Telomere Length, Mitochondrial DNA, and Micronucleus Yield in Response to Oxidative Stress in Peripheral Blood Mononuclear Cells.

Telomere shortening, chromosomal damage, and mitochondrial dysfunction are major initiators of cell aging and biomarkers of many diseases. However, the underlying correlations between nuclear and mitochondrial DNA alterations remain unclear. We investigated the relationship between telomere length (TL) and micronucleus (MN) and their association with mitochondrial DNA copy number (mtDNAcn) in peripheral blood mononuclear cells (PBMCs) in response to 100 µM and 200 µM of hydrogen peroxide (H2O2) at 44, 72, and 96 h. Significant TL shortening was observed after both doses of H2O2 and at all times (all p < 0.05). A concomitant increase in MN was found at 72 h (p < 0.01) and persisted at 96 h (p < 0.01). An increase in mtDNAcn (p = 0.04) at 200 µM of H2O2 was also found. In PBMCs treated with 200 µM H2O2, a significant inverse correlation was found between TL and MN (r = -0.76, p = 0.03), and mtDNA content was directly correlated with TL (r = 0.6, p = 0.04) and inversely related to MN (r = -0.78, p = 0.02). Telomere shortening is the main triggering mechanism of chromosomal damage in stimulated T lymphocytes under oxidative stress. The significant correlations between nuclear DNA damage and mtDNAcn support the notion of a telomere-mitochondria axis that might influence age-associated pathologies and be a target for the development of relevant anti-aging drugs.

Influence of Chromosome 9p21.3 rs1333049 Variant on Telomere Length and Their Interactive Impact on the Prognosis of Coronary Artery Disease.

BACKGROUND: Both telomere shortening and the chromosome 9p21.3 (Chr9p21) rs1333049 (G/C) variant are involved in coronary artery disease (CAD) risk, likely affecting mechanisms related to cell cycle arrest and vascular senescence. The aim of the study was to examine the link between Chr9p21 rs1333049 variant and leucocyte telomere length (LTL), as well as their interactive effect on the risk of major adverse cardiovascular events (MACEs). METHODS: A cohort of 472 patients with angiographically proven and clinically stable CAD were included in the study. At baseline, the LTL, biochemical parameters, and genotype analysis of Chr9p21 rs1333049 variant were measured in all patients. The primary endpoint of this study was the occurrence of MACE defined as a composite of coronary-related death, nonfatal MI, and coronary revascularization. RESULTS: On multivariable linear regression analysis, age (p = 0.02) and Chr9p21 rs1333049 variant (p = 0.002) were the only independent predictors of LTL levels. Carriers of the CC genotype of this SNP had shorter telomeres than GC carriers (p = 0.02) and GG carriers (p = 0.0005). After a follow-up with a mean period of 62 ± 19 months, 90 patients (19.1%) had MACE. Short LTL was an independent prognostic factor of MACE incidence (HR:2.2; 95% CI: 1.3-3.7; p = 0.005) after adjustment for potential confounders. There was a significant interaction (p = 0.01) between the LTL and rs1333049 variant, with patients with risk-allele C and short LTL having a higher risk (HR:5.8; 95% CI: 1.8-19.2; p = 0.004). CONCLUSION: A strong relationship between LTL and Chr9p21 rs1333049 variant was identified, and they interactively affect the risk of poor prognosis in CAD patients.

An extensive evaluation of hepatic markers of damage and regeneration in controlled and uncontrolled donation after circulatory death.

Livers from donations after circulatory death (DCDs) are very sensitive to ischemia/reperfusion injury and thus need careful reconditioning, such as normothermic regional perfusion (NRP). So far, its impact on DCDs has not been thoroughly investigated. This pilot cohort study aimed to explore the NRP impact on liver function by evaluating dynamic changes of circulating markers and hepatic gene expression in 9 uncontrolled DCDs (uDCDs) and 10 controlled DCDs. At NRP start, controlled DCDs had lower plasma levels of inflammatory and liver damage markers, including α-glutathione s-transferase, sorbitol-dehydrogenase, malate dehydrogenase 1, liver-type arginase-1, and keratin-18, but higher levels of osteopontin, sFas, flavin mononucleotide, and succinate than uDCDs. During 4-hour NRP, some damage and inflammatory markers increased in both groups, while IL-6, HGF, and osteopontin increased only in uDCDs. At the NRP end, the tissue expression of early transcriptional regulators, apoptosis, and autophagy mediators was higher in uDCDs than in controlled DCDs. In conclusion, despite initial differences in liver damage biomarkers, the uDCD group was characterized by a major gene expression of regenerative and repair factors after the NRP procedure. Correlative analysis among circulating/tissue biomarkers and the tissue congestion/necrosis degree revealed new potential candidate biomarkers.

Classes of Lipid Mediators and Their Effects on Vascular Inflammation in Atherosclerosis.

