Band 49, Heft 12

In a state of caloric excess, adipose tissue plays an essential role by storing lipids. Its expandability determines the onset of metabolic syndrome (central obesity, dyslipidemia, glucose intolerance and hypertension). When the adipocyte endoplasmic reticulum is no longer capable of processing the excess nutrients, the so-called “endoplasmic reticulum stress” develops. This triggers efflux of free fatty acids from adipocytes into the circulation and causes triglyceride overload in skeletal muscle, liver and pancreas. Adipose tissue hypoxia then develops, due to the failure of vasculature to expand with adipocyte hypertrophy. Increased catabolism in mitochondria leads there to oxidative stress. Both phenomena cause deranged adipokine secretion and low-grade inflammation. Inflammatory cytokines, reactive oxygen species and ectopic lipid deposition are the main mediators of insulin resistance and vascular impairment, which both lead finally to diabetes type 2 and cardiovascular disease. Recently, fibrosis of adipose tissue was also demonstrated in obesity, contributing to the interplay of deleterious factors forcing inflammation. The present paper reviews recent evidence for adipose tissue dysfunction, trying to define causes and consequences. In conclusion, insulin resistance and associated complications originate from excess lipids, which cannot be stored without limit in adipose tissue, thus affecting its integrity and adipokine secretion.

Cardiotoxicity is a serious adverse effect of anticancer drugs, impacting on quality of life and overall survival of cancer patients. According to the current standard for monitoring cardiac function, cardiotoxicity is usually detected only when a functional impairment has already occurred, precluding any chance of preventing its development. Over the last decade, however, a new approach, based on the use of cardiac biomarkers, has emerged, and has proven to be an effective alternative strategy for early detection of subclinical cardiac injury. In particular, the role of troponin I in identifying patients at risk of cardiotoxicity and of angiotensin-converting enzyme inhibitors in preventing left ventricular ejection fraction reduction and late cardiac events represent an effective tool for the prevention of this complication.

Recent studies demonstrated that large amounts of the pro-hormone peptide of brain natriuretic peptide (proBNP) can be detected in plasma of healthy subjects and in particular of patients with heart failure. As a result, a great part of B-type natriuretic peptides measured in patients with cardio-vascular disease may be devoid of biological activity. These findings stimulated the set up of specific immunoassay methods for the measurement of the intact proBNP peptide. The aim of this review article is to discuss the methodological characteristics and the possible clinical relevance of specific immunoassay methods for the measurement of the proBNP peptide. From an analytical point of view, a fully automated immunoassay of proBNP has some theoretical advantages (e.g., a more stable molecule with higher molecular weight than the derived peptides) compared to the active hormone BNP. Recent studies supported the concept that the precursor proBNP might be actually considered a circulating prohormone, which can be cleaved by specific plasma proteases in BNP, the active hormone, and NT-proBNP, an inactive peptide. The peripheral processing of circulating proBNP could likely be submitted to regulatory rules, which might be impaired in patients with heart failure, opening new perspectives in the treatment of heart failure (e.g., by studying drugs inducing the cleavage of the prohormone into active BNP). Furthermore, as a future perspective, the specific assay in the same plasma sample of the intact precursor proBNP and of the biologically active peptide BNP, could allow a more accurate estimation of the production/secretion of B-type related peptides from cardiomyocytes and of the global cardiac endocrine function.

Evaluating patients with acute chest pain presenting to the emergency department remains an ongoing challenge. The spectrum of etiologies in acute chest pain ranges from minor disease entities to life-threatening diseases, such as pulmonary embolism, acute aortic dissection or acute myocardial infarction (MI). The diagnosis of acute MI is usually made integrating the triad of patient history and clinical presentation, readings of 12-lead ECG and measurement of cardiac troponins (cTn). Introduction of high-sensitivity cTn assays substantially increases sensitivity to identify patients with acute MI even at the time of presentation to the emergency department at the cost of specificity. However, the proportion of patients presenting with cTn positive, non-vascular cardiac chest pain triples with the implementation of new sensitive cTn assays increasing the difficulty for the emergency physician to identify those patients who are at need for invasive diagnostics. The main objectives of this mini-review are 1) to discuss elements of disposition decision made by the emergency physician for the evaluation of chest pain patients, 2) to summarize recent advances in assay technology and relate these findings into the clinical context, and 3) to discuss possible consequences for the clinical work and suggest an algorithm for the clinical evaluation of chest pain patients in the emergency department.

