Carbohydrate-deficient transferrin: utility of HPLC in handling atypical samples uninterpretable by capillary electrophoresis.

AIMS: Carbohydrate-deficient transferrin (CDT) is a marker of chronic alcohol abuse. Uninterpretable (atypical) CDT patterns have been detected by both capillary electrophoresis (CE) and HPLC. The aim of this study was to evaluate the performance of HPLC as a second-line test for the interpretation of most frequent atypical CDT profiles detected by CE. METHODS: CDT was analyzed by CE (Capillarys 2, Sebia) on 9120 consecutive samples in a routine laboratory setting during a 2-year period. A commercial method (ClinRep CDT kit, Recipe) was employed to retest 123 (1.4%) samples with atypical CDT patterns on a Prominence LC-20AT HPLC (Shimadzu). RESULTS: CE-uninterpretable samples were categorized as having low transferrin (Tf) concentration (LT; n = 42, 0.5%), di-trisialotransferrin bridging (D-TB; n = 63, 0.7%) or atypical peak profile (APP; n = 18, 0.2%). CDT was detectable by HPLC in 58 of 123 (47%) samples including 21of 42 (50%) with LT, 27 of 63 (43%) with D-TB and 10 of 18 (56%) with APP. CONCLUSIONS: Second-line HPLC testing reduced uninterpretable samples by 47%, with similar rates of improvement regardless of the type of CDT pattern. The usefulness of HPLC as a second-line test for CDT should be evaluated according to cost-benefit considerations in the context of each laboratory.

Severe Spontaneous Echo Contrast/Auricolar Thrombosis in "Nonvalvular" AF: Value of Thromboembolic Risk Scores.

BACKGROUND: Patients with atrial fibrillation (AF) have an increased thromboembolic risk that can be estimated with risk scores and sometimes require oral anticoagulation therapy (OAT). Despite correct anticoagulation, some patients still develop left atrial spontaneous echo contrast (SEC) or thrombosis. The value of traditional risk scores (R2 CHADS2 , CHADS2 , and CHA2 DS2 -VASc) in predicting such events remains controversial. METHODS AND RESULTS: The aim of our study was to explore variables linked to severe SEC or atrial thrombosis and evaluate the performance of traditional risk scores in identifying these patients. In order to do this, we retrospectively analyzed 568 patients with nonvalvular nonparoxysmal AF who underwent electrical cardioversion from January 2011 to December 2016 after OAT for a minimum of 4 weeks. A transesophageal echocardiogram was performed in 265 patients for various indications, and 24 exhibited left atrial SEC or thrombosis. Female gender, history of heart failure or left ventricular ejection fraction <40%, and high levels (>1 mg/dL) of C-reactive protein (CRP) were independently associated with left atrial SEC/thrombosis. A score composed by these factors (denominated HIS [Heart Failure, Inflammation, and female Sex]) showed a sensitivity of 79% and a specificity of 60% (area under receiver operating characteristic curve 0.695, P = 0.002) in identifying patients with a positive transesophageal echo; traditional risk scores did not perform as well. CONCLUSIONS: In patients with persistent AF and suboptimal anticoagulation, a risk score composed by history of heart failure, high CRP, and female gender identifies patients at high risk of left atrial SEC/thrombosis when its value is >1.

The STRATEGY Study (Stress Cardiac Magnetic Resonance Versus Computed Tomography Coronary Angiography for the Management of Symptomatic Revascularized Patients): Resources and Outcomes Impact.

BACKGROUND: Computed tomography coronary angiography (cTCA) and stress cardiac magnetic resonance (stress-CMR) are suitable tools for diagnosing obstructive coronary artery disease in symptomatic patients with previous history of revascularization. However, performance appraisal of noninvasive tests must take in account the consequent diagnostic testing, invasive procedures, clinical outcomes, radiation exposure, and cumulative costs rather than their diagnostic accuracy only. We aimed to compare an anatomic (cTCA) versus a functional (stress-CMR) strategy in symptomatic patients with previous myocardial revascularization procedures. METHODS AND RESULTS: Six hundred patients with chest pain and previous revascularization included in a prospective observational registry and evaluated by clinically indicated cTCA (n=300, mean age 68.2±9.7 years, male 255) or stress-CMR (n=300, mean age 67.6±9.7 years, male 263) were enrolled and followed-up in terms of subsequent noninvasive tests, invasive coronary angiography, revascularization procedures, cumulative effective radiation dose, major adverse cardiac events, defined as a composite end point of nonfatal myocardial infarction and cardiac death, and medical costs. The mean follow-up for cTCA and stress-CMR groups was similar (773.6±345 versus 752.8±291 days; P=0.21). Compared with stress-CMR, cTCA was associated with a higher rate of subsequent noninvasive tests (28% versus 17%; P=0.0009), invasive coronary angiography (31% versus 20%; P=0.0009), and revascularization procedures (24% versus 16%; P=0.007). Stress-CMR strategy was associated with a significant reduction of radiation exposure and cumulative costs (59% and 24%, respectively; P<0.001). Finally, patients undergoing stress-CMR showed a lower rate of major adverse cardiac events (5% versus 10%; P<0.010) and cost-effectiveness ratio (119.98±250.92 versus 218.12±298.45 Euro/y; P<0.001). CONCLUSIONS: Compared with cTCA, stress-CMR is more cost-effective in symptomatic revascularized patients.

