chem Lab Med 2007;45:1339–46.

Background : The lifespan of patients with chronic renal failure (CRF) is reduced, and coronary artery disease is the leading cause of morbidity and mortality in these patients. The progression of atherosclerosis is accelerated and angiogenesis is impaired in CRF. Risk factors that could contribute to further understanding of vascular pathology include markers of inflammation and growth factors. The purpose of this study was to determine the levels of cytokines (IL-2, IL4, IL-6, IL-8, IL-10, IL-1α, IL-1β), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interferon-γ (IFNγ), tumor necrosis factor-α (TNFα) and monocyte chemotactic protein-1 (MCP-1) in patients on chronic hemodialysis (HD; n=75), and to compare values with those of control subjects (n=113). Methods : Evidence ® biochip array analyzer was used for quantification of plasma concentrations in samples. Results : Significant differences were found between the control subjects and HD patients. IL-2 (p<0.001), IL-4 (p<0.001) and EGF (p<0.001) levels were higher in controls than in HD patients, while IL-6 (p<0.001), IL-8 (p=0.081), IL-10 (p=0.008), TNFα (p<0.001), IL-1β (p<0.001) and MCP-1 (p<0.001) levels were higher in HD patients. We also found IL-2 (p=0.015) and IL-1α (p=0.035) levels to be significantly higher in males than females, while IL-4 (p=0.025) and IL-1β (p=0.049) levels were significantly higher in females. Among HD patients, IL-2 levels were higher in patients under the age of 50 years (p<0.048). It was also higher in female than in male patients (p<0.035) and in patients on HD for more than 10 years (p<0.009). IL-6 levels were higher in patients over the age of 50 years (p<0.047). Patients with previous glomerulonephritis had the highest level of IL-6 compared to patients with previous pyelonephritis and diabetes mellitus (p<0.063). IL-6 levels were higher in patients with concomitant hepatitis C virus (HCV) infection (p<0.036) and in patients with developed atherosclerosis (p<0.003). IL-8 levels were higher in patients over the age of 50 years (p<0.003) and in the group with previous glomerulonephritis (p<0.031). IL-10 levels were higher in the group with developed atherosclerosis (p<0.045). EGF was the highest in the group of patients with previous diabetes mellitus compared to pyelonephritis and glomerulonephritis groups (p<0.073). TNFα levels were higher in the patient population on HD for more than 10 years (p<0.032) and in the concomitant HCV group (p<0.073). IL-1β levels were higher in the HCV group (p<0.088). Conclusions : Plasma concentrations of some cytokines and growth factors could serve as useful diagnostic and prognostic parameters for patients with CRF on HD. Clin Chem Lab Med 2007;45:1347–52.

Background : Circulating immunoreactive B-type natriuretic peptide-32 (ir-BNP-32) has diagnostic and prognostic values in heart failure. We compared, in parallel, a point-of-care (POC) test (Triage ® BNP Test) of whole plasma and radioimmunoassay (RIA) of solid-phase extracted (SPE) plasma (SPE/RIA) utilizing a novel copolymer column, in the measurement of patient ir-BNP-32 concentrations. Methods : Approximately 0.25 mL thawed plasma was transferred to a BNP test device and inserted in a Triage ® Meter Plus, which gave ir-BNP-32 concentration in pg/mL. Concurrently, for the SPE/RIA measurement, 1.0 mL plasma was acidified and extracted with an OASIS ® column; eluate dried, reconstituted and quantified by RIA. Results : Inter-day coefficient of variation for both methods were <15%. Plasma SPE recovery was 75.2%. POC correlated with recovery corrected SPE/RIA for ir-BNP-32, r=0.843 (p<0.0001) and the Passing-Bablok model was POC ir-BNP-32=1.43× (recovery corrected SPE/RIA ir-BNP-32)+9.75 ng/L (n=81). A proportional bias was also evident from the Bland-Altman plot, r=0.716 (p<0.0001). Conclusions : A proportional bias is responsible for plasma ir-BNP-32 concentration differences between whole plasma POC test and recovery corrected SPE/RIA measurements. Ir-BNP-32 assays are influenced by plasma matrix and antibody multispecificity. Consequently, consistent analytical accuracy between immunoassays is necessary to attain a single ir-BNP-32 concentration threshold for diagnosis. Clin Chem Lab Med 2007;45:1353–9.