A revised framework for the regulation of in vitro diagnostic devices (IVDs) came into force in Australia on July 1, 2010 that aims to ‘ensure that public and personal health are adequately protected’, but which instead may lead to adverse outcomes in clinical diagnosis and management. The regulatory process aims to regulate all IVDs, including those used by clinical diagnostic laboratories, which are already subject to scrutiny as part of the current laboratory accreditation process. The IVD regulatory process initiated in Australia is similar to that used in Canada, but different to that currently operating in the USA and Europe. However, it is feasible that other countries will in time adopt a similar regulatory framework, given that many countries are involved in the development process. In this opinion paper, the regulatory process for IVDs across several geographies are outlined, as are some benefits and weaknesses of the new regulatory process now applied to Australia, as potentially planned for other regions of the world.

In Greece, there is no officially organized training in clinical chemistry for scientists. The Greek Society of Clinical Chemistry-Clinical Biochemistry (GSCC-CB), following the encouragement of the EC4/RC decided to organize a voluntary Register for specialists in clinical chemistry. The following criteria for registration were defined: 1) University degree in Chemistry, Biochemistry, Biology, Medicine, Pharmacy or other relevant subject. 2) A total of 9 years of university studies and postgraduate specialization in clinical chemistry-clinical biochemistry. 3) A minimum of 4 years of postgraduate specialization in clinical chemistry-clinical biochemistry on the job. 4) The candidate must be practicing clinical chemistry-clinical biochemistry in a laboratory in a medical environment in Greece. The postgraduate specialization in clinical chemistry-clinical biochemistry includes the laboratory training and the theoretical education. The laboratory training is organized by the GSCC-CB according to the Professional Training Dossier. The theoretical education was organized in a series of 18 “Seminars” which was the content of the “Educational program” of the GSCC-CB. Successful completion of the Educational program leads to a Certificate of Competence. The Greek Register has gained equivalence with the EC4 Register and it has 218 members, more than 80 of whom are European clinical chemists.

Background: Human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. The B27 allele is present in 90% of patients with this disease, whereas it is present in only 9% of Caucasians. Molecular detection of HLA-B27 is traditionally based on allele specific amplification of exon 2 (Olerup method) or exon 3 (Dominguez method) by PCR, followed by gel analysis. Methods: We developed a real-time TaqMan PCR based on the Dominguez method with a β-Globin PCR as internal control. Results: A total of 544 clinical samples were used to compare the real-time TaqMan PCR with the traditional Dominguez PCR, the traditional Olerup PCR and a commercial Olerup based HLA-B27 detection kit ( Olerup SSP TM HLA-B27, GenoVision). While 542 samples gave concordant results, two samples showed discrepancies and were further analyzed. One sample that showed a discrepancy was negative with the traditional Olerup method and positive with the three other procedures. Sequencing analysis showed the presence of HLA-B*2712 in this sample. The other sample, positive with both Olerup based PCRs and negative with both Dominguez based methods, turned out to be positive for HLA-B*2707 by sequence analysis. Conclusions: With a correct result for 543 out of 544 samples (99.8%), we consider our real-time HLA-B27 PCR is a reliable method to detect HLA-B27 in the Dutch population, with reduced hands-on time and contamination risk compared to traditional PCR methods.