Prognostic Benefit of Cardiac Magnetic Resonance Over Transthoracic Echocardiography for the Assessment of Ischemic and Nonischemic Dilated Cardiomyopathy Patients Referred for the Evaluation of Primary Prevention Implantable Cardioverter-Defibrillator Therapy.

BACKGROUND: The aim of this study was to determine the prognostic benefit of cardiac magnetic resonance (CMR) over transthoracic echocardiography (TTE) in ischemic cardiomyopathy and nonischemic dilated cardiomyopathy patients evaluated for primary prevention implantable cardioverter-defibrillator therapy. METHODS AND RESULTS: We enrolled 409 consecutive ischemic and dilated cardiomyopathy patients (mean age: 64±12 years; 331 men). All patients underwent TTE and CMR, and left ventricle end-diastolic volume, left ventricle end-systolic volume, and left ventricle ejection fraction (LVEF) were evaluated. In addition, late gadolinium enhancement was also assessed. All patients were followed up for major adverse cardiac events (MACE) defined as a composite end point of long runs of nonsustained ventricular tachycardia, sustained ventricular tachycardia, aborted sudden cardiac death, or sudden cardiac death. The median follow-up was 545 days. CMR showed higher left ventricle end-diastolic volume (mean difference: 43±22.5 mL), higher left ventricle end-systolic volume (mean difference: 34±20.5 mL), and lower LVEF (mean difference: -4.9±10%) as compared to TTE (P<0.01). MACE occurred in 103 (25%) patients. Patients experiencing MACE showed higher left ventricle end-diastolic volume, higher left ventricle end-systolic volume, and lower LVEF with both imaging modalities and higher late gadolinium enhancement per-patient prevalence as compared to patients without MACE. At multivariable analysis, CMR-LVEF ≤35% (hazard ratio=2.18 [1.3-3.8]) and the presence of late gadolinium enhancement (hazard ratio=2.2 [1.4-3.6]) were independently associated with MACE (P<0.01). A model based on CMR-LVEF ≤35% or CMR-LVEF ≤35% plus late gadolinium enhancement detection showed a higher performance in the prediction of MACE as compared to TTE-LVEF resulting in net reclassification improvement of 0.468 (95% confidence interval, 0.283-0.654; P<0.001) and 0.413 (95% confidence interval, 0.23-0.63; P<0.001), respectively. CONCLUSIONS: CMR provides additional prognostic stratification as compared to TTE, which may have direct impact on the indication of implantable cardioverter-defibrillator implantation.

Carbohydrate-Deficient Transferrin Determination in a Clinical Setting: Consistency Between Capillary Electrophoresis Assays and Utility of HPLC as a Confirmatory Test.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) is used to assess chronic alcohol consumption in administrative and forensic context. The aim of the present study was the optimization of the diagnostic strategy for CDT determination in a clinical laboratory setting. METHODS: Two capillary zone electrophoresis (CZE) assays, the CEofix CDT (Analis, Suarlée, Belgium) run on single capillary MDQ instrument and the muticapillary (Sebia, Lisses, France), were compared as screening methods and a commercial high-performance liquid chromatography (HPLC) assay (Recipe, Munich, Germany) was used for confirmation. RESULTS: In total, 367 serum samples were analyzed by both CZE assays with concordant classification in 92% of cases. All discordant samples were classified as negative by HPLC, as did 2/3 of those that could not be classified by either CZE assay. Classification of samples with CDT values close to cut-off by CZE was confirmed by HPLC in 95-100% of negative samples but only in 28.6-33.3% of positive samples. CONCLUSIONS: Both CZE assays proved suitable for CDT screening. HPLC was useful for discriminating CDT value in most of samples that could not be interpreted by CZE due to analytical interferences. Considering the implication of CDT testing, HPLC assay may also be helpful for the confirmation of positive results close to the cut-off value of CZE assays.