Background : Atherosclerotic plaques prone to cause thrombotic complications and plaque rupture account for the majority of fatal myocardial infarctions (MI), which may be complicated by ventricular fibrillation (VF). Matrix-degrading metalloproteinases (MMPs) and their inhibitors (TIMPs) are expressed in atherosclerotic lesions and contribute to plaque vulnerability. Interleukin-8 (IL-8) is one of the predominant chemokines interacting with MMPs and TIMPs and the coagulation system. The aim of the present study was to assess potential differences of levels of MMP-9, TIMP-1 and IL-8 in postmyocardial infarction patients with or without VF complicating acute MI. Methods : Blood samples were taken from 45 patients with VF complicating acute MI and from 88 patients without VF. All samples were collected during a symptom-free interval remote from the acute ischemic event with a median of 556 days. The markers of interest were TIMP-1, MMP-9 and IL-8. Results : IL-8 and TIMP-1 levels were significantly higher among patients with VF than among patients without VF (p<0.001). In a logistic regression approach IL-8 was an independent indicator of patients prone to VF during MI (p=0.03). High levels of TIMP-1 (p=0.05), MMP-9 (p=0.03), the MMP-9/TIMP-1 ratio (p=0.049) and hypertension (p=0.02) were found to be indicators in patients with reinfarction or unstable angina pectoris during follow-up. Hypertension (p=0.02) and MMP-9 (p=0.03) were the only significant indicators characterizing patients undergoing coronary reinterventions, such as percutaneous coronary interventions and coronary bypass surgery. Conclusions : Higher TIMP-1 and IL-8 levels are present in patients with VF complicating MI. High TIMP-levels may be related to the degree of fibrosis which is a substrate for electrical instability and may contribute to the occurrence of VF. Patients prone to develop VF during MI seem to have an increased proinflammatory condition compared to patients without VF. Clin Chem Lab Med 2007;45:1360–5.

Background : This study evaluated the effects of methylprednisolone on cortisol and cell-mediated immune response (T-lymphocytes and HLA-DR+ monocytes) in peripheral blood after open-heart surgery with cardiopulmonary bypass (CPB) for ventricular septal defect. Methods : A prospective observational study was carried out in a tertiary multidisciplinary neonatal and paediatric intensive care unit. Ten children under 2 years of age received methylprednisolone succinate (30 mg/kg body weight) in CPB priming solutions before the CPB system was connected to the patient during surgery. Before and immediately after and at 24 and 96 h after the operation, T-lymphocytes and HLA-DR+ monocytes were measured by flow cytometry, and methylprednisolone, methylprednisolone succinate and cortisol in blood plasma were assayed by liquid chromatography-mass spectrometry. Results : The children were divided into groups with normal cardiac index (CI) and low CI. No significant differences in methylprednisolone and cortisol concentrations before and after surgery were found between the two groups. The normal CI group exhibited more than a three-fold decrease in T-lymphocytes 24 h after surgery and a two-fold decrease in HLA-DR+ monocyte fluorescence immediately after surgery. Conclusions: Children with normal and low CI were differentiated by T-lymphocytes and HLA-DR+ monocytes. Since no differences in methylprednisolone exposure and cortisol plasma levels between the low-CI and normal-CI groups were found, it can be concluded that factors other than methylprednisolone must contribute to differences in the cell-mediated response. Clin Chem Lab Med 2007;45:1366–72.