Background: Serum creatinine is important for detecting the beginning of a decline in kidney function. The Beckman Coulter Jaffe reagents for measuring creatinine have been standardized to the internationally accepted reference method: isotope dilution mass spectrometry (IDMS). The impact of this recalibration on the Beckman Coulter ® modular or cup (stat) Jaffe method is studied. Methods: Recalibrated Jaffe (calibrator set points IDMS traceable) and classic National Institute of Standards and Technology (NIST) creatinine methods (traceable to NIST 914a) were compared with an enzymatic method (Sentinel, traceable to NIST SRM 967). All measurements were performed on Synchron DxC 800 systems. Imprecision of the routine methods was studied using the Clinical and Laboratory Standards Institute (CLSI) protocols and laboratory quality survey. Thirteen plasma pools, with concentrations <354 μmol/L, were analyzed with a gas chromatography isotope dilution mass spectrometry (GC-IDMS) method and routine methods. Total error of 8.2% based on biological variability, set on the GC-IDMS values and acceptance criteria (bias <5%, imprecision <8% at concentrations ≥88.4 μmol/L and a maximum 10% increase in the relative error of estimated glomerular filtration rate (eGFR) of the National Kidney Disease Educational Program (NKDEP) were used for evaluating analytical performance of the routine methods studied. Results: After recalibration of the Jaffe method, median bias (μmol/L) decreased from 12.4 (95% CI: 9.1–21.2) to 7.3 (95% CI: 1.5–10.5). Imprecision of the Jaffe method is in agreement with the claim of the manufacturer, namely <9 μmol/L or <3% below or above 292 μmol/L. Below creatinine of 88.4 μmol/L, imprecision of the recalibrated Jaffe and enzymatic methods is between 4.1% and 6.9%, and 5.0% and 7.1%, respectively, and significantly different (p=0.02 for both the Jaffe and enzymatic methods) from the goal, based on biological variability, of 2.7%. For the adult pools, all recalibrated Jaffe and enzymatic results fall within the total error of 8.2%. In the pediatric samples, one-third of the recalibrated Jaffe and three of the six enzymatic results fall within this total error goal. Conclusions: Recalibration significantly reduced bias of the Jaffe method. For pediatric samples, recalibrated Jaffe results do not comply with either the imprecision goal or the total error based on biological variability. Adult recalibrated Jaffe results are in compliance with the goals based on biological variability and with the acceptance criteria from the NKDEP.

Background: Heterophilic antibodies are still an important source of interference in immunoassays. We have conducted a screening study for interference in a panel of commercially available assays using two sera known to contain high titer Fc-reactive heterophilic antibodies. Methods: The sera were distributed to laboratories participating in the Nordic External Quality Assessment cooperation (EQANord). Duplicate samples pre-blocked with aggregated murine monoclonal MAK33 were also supplied. Discrepancies (>50%) between the results for native and blocked samples were used to classify the tested assays as susceptible to interference. A total of 170 different assay kits covering 91 analytes were tested. Results: We found that 21 assays, covering 19 different analytes, were susceptible to interference from the heterophilic antibodies in the two sera. Many of these are clinically and commercially important assays. Some of the false results were grossly elevated and could have been detrimental to patient care in a clinical setting. Conclusions: Heterophilic antibodies with Fc-reactivity remain a threat. A more widespread use of antibody fragments and aggregated immunoglobulin could potentially improve the heterophilic antibody resistance of assays intended for clinical use.

Background: Oxidative stress has a critical role in inflammatory responce against tobacco smoke (TS). Testing exhaled breath condensate (EBC) samples is one of the methods used for assessment of airway inflammation caused by TS. We aimed to investigate oxidative stress in the lungs associated with TS and to evaluate the effect of this stress with pulmonary function tests (PFTs). Methods: We included 69 subjects as three groups into the study (Group I; 26 smokers, Group II; 21 passive smokers, Group III; 22 non-smokers without TS exposure). Levels of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), nitrite/nitrate ;ibindex of nitric oxide (NO) production;ic, vitamin C, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were measured in EBC samples collected using a condenser and PFTs were performed. Results: The levels of MDA, 8-OHdG, SOD and GSH-Px were higher in smokers. NO levels gradually increased from Group I to Group III. MDA levels were lower in Group III than Group II. The levels of vitamin C were similar in all groups. We determined negative correlation between 8-OHdG levels and forced expiratory volume in one second (FEV 1 ), and maximum mean expiratory flow (MMEF), and a positive correlation between SOD levels and FEV 1 . Conclusions: TS exposure affected the balance between oxidative stress and antioxidant capacity of lungs. Preventing environmental TS exposure might decrease oxidative damage. Increased levels of 8-OHdG and SOD levels could be assessed as an early sign of airway damage.