A serially coupled stationary phase method for the determination of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine by liquid chromatography ion trap tandem mass spectrometry.

Oxidative attack to DNA is of particular interest since DNA modifications can lead to heritable mutations. The most studied product of DNA oxidation is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). While 8-oxodG determination in blood and tissue cells is prone to artifacts, its measurement in urine employing liquid chromatography tandem mass spectrometry (LC-MS/MS) has gained more and more interest for increased reliability. LC-MS/MS can be affected by matrix effects and this is particularly true when ion trap is used as MS analyzer, due to ion accumulation in the trap and related space charge effect. In the present work, we have developed a LC-MS/MS method where the combination of cation exchange and reverse phase solid phases resulted in LC separation optimization. This together with the employment of an isotopically labeled internal standard, allowed the usage of ion trap LC-MS/MS, typically not employed for quantitative measurement in biological samples, for the measurement of 8-oxodG in urine samples from control populations. Four different urine matrices were employed for method validation. Limit of quantitation was set at least at 0.5 ng/ml. While analyzing urine samples from healthy volunteers, 8-oxodG levels reported as ng/ml were statistically different comparing males with females (p<0.05, Mann Whitney test); while comparing results normalized for creatinine no statistical significant difference was found. Mean urinary 8-oxodG level found in healthy volunteers was 1.16±0.46 nmol/mmol creatinine. The present method by enhancing at best the chromatographic performances allows the usage of ion trap LC-MS/MS for the measurement of 8-oxodG in urine samples from control populations.

HLA and killer immunoglobulin-like receptor genes as outcome predictors of hepatitis C virus-related hepatocellular carcinoma.

PURPOSE: We evaluated the impact of the killer immunoglobulin-like receptors (KIR) of natural killer (NK) cells and of their HLA ligands over the clinical outcome of hepatitis C virus (HCV)-related hepatocellular carcinoma after curative treatment by either surgical resection or radiofrequency thermal ablation (RTA). EXPERIMENTAL DESIGN: Sixty-one consecutive patients with HCV-related hepatocellular carcinoma underwent KIR genotyping and HLA typing. A phenotypic/functional characterization of NK cells was carried out in patients with different KIR/KIR-ligand genotype. RESULTS: Activating KIR2DS5 was associated with significantly longer time to recurrence (TTR) and overall survival (OS; P < 0.03 each). Homozygous HLA-C1 (P < 0.02) and HLA-Bw4I80 (P < 0.05) were expressed by patients with significantly better OS, whereas HLA-C2 (P < 0.02) and HLA-Bw4T80 (P < 0.01) were associated with a worse OS. Multivariate analysis identified as parameters independently related to TTR the type of treatment (surgical resection vs. RTA; P < 0.03) and HLA-C1 (P < 0.03), whereas only KIR2DS5 was an independent predictor of longer OS (P < 0.05). Compound KIR2DL2-C1 and KIR3DS1-Bw4T80 genotypes were associated with better TTR (P < 0.03) and worse OS (P = 0.02), respectively. A prevalent cytotoxic (CD56(dim)) NK phenotype was detected in patients with both longer TTR and OS. Cytotoxic capacity measured by upregulation of CD107a was significantly higher in subjects with HLA-C1 alone or combined with KIR2DL2/KIR2DL3. CONCLUSIONS: These results support a central role of NK cells in the immune response against hepatocellular carcinoma, providing a strong rationale for therapeutic strategies enhancing NK response and for individualized posttreatment monitoring schemes.

Immunological and molecular correlates of disease recurrence after liver resection for hepatocellular carcinoma.