Background : The improvement of the consistency of γ-glutamyltransferase (GGT) activity results among different assays after calibration with a common material was estimated. We evaluated if this harmonization could lead to reference limits common to different routine methods. Methods : Seven laboratories measured GGT activity using their own routine analytical system both according to the manufacturer's recommendation and after calibration with a multi-enzyme calibrator [value assigned by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference procedure]. All samples were re-measured using the IFCC reference procedure. Two groups of subjects were selected in each laboratory: a group of healthy men aged 18–25 years without long-term medication and with alcohol consumption less than 44 g/day and a group of subjects with elevated GGT activity. Results : The day-to-day coefficients of variation were less than 2.9% in each laboratory. The means obtained in the group of healthy subjects without common calibration (range of the means 16–23 U/L) were significantly different from those obtained by the IFCC procedure in five laboratories. After calibration, the means remained significantly different from the IFCC procedure results in only one laboratory. For three calibrated methods, the slope values of linear regression vs. the IFCC procedure were not different from the value 1. The results obtained with these three methods for healthy subjects (n=117) were gathered and reference limits were calculated. These were 11–49 U/L (2.5th–97.5th percentiles). The calibration also improved the consistency of elevated results when compared to the IFCC procedure. Conclusions : The common calibration improved the level of consistency between different routine methods. It permitted to define common reference limits which are quite similar to those proposed by the IFCC. This approach should lead to a real benefit in terms of prevention, screening, diagnosis, therapeutic monitoring and for epidemiological studies. Clin Chem Lab Med 2007;45:1373–80.

Background : Stress fractures are frequent injuries among athletes. In vitro, decreases in pH stimulate osteoclasts and inhibit osteoblasts. We hypothesized that exercise-induced lactacidosis stimulates osteoclasts and reduces osteoblast activity in vivo. Methods : A total of 32 volunteers (MA, male athletes; MCo, male controls; female athletes; and female controls) performed three 60-min cycle ergometer tests at 75%, 95% and 110% of their individual anaerobic threshold (IAT). Blood was taken before and at 3 and 24 h after exercise. Osteocalcin (OC), pro-collagen type I N-terminal peptide (PINP), C-terminal telopeptides of collagen I (CTx) and tartrate-resistant acid phosphatase (TRAP) were measured. Results : At 75% and 95% IAT, pH did not change. At 110% IAT, pH decreased in MA by 0.08 units (p=0.041) and in MCo by 0.03 units (p=0.017). The pH results were substantiated by circulating lactate concentrations. The bone resorption markers TRAP and CTx were not consistently modified by any of the exercise tests. Exercise at 75% decreased OC and PINP in all groups. Exercise at 95% and 110% did not induce homogeneous effects. Conclusions : Anaerobic exercise does not systemically affect bone turnover, suggesting that exercise-induced acidosis is not involved in the pathogenesis of stress fractures. Clin Chem Lab Med 2007;45:1381–9.

Background : One of the main issues in biobanking is the establishment of standard operating procedures for specimen collection, preparation and storage to control for pre-analytical variation. For biological fluids such as serum, there is currently a lack of sensitive biomarkers for the quality control of cryopreservation conditions. Methods : The process approach was used to establish an ISO 9001:2000 quality management system. Immunoenzymatic and functional assays were used to assess the stability of the following candidate quality control biomarkers: secretory phospholipase A2, matrix metalloprotease 7, transforming growth factor β1 and anti-HBs immunoglobulin. Results : Five product processes and their corresponding indicators were identified. In the preparation-aliquoting-storage process, no quality control indicator for serum was identified. Only matrix metalloprotease 7 showed moderate susceptibility to freeze-thaw cycles. Conclusions : Biomarkers that have an on-off response to temperature variation could serve as quality indicators for the core processes of biobanking, which are the preparation and storage of biological fluids. The identification of such biomarkers is needed. Clin Chem Lab Med 2007;45:1390–5.