Background: Cortisol level in human hair would be an endogenous biomarker for the retrospective assessment of long-term central hypothalamo-pituitary-adrenal activity. However, no direct evidence supports that blood-related diffusion is a biologically endogenous source of hair cortisol in humans. The present study aims to validate the direct correlation between cortisol in 1-cm hair segments and salivary cortisol in healthy humans. Methods: We collected three saliva samples from the same participant at Time 1, Time 2 (1 week later) and Time 3 (2 weeks later), and hair 4 weeks later. Cortisol levels in 1-cm hair segments and saliva were determined with high performance liquid chromatography tandem mass spectrometry. Results: Salivary cortisol at Time 1 was significantly associated with that at Time 2 (r=0.514, p=0.003), but not with that at Time 3 (r=0.187, p=0.305); and the one at Time 2 was significantly associated with that at Time 3 (r=0.380, p=0.032). Hair cortisol was significantly correlated with salivary cortisol at Time 2 (r=0.389, p<0.05) and average salivary cortisol (r=0.383, p<0.05) from three sampling. Conclusions: Our results confirmed that blood-related diffusion mechanism is a biologically endogenous source of hair cortisol.

Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for therapeutic drug monitoring of immunosuppressants given to transplant recipients. This study evaluated the performance of the newly introduced MassTrak Immunosuppressant XE Kit (Waters Corporation; “the Kit”) in the determination of everolimus and cyclosporin A (CsA) using LC-MS/MS. Methods: The linearity, precision, detection limit, carryover and matrix effect of the Kit and comparison of the in-house method and Kit procedure were evaluated according to Clinical and Laboratory Standards Institute guidelines. Results: The Kit afforded good linearity in the measurement of everolimus from 2 to 26 ng/mL (R 2 =0.996) and of CsA from 42.6 to 1407 ng/mL (R 2 =0.995). The between- and within-run assay coefficients of variation were 2.3%–8.0% for everolimus and 1.1%–3.1% for CsA. The lower limit of quantitation of everolimus and CsA were 1.1–4.1 ng/mL, respectively. A comparison of CsA values obtained using in-house reagents indicated good correlation with Kit results (R=0.9909). The average matrix effect ranged from 15%–39% for everolimus and 3%–10% for CsA, and ion enhancement was mostly observed for the matrix effect. The effect on analyte/internal standard ratios ranged from 1%–8% for everolimus and 1%–16% for CsA. Conclusions: The Kit employing isotopically labeled internal standards provides reliable measurements of immunosuppressant levels over a broad range of concentrations.

Background: A pharmacogenomics study of cyclophosphamide in systemic lupus erythematosus patients is being conducted in our laboratory in which the plasma concentrations of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide should be assayed rapidly and sensitively. Methods: A rapid, stable and sensitive liquid chromato-graphy/electrospray ionization tandem mass spectrometry method was developed to simultaneously determine cyclophosphamide and 4-hydroxycyclophosphamide in human plasma with ifosfomide as an internal standard. After a protein precipitation with cold acetonitrile and stabilization of 4-hydroxycyclophosphamide by ansyldrazine and extraction with ethyl acetate, separation was performed on a C18 3.5 μm 2.1×50 mm column with mobile phase of acetonitrile and water (50:50, v/v) with 0.1% formic acid at 200 μL/min. The chromatographic run time was 3 min. Results: The linear calibration curves ranged from 5 to 5000 ng/mL for cyclophosphamide and 5–500 ng/mL for 4-hydroxycyclophosphamide. The recoveries of the liquid extraction were 54.5%–58.5% for cyclophosphamide and 103.5%–105.5% for 4-hydroxycyclophosphamide. The lower limit of quantification was 5 ng/mL for both analytes. The intra- and inter-day precision was <15% for quality control samples at 4000, 500, 50 ng/mL for cyclophosphamide and 4-hydroxycyclophosphamide at 400, 100, 20 ng/mL. The method was applied in this pharmacogenomics study in Chinese systemic lupus erythematosus patients treated with low-dose cyclophosphamide. Conclusions: The method was efficient with shorter running time and lower limit of quantification compared to previous reports and has been successfully applied in this pharmacogenomics study.