The definition of the risk of hepatocellular carcinoma (HCC) recurrence after resection represents a central issue to improve the clinical management of patients. In this study we examined the prognostic relevance of infiltrating immune cell subsets in the tumor (TIL) and in nontumorous (NT) liver (LIL), and the expression of immune-related and lineage-specific mRNAs in HCC and NT liver derived from 42 patients. The phenotype of infiltrating cells was analyzed by flow cytometry, and mRNA expression in liver tissue was examined by real-time reverse transcription (RT)-PCR. The tumor immune microenvironment was enriched in inhibitory and dysfunctional cell subsets. Enrichment in CD4+ T-cells and in particular CD4 and CD8+ memory subsets within TIL was predictive of better overall survival (OS) and time to recurrence (TTR). Increased programmed death ligand 1 (PDL1) mRNA content and higher prevalence of invariant NKT (iNKT) cells were associated with shorter OS and TTR, respectively. By combined evaluation of infiltrating cell subsets along with mRNA profiling of immune and tumor related genes, we identified the intratumoral frequency of memory T-cells and iNKT-cells as well as PDL1 expression as the best predictors of clinical outcome. HCC infiltrate is characterized by the expression of molecules with negative regulatory function that may favor tumor recurrence and poor survival.

A system consisting of cation-exchange chromatography, combined surface-activated chemical and electrospray ionization, and ion-trap analysis for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine.

RATIONALE: 8-Hydroxy-2'-deoxyguanosine has served as a biomarker for oxidative damage to DNA from different types of biological samples, and various techniques have been used to analyze it. In particular, liquid chromatography coupled to mass spectrometry has been used to identify 8-hydroxy-2'-deoxyguanosine in urine samples. Usually, a triple quadrupole analyzer and multiple reaction monitoring have been employed for its detection. Only a few studies have used a less expensive ion-trap analyzer instead. METHODS: We have developed a new liquid chromatography/mass spectrometry procedure that incorporates cation-exchange chromatography in conjunction with surface-activated and electrospray ionization with an ion trap analyzer for the mass spectral step. RESULTS: The combination of two ionization sources reduced the matrix effect arising from in-source reactions, thus increasing the sensitivity to levels comparable with those obtained by triple quadrupole analyzers. CONCLUSIONS: This new method for 8-hydroxy-2'-deoxyguanosine detection provided increased sensitivity and reduced chemical noise, using a less expensive, stable and accurate mass spectrometric technology.

Interleukin 28B genotype determination using DNA from different sources: A simple and reliable tool for the epidemiological and clinical characterization of hepatitis C.

Recent studies reported a close correlation between polymorphisms in the Interleukin (IL)28B gene and rates of resolution of hepatitis C virus infection occurring spontaneously or induced by treatment. The diagnostic utility of IL28B genotype, however, is not understood completely. For rapid data collection on the natural history of HCV infection in patients with different IL28B genotype, simple, sensitive and rapid methods suitable for non-invasive and archival clinical samples are needed urgently. A real-time polymerase chain reaction (PCR) method for IL28B typing (rs12979860) was developed using very small DNA quantities extracted from different biological specimens. Consistent IL28B genotyping of at least two DNA samples obtained from different sources such as whole blood, buccal swab, serum, and formalin fixed paraffin-embedded liver tissue was obtained from 58 patients with liver disease of mixed etiology. IL28B genotype prevalence in 170 patients with liver disease in this region of Italy was consistent with data reported in Caucasian populations. Differential distribution of genotypes was observed according to response to treatment in 68 patients infected with HCV, with higher prevalence of CC genotype in responders (50%) compared to non-responders (17.85%; p=0.015). These results indicate that the possibility of reliable IL28B genotyping using different DNA sources may represent a useful tool for both clinical research and characterization of patients with hepatitis C.

Evaluation of Aution Max AX-4030 and 9UB Uriflet, 10PA Aution Sticks urine dipsticks in the automated urine test strip analysis.

BACKGROUND: Aution Max AX-4030, a test strip analyzer recently introduced to the market, represents an upgrade of the Aution Max AX-4280 widely employed for urinalysis. This new instrument model can allocate two different test strips at the same time. In the present study the two instruments have been compared together with the usage of Uriflet 9UB and the recently produced Aution Sticks 10PA urine strips, the latter presenting an additional test area for the measurement of urinary creatinine. METHODS: Imprecision and correlation between instruments and strips have been evaluated for chemical-physical parameters. Accuracy was evaluated for protein, glucose and creatinine by comparing the semi-quantitative results to those obtained by quantitative methods. The well-known interference effect of high ascorbic acid levels on urine glucose test strip determination was evaluated, ascorbic acid influence was also evaluated on protein and creatinine determination. RESULTS: The two instruments have demonstrated comparable performances: precision and correlation between instruments and strips, evaluated for chemical-physical parameters, were always good. Furthermore, accuracy was always very good: results of protein and glucose semi-quantitative measurements resulted to be highly correlated with those obtained by quantitative methods. Moreover, the semi-quantitative measurements of creatinine, employing Aution Sticks 10PA urine strips, were highly comparable with quantitative results. CONCLUSIONS: 10PA urine strips are eligible for urine creatinine determination with the possibility of correcting urinalysis results for urinary creatinine concentration, whenever necessary and calculating the protein creatinine ratio. Further studies should be carried out to evaluate effectiveness and appropriateness of the usage of creatinine semi-quantitative analysis.