Background : The high specificity of anti-cyclic citrullinated peptide antibodies (anti-CCP) helps substantially in the diagnosis of rheumatoid arthritis (RA), combined with classical markers such as rheumatoid factor (RF). The recent introduction of third-generation methods (anti-CCP3) for anti-CCP detection could further improve diagnostic efficiency. The aim of this study was to determinate the diagnostic efficiency (sensitivity and specificity) of anti-CCP using a new anti-CCP3 method and to compare it with the previous second-generation method (anti-CCP2). Methods : Anti-CCP were studied in sera of 234 patients with recent-onset polyarthritis (ROP) (age ≥16 years; evolution time ≥4 weeks ≤1 year; 2 or more inflamed joints, without drug therapy). After 1 year, 124 patients were diagnosed with RA (American College of Rheumatology criteria). Two ELISAs, an anti-CCP2 and an anti-CCP3, were performed. Results : The best sensitivity according to receiver operating characteristic curves was 51.5% and 54.8% for anti-CCP3 and anti-CCP2, with a specificity of 96.2% and 98.1%, respectively (optimal cutoffs 14.2 and 18.7 U/mL). Significant correlations were obtained (p<0.001) when the methods were compared to each other and to RF. Conclusions : Testing with both types of anti-CCP kit is highly specific for the presence of RA. In our ROP group, anti-CCP2 and anti-CCP3 exhibited similar diagnostic efficiency. Clin Chem Lab Med 2007;45:1396–401.

Background : Low serum copper is often indicative of copper deficiency. Acquired copper deficiency can cause hematological/neurological manifestations. Wilson disease (copper toxicity) is associated with neurological manifestations and low serum copper, with copper deposited in tissues responsible for the toxicity. Low serum copper can also be observed in some carriers of the Wilson disease gene and aceruloplasminemia. This study was undertaken to determine the clinical significance of low serum copper. Methods : The Mayo Medical Laboratories', Metals Laboratory database was reviewed over a 9-month period to identify patients who received their care at the Mayo Clinic and had low serum copper. The medical records were analyzed to determine the significance of the low copper. Results : In six of the 57 patients with low serum copper, the low copper was due to Wilson disease. In the remaining 51 patients, copper deficiency due to an underlying cause was identified in 38 as a reason for the low serum copper. The most commonly identified neurological manifestation of copper deficiency was myeloneuropathy. Coexisting nutrient deficiencies and hematological manifestations of copper deficiency were often but not invariably present. Conclusions : Copper deficiency, Wilson disease (or a carrier state), and aceruloplasminemia are all associated with low serum copper. The presence of coexisting neurological or hematological manifestations that are recognized sequelae of copper deficiency should be considered prior to making a diagnosis of copper deficiency. Gastrointestinal disease or surgery is a common cause of acquired copper deficiency. Even in patients in whom low serum copper is indicative of copper deficiency, the cause of the copper-deficient state may not be evident. Clin Chem Lab Med 2007;45:1402–10.

Background: Critical values' reporting is an essential requisite for clinical laboratories. Local policies were investigated within an indicative cohort of Italian laboratories to monitor the situation and establish a performance benchmark. Methods: A five-point questionnaire was administered to 150 laboratory specialists attending the SIMEL (Italian Society of Laboratory Medicine) National Meeting in June 2006. Results: A total of 107 questionnaires (71.3%) were returned with a 100% individual question response rate. Only 55% of the participants acknowledge critical values reporting as an essential practice, 80% admit that a comprehensive list of critical values is unavailable in the laboratory and 4% do not promptly communicate critical values. The list of critical values is variable among laboratories, ranging from none to 20 analytes included. The requesting physician or his/her office staff receives the great majority (97%) of notifications by telephone for outpatients. Critical values for inpatients are notified directly by telephone (81%) and in a minority of cases by either fax or computer (19%). In the inpatient setting, the information is notified to physicians (77%), nurses (15%) or other healthcare staff in the clinic (8%). It was found that 49% of the participants adopt a standard (digital or written) policy for routine recording of notifications; in 32% of the cases the registration is left to individual attitudes, whereas in 20% of the cases the notification is not recorded. No laboratory has yet adopted a read-back verification of the complete test result by the person receiving the information. Conclusions: The importance of critical value reporting is still poorly recognized in Italy and uniform or internationally accredited practices for communication and recording are not currently implemented. Clin Chem Lab Med 2007;45:1411–3.