Background: This study reports our experience over the last six years in the diagnosis of Smith-Lemli-Opitz syndrome and other inborn errors of cholesterol biosynthesis. Methods: Gas chromatography/mass spectrometry was used to obtain sterol profiles in plasma and erythrocyte membranes of suspected patients. Results: Plasma sterol reference values calculated in unaffected subjects (n=276) were in agreement with those previously reported. Among patients investigated from 2005 to 2010, we report 16 patients affected by Smith-Lemli-Opitz syndrome, three of whom represent new cases and 13 of whom were follow-up patients. In this period we also identified a new case of chondrodysplasia punctata 2 X-linked. The estimated incidence obtained for Smith-Lemli-Opitz syndrome was 1:93 suspected patients (1.08%). We also studied the effect of storage on the dehydrocholesterols/cholesterol ratio in plasma and erythrocyte membranes of patients affected by Smith-Lemli-Opitz syndrome stored at –20°C for up to 22 and 20 months, respectively. A significant negative linear correlation between storage time and the dehydrocholesterols/cholesterol ratio was identified in both plasma and erythrocyte membranes. The decrease in the dehydrocholesterols/cholesterol ratio in erythrocyte membranes was at least two-fold higher than in plasma. Conclusions: The results of this study may be helpful for diagnosis and interpretation of data in patients with findings suggestive of a cholesterol biosynthesis defect.

Background: Recent publications report that up to 70% of total laboratory errors occur in the pre-analytical phase. Identification of specific problems highlights pre-analytic processes susceptible to errors. The rejection of unsuitable samples can lead to delayed turnaround time and affect patient care. Methods: A retrospective audit was conducted investigating the rejection rate of routine blood specimens received at chemistry and haematology laboratories over a 2-week period. The reasons for rejection and potential clinical impact of these rejections were investigated. Thirty patient files were randomly selected and examined to assess the impact of these rejections on clinical care. Results: A total of 32,910 specimens were received during the study period, of which 481 were rejected, giving a rejection rate of 1.46%. The main reasons for rejection were inappropriate clotting (30%) and inadequate sample volume (22%). Only 51.7% of rejected samples were repeated and the average time for a repeat sample to reach the laboratory was about 5 days (121 h). Of the repeated samples, 5.1% had results within critical values. Examination of patient folders showed that in 40% of cases the rejection of samples had an impact on patient care. Conclusions: The evaluation of pre-analytical processes in the laboratory, with regard to sample rejection, allowed one to identify problem areas where improvement is necessary. Rejected samples due to factors out of the laboratory’s control had a definite impact on patient care and can thus affect customer satisfaction. Clinicians should be aware of these factors to prevent such rejections.

Background: Mucositis is a painful ulcerative condition of the oral cavity and gastrointestinal tract, occurring in association with chemotherapy and radiotherapy regimes. Trefoil factor family peptides (TFF, trefoil peptides), present in saliva, contribute to epithelial restitution and repair and are therefore potentially important in the healing phase of mucositis. This study aimed to assess any changes in the levels of trefoil peptides in oncology patients with and without mucositis. Methods: Saliva was collected from healthy children, pre-treatment oncology patients, neutropenic patients on treatment with no oral disease and mucositic patients. TFF1, 2 and 3 were quantified using ELISA. Results: In healthy children TFF2 and 3 were positively correlated with age (ρ=0.454, p=0.01 for TFF2; ρ=0.410, p=0.05 for TFF3 Spearman rank correlation). TFF3 was higher in mucositis compared to all other groups. A linear regression prediction model indicated that TFF3, but not TFF1 and TFF2, was significantly different in mucositic and healthy controls, suggesting an altered pattern of trefoil peptide secretion (p=0.021). Conclusions: This study is the first to focus on trefoil peptides in paediatric saliva. It shows the correlation between TFF2, TFF3 and age in healthy children. Paediatric mucositis disease occurs in the presence of increased concentrations and an altered pattern of trefoil peptides.

Background: Trefoil peptides (TFF1, TFF2 and TFF3) are 7–12 kDa molecules, secreted by mucin-producing epithelial cells. Increased serum concentrations have been reported in a number of pathological conditions, which warrants the need for validated commercially available assays. Methods: We validated commercial assays for TFF1-3 and compared results obtained with our in-house assays, using serum from blood donors. Results: Level of detection was: ≤0.008 nmol/L. Measuring ranges were: 0.032–0.51 (TFF1), 0.038–0.76 (TFF2) and 0.019–0.15 (TFF3) nmol/L. Imprecision (CV), judged from the measurement of serum pools in two levels, was below 9% (TFF2 and TFF3) but up to 18% (mean 0.41 nmol/L) for TFF1. No cross reactivity between the TFFs (concentrations >100 nmol/L) was observed. The 95% non-parametric reference intervals were: <0.0032–0.53 (TFF1), 0.099–1.4 (TFF2) and 0.086–0.87 (TFF3) nmol/L. Comparing commercial to in-house assays (n=132), showed biases explained by differences in the calibrators (TFF1 and TFF2). A number of samples showed markedly different results. Conclusions: The commercial assays for TFF2 and TFF3 are acceptable for use on serum samples, while the TFF1 assay revealed a poor imprecision and a too narrow measuring range. Results obtained with the commercial and the in-house assays differed, partly because of differences in the calibrators employed.