Translating pharmacogenetics into clinical practice: interleukin (IL)28B and inosine triphosphatase (ITPA) polymophisms in hepatitis C virus (HCV) infection.

Hepatitis C virus (HCV) infection is frequently characterized by evolution to chronicity and by a variable clinical course of the disease. The clinical heterogeneities of HCV infection and the imperfect predictability of the response to interferon (IFN) have suggested the need to search for a genetic basis of clinical features. This led to the discovery of genetic polymorphisms playing a major role in the evolution of infection, as well as on treatment response and adverse effects. This review will cover recent reports on the subject, focusing on the potential use of the new genetic markers in the diagnostic algorithm for the stratification of patients for personalized antiviral regimens.

Effectiveness of the biological monitoring of chemical exposure in the workplace: results of a survey in an area of Northern Italy.

A survey of biological monitoring results from 2000 to 2008 was carried out as a part of institutional activities performed by the Workplace Prevention and Safety Service (SPSAL) of the south area of Modena district (Italy). The survey involved 18 companies employing 945 workers altogether. According to workplace exposure, routine biological monitoring had been carried out yearly or 6-monthly in private laboratories chosen by each company. Analysis of consecutive data from six plants showed fluctuating biomarker levels inconsistent with expected occupational exposure and sometimes exceeding biological exposure indices. Surveillance samplings were thus organized for comparison to be carried out in a public reference laboratory. Overall results showed systematic underestimation of marker levels by the laboratories in charge of routine biomonitoring that, together with inappropriate biomarker selection and timing of sample collection, led to insufficient and/or ineffective corrective measures. These results raise major concerns on the reliability of laboratory assessments performed for the routine biomonitoring of exposed workers. Continuous surveillance of the consistency between biological and ambient monitoring data, together with the systematic evaluation of the analytical quality of laboratory results, would be essential for a reliable assessment of health risk in the workplace.

[Analytical quality in biological monitoring of workers exposed to chemicals: experience of the Prevention and Safety at the Workplace Service in Modena]. / Qualità del dato analitico nel monitoraggio biologico dei lavoratori esposti a sostanze chimiche: esperienza del Servizio Prevenzione e Sicurezza Ambienti di Lavoro della Azienda USL di Modena.

BACKGROUND: The quality of laboratory data is one of the main factors in guaranteeing efficacy of biological monitoring. OBJECTIVES: To analyze the quality of laboratory data used for biological monitoring of exposed workers. METHODS: A survey involving 18 companies employing 945 workers in the area of Modena, Italy, was carried out in 2008. RESULTS: Most of the 9 private laboratories receiving biological samples did not perform directly part or all of the laboratory assessments requested, but this was not indicated in the final report. Major problems were observed in the application of internal quality control, and only one laboratory participated in external quality assessment for blood lead measurements. CONCLUSIONS: Our results raise major concerns on the traceability and reliability of laboratory assessments performed for biomonitoring of exposed workers. Systematic evaluation of the quality of analytical data would be highly recommendable.

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury.

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis.

Alpha-tocopherol amplifies benzoyl peroxide free radical decomposition in a chemical system.

Benzoyl peroxide is commonly used in the treatment of acne, even though some adverse effects have been reported, probably mediated by the formation of peroxide-derived free radicals and the depletion of antioxidants. In the present work we have studied, in a chemical system, the effect of alpha-tocopherol on benzoyl peroxide radical decomposition to analyse the presence of an interaction between these two compounds, leading to an enhanced peroxide-cytotoxicity, as we have previously reported. Under our experimental conditions alpha-tocopherol strongly amplified the peroxide free radical decomposition occurring either in the presence or in the absence of UV irradiation, and lead to the formation of an unknown radical species in addition to benzoyloxy, phenyl and tocopheroxyl free radicals. The results of this study show that the enhancement of benzoyl peroxide toxicity in cells exposed simultaneously to this peroxide and alpha-tocopherol, is likely due to the generation of the detected radical species.