Background: There are recommendations to mix venous blood samples by inverting the tubes immediately after venipuncture. Though mixing allows efficient anticoagulation in plasma tubes and fast initiation of coagulation in serum tubes, the effect on laboratory analyses and risk of haemolysis has not been thoroughly evaluated. Methods: Venous blood samples were collected by venipuncture in vacuum tubes from 50 patients (10 or 20 patients in each group). Four types of tubes and 18 parameters used in routine clinical chemistry were evaluated. For each patient and tube, three types of mixing strategies were used: instant mixing, no mixing and 5 min of rest followed by mixing. Results: Most analyses did not differ significantly in samples admitted to different mixing strategies. Plasma lactate dehydrogenase and haemolysis index showed a small but significant increase in samples omitted to instant mixing compared to samples without mixing. However, in one out of twenty non-mixed samples, activated partial thromboplastin time was seriously affected. Conclusions: These results indicate that mixing blood samples after venipuncture is not mandatory for all types of tubes. Instant mixing may introduce interference for those analyses susceptible to haemolysis. However, tubes with liquid-based citrate buffer for coagulation testing should be mixed to avoid clotting.

Background: The exclusion of heparin induced thrombocytopenia (HIT) is required for selecting the most appropriate anticoagulation therapy in affected patients. It requires the combination of clinical data with the detection of antibodies directed against platelet factor 4 (PF4) in complex with polyanions (PA) such as heparin. Methods: We developed a lateral flow immunoassay (LFIA) for PF4/PA complex specific IgG antibodies based on gold nanoparticles. Unlike most other assays, the initial immune reaction takes place in the liquid phase. The sensitivity of the assay has been adjusted with clinical samples aiming in the reliable detection of sera which are positive in a functional platelet activation assay. Results: Sera from 60 patients with suspected HIT were investigated. LFIA identified correctly all samples (n=20) which were positive in a functional assay (HIPA) and an IgG specific ELISA. It correlated with ELISA, but false positive results were less frequent (7 samples were negative with LFIA and HIPA but positive with ELISA). Conclusions: The LFIA may be a suitable tool for the rapid exclusion of HIT within 10 min.

Background: Interpreting hemoglobin high performance liquid chromatograms with borderline HbA 2 values is often problematic, especially in antenatal cases if the partner is a known thalassemia trait. Methods: We tested for underlying β-thalassemia mutations in 25 subjects with borderline HbA 2 values (between 3.0%–4.0%). Amplification refractory mutation system (ARMS-PCR) was used to detect the five common Indian β-thalassemia mutations: (IVS-I-5 (G>C), IVS-I-1 (G>T), codons 8/9 (+G), codons 41/42 (-TTCT) and 619 bp deletion). β-Globin gene sequencing was performed if no mutation was detected. Results: A β-globin gene defect was identified in 8 (32%) of the 25 cases with HbA 2 levels ranging from 3.5%–3.9%. ARMS-PCR revealed IVS-I-5 (G>C) in three, 619 bp deletion in two and codons 41/42 (-TTCT) in one case. Two cases had CAP +1 (A>C) mutation on gene sequencing. IVS-I-1 (G>T) and codons 8/9 (+G) were not found in this small cohort. Conclusions: Mutation analysis should be offered to all at-risk couples with borderline HbA 2 , especially those with values between 3.5% and 4.0% and microcytic hypochromic indices. Significant mutations different from those in other ethnic populations were seen in this small institution-based study.

Background: The presence of sequence variants in miRNA genes may influence their processing, expression and binding to target mRNAs. Since single miRNA can have a large number of potential mRNA targets, even minor variations in its expression can have influences on hundreds of putative mRNAs. Methods: Here, we evaluated 101 paired samples (cancer and normal tissues) from non-small cell lung carcinoma (NSCLC) patients to study the genotype distribution of single nucleotide polymorphisms (SNPs) in miR-146a (rs2910164 C-G), miR-149 (rs2292832 C-T), miR-196a2 (rs11614913 C-T) and miR-499 (rs3746444 G-A) and their influence on the expression of respective miRNAs. Results: Relative expression of miR-146a, miR-149 and miR-499 were comparable in NSCLC and in paired control tissues. On the contrary, we clearly detected a significant increase (p<0.001) of miR-196a2 expression in NSCLC. In particular we found a significant association between miR-196a2 CC genotype and high expression, whereas TT geno-type showed a very low expression in comparison to both CT (p<0.005) and CC patients (p<0.01). We did not find any association between miR-149, miR-196a2 and miR-499 genotype and risk of NSCLC. Conversely, CG genotype of miR-146a appeared associated to an increased risk for NSCLC (p=0.042 and 1.77 OR). Conclusions: Our results seem to demonstrate that sequence variants of miR-196a2 can have an influence on its expression, while miR-146a can have a role in increasing the risk of NSCLC.

Background: Cancer antigen 125 (CA125) is the best known single tumor marker for ovarian cancer (OC). We investigated whether the additional information of the human epididymis protein 4 (HE4) improves diagnostic accuracy. Methods: We retrospectively analyzed preoperative sera of 109 healthy women, 285 patients with benign ovarian masses (cystadenoma: n=78, leimyoma: n=66, endometriosis: n=52, functional ovarian cysts: n=79, other: n=10), 16 low malignant potential (LMP) ovarian tumors and 125 OC (stage I: 22, II: 15, III: 78, IV: 10). CA125 was analyzed using the ARCHITECT system, HE4 using the ARCHITECT(a) system and EIA(e) technology additionally. Results: The lowest concentrations of CA125 and HE4 were observed in healthy individuals, followed by patients with benign adnexal masses and patients with LMP tumors and OC. The area under the curve (AUC) for the differential diagnosis of adnexal masses of CA125 alone was not significantly different to HE4 alone in premenopausal (CA125: 86.7, HE4(a): 82.6, HE4(e): 81.6% p>0.05) but significantly different in postmenopausal [CA125: 93.4 vs. HE4(a): 88.3 p=0.023 and vs. HE4(e): 87.8% p=0.012] patients. For stage I OC, HE4 as a single marker was superior to CA125, which was the best single marker in stage II-IV. The combination of CA125 and HE4 using risk of malignancy algorithm (ROMA) gained the highest sensitivity at 95% specificity for the differential diagnosis of adnexal masses [CA125: 70.9, HE4(a): 67.4, HE4(e): 66.0, ROMA(a): 76.6 and ROMA(e): 74.5%], especially in stage I OC [CA125: 27.3, HE4(a): 40.9, HE4(e): 40.9, ROMA(a): 45.5 and ROMA(e): 45.5%]. Conclusions: CA125 is still the best single marker in the diagnosis of OC. HE4 alone and even more the combined analysis of CA125 and HE4 using ROMA improve the diagnostic accuracy of adnexal masses, especially in early OC.

Background: Sepsis is a costly diagnosis in hospitalized patients. Failure to diagnose sepsis in a timely manner creates a potential financial and safety hazard. The use of transthyretin, C-reactive protein and procalcitonin measurement as early markers of systemic inflammatory response syndrome (SIRS) and sepsis in association with admission of emergency department patients to the intensive care unit (ICU) has been studied. In these studies the SIRS criteria as well as the use of an elevated neutrophil count with granulocyte precursors (left shift) have proved to be problematic. Despite the validity of procalcitonin measurement (PCT, Brahms) in the early diagnosis of SIRS the cost and time for testing are limiting considerations. Immature granulocyte (IG) measurement has been proposed as a more readily available indicator of the presence of granulocyte precursors (left shift). Methods: This study calibrates and validates the measurement of granulocyte maturation [Immature granulocytes (IG)] to the identification of sepsis, a study carried out on a Sysmex Analyzer, model XE 2100 (Kobe, Japan). The Sysmex IG parameter is a crucial measure of immature granulocyte counts and includes metamyelocytes and myelocytes, but not band neutrophils. Results and conclusions: We found agreement with previous work that designated an IG measurement cut-off of 3.2 as optimal. The analysis was then carried a step further with a multivariable discriminator.

Background: Hemolyzed specimens are the leading pre-analytical problem in the laboratory practice, and exert a negative impact on test results. We assessed the reliability of Beckman Coulter UniCel Dxl 800 Accu-TnI testing on hemolyzed specimens. Methods: Twelve non-hemolyzed K 2 EDTA-anticoagulated samples displaying Accu-TnI values >0.20 μg/L and nine with values <0.04 μg/L were selected and three aliquots were obtained from each. The first (“#A”) was processed without further manipulation, whereas the second (“#B”) and third (“#C”) were hemolyzed by aspirating anticoagulated blood through a thin needle. Plasma was separated and tested for hemolysis index (HI) and Accu-TnI. Results: As compared with aliquot #A (HI: 0), a progressive increase of hemolysis occurred in aliquots #B (HI: 25) and #C (HI: 45). The concentration of Accu-TnI gradually decreased from aliquots #A (0.89 μg/L, 0.20–20.16 μg/L), to aliquots #B (0.81 μg/L, 95% CI 0.17–18.37 μg/L; p=0.041) and #C (0.78 μg/L, 95% CI 0.15–17.48 μg/L; p=0.026). In 0/12 (aliquots #B) and 3/12 cases (aliquots #C) the percent decrease exceeded 20% variation. The values remained unchanged in nine samples with Accu-TnI <0.04 μg/L. Conclusions: Accu-TnI values decrease in hemolyzed samples, but the bias might not be clinically significant in samples with hemoglobin <14.5 g/L.

Background: As a key enzyme in folate metabolism, 5,10-methylenetetrahydrofolate reductase (MTHFR) regulates the homeostasis between DNA synthesis and methylation. Data on the association between the MTHFR C677T polymorphism and congenital heart disease (CHD) are conflicting. Methods: To assess the relationship between the MTHFR 677TT genotype and the risk of CHD, we performed a meta-analysis, searching in Pubmed for studies on this topic published in the English language up to 1 December 2010. For each study, we calculated odds ratios (ORs) and 95% confidence intervals (CIs), assuming frequency of allele comparison, homozygote comparison, dominant, and recessive genetic models. We then calculated pooled ORs and 95% CIs. Thirteen studies were included in the meta-analysis. Results: The MTHFR T allele was associated with a border-line significantly increased risk of CHD in the frequency of allele comparison (T vs. C: OR=1.160; 95% CI=0.990–1.359; p=0.001 for heterogeneity). The MTHFR TT genotype was not associated with the risk of CHD in the homozygote comparison (TT vs. CC: OR=1.272; 95% CI=0.947–1.707; p=0.028 for heterogeneity), the dominant genetic model (TT+CT vs. CC: OR=1.127; 95% CI=0.937–1.355; p=0.034 for heterogeneity) and the recessive genetic model (TT vs. CT+CC: OR=1.272; 95% CI=0.975–1.659; p=0.030 for heterogeneity). However, a stratification analysis showed that the association between the MTHFR C677T polymorphism and the risk of CHD was evident among Caucasians instead of Asians. Conclusions: Our meta-analysis suggests that genotypes for the MTHFR C677T polymorphism might be associated with the risk of CHD among Caucasians.

Background: Asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of nitric oxide synthase plays an important role in endothelial dysfunction processes. Recent studies have linked high ADMA levels with several pathological conditions. The interest as a marker of endothelial dysfunction has increased in the last few years. In this paper, a method for serum ADMA quantification by liquid chromatography tandem mass spectrometry has been described. To test the utility in a pathological condition ADMA levels in hypertensive subjects have been measured. Methods: HPLC separation was performed by hydrophilic interaction chromatography using acetonitrile/water containing 0.1% formic acid and 20 mmol/L ammonium formate. Selected reaction monitoring was performed following the transitions m/z 203.1→46.4 for ADMA and 210.1→46.3 for the internal standard [ 2 H 7 ]ADMA. Results: The method was linear up to 10 μmol/L, limit of detection and limit of quantification were 0.005 μmol/L and 0.01 μmol/L, respectively. Recovery was higher than 96%. Intra- and inter-assay imprecision were lower than 6%. The accuracy, expressed as bias %, was <2.5. ADMA in “healthy” subjects ranged from 0.343 to 0.608 μmol/L and resulted significantly lower than that measured in hypertensive subjects (p<0.001). Conclusions: The method developed is selective and sensitive, thus suitable not only for research purposes, but also for routinely work.