It is commonly believed that the inactivation of inflammation is mainly due to the decay or cessation of inducers. In reality, in connection with the development of atherosclerosis, spontaneous decay of inducers is not observed. It is now known that lipid mediators originating from polyunsaturated fatty acids (PUFAs), which are important constituents of all cell membranes, can act in the inflamed tissue and bring it to resolution. In fact, PUFAs, such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are precursors to both pro-inflammatory and anti-inflammatory compounds. In this review, we describe the lipid mediators of vascular inflammation and resolution, and their biochemical activity. In addition, we highlight data from the literature that often show a worsening of atherosclerotic disease in subjects deficient in lipid mediators of inflammation resolution, and we also report on the anti-proteasic and anti-thrombotic properties of these same lipid mediators. It should be noted that despite promising data observed in both animal and in vitro studies, contradictory clinical results have been observed for omega-3 PUFAs. Many further studies will be required in order to clarify the observed conflicts, although lifestyle habits such as smoking or other biochemical factors may often influence the normal synthesis of lipid mediators of inflammation resolution.

Lipidomic Analysis to Assess the Correlation between Ceramides, Stress Hyperglycemia, and HbA1c in Acute Myocardial Infarction.

Ceramides have been associated with cardiometabolic disease (e.g., acute myocardial infarction (AMI) and type 2 diabetes (T2D)) and adverse outcomes. Acute admission hyperglycemia (AH) is a transient glucose alteration in response to stress. As glycated hemoglobin (HbA1c) reflects the glycemia over a longer period of time, its use may be helpful in distinguishing between the AH and hyperglycemia associated with T2D in the AMI setting. The aim was to assess the correlation of ceramides with both AH (defined as an admission glucose level ≥140 mg/dL in the absence of T2D) and HbA1c-T2D and other demographic, clinical, and inflammatory-related biomarkers in AMI. High-performance liquid chromatography-tandem mass spectrometry was used to identify nine ceramide species, and their three ratios, in 140 AMI patients (FTGM coronary unit, Massa, Italy). The ceramides did not correlate with stress hyperglycemia, but specific species were elevated in T2D-AMI. Moreover, some ceramides were associated with other cardiometabolic risk factors. Ceramides assessment may be helpful in better understanding the pathogenic molecular mechanisms underlying myocardial acute events and cardiometabolic risk, as a basis for the future evaluation of their role as prognostic predictors and therapeutic targets in T2D-AMI patients.

Ceramides as Mediators of Oxidative Stress and Inflammation in Cardiometabolic Disease.

Ceramides, composed of a sphingosine and a fatty acid, are bioactive lipid molecules involved in many key cellular pathways (e.g., apoptosis, oxidative stress and inflammation). There is much evidence on the relationship between ceramide species and cardiometabolic disease, especially in relationship with the onset and development of diabetes and acute and chronic coronary artery disease. This review reports available evidence on ceramide structure and generation, and discusses their role in cardiometabolic disease, as well as current translational chances and difficulties for ceramide application in the cardiometabolic clinical settings.

Ceramides and Cardiovascular Risk Factors, Inflammatory Parameters and Left Ventricular Function in AMI Patients.

Background: Ceramides, biologically active lipids correlated to oxidative stress and inflammation, have been associated with adverse outcomes in acute myocardial infarction (AMI). The purpose of this study was to assess the association between ceramides/ratios included in the CERT1 score and increased cardiovascular (CV) risk, inflammatory and left ventricular function parameters in AMI. Methods: high performance liquid chromatography-tandem mass spectrometry was used to identify Cer(d18:1/16:0), Cer(d18:1/18:0), and Cer(d18:1/24:1) levels and their ratios to Cer(d18:1/24:0), in 123 AMI patients (FTGM coronary unit, Massa, Italy). Results: Cer(d18:1/16:0): higher in female patients (<0.05), in patients with dyslipidemia (<0.05), and it directly and significantly correlated with aging, brain natriuretic peptide-BNP, erythrocyte sedimentation rate-ESR and fibrinogen. Cer(d18:1/18:0): higher in females (<0.01) and patients with dyslipidemia (<0.01), and increased according to the number of CV risk factors (considering hypertension, dyslipidemia and diabetes). Moreover, it significantly correlated with BNP, troponin at admission, ESR, C reactive protein-CRP, and fibrinogen. Cer(d18:1/24:1): significantly correlated with aging, BNP, fibrinogen and neutrophils. Cer(d18:1/16:0)/Cer(d18:1/24:0): higher in female patients (<0.05), and in patients with higher wall motion score index-WMSI (>1.7; ≤0.05), and in those with multivessel disease (<0.05). Moreover, it significantly correlated with aging, BNP, CRP, ESR, neutrophil-to-lymphocyte ratio-NRL, and fibrinogen. Cer(d18:1/18:0)/Cer(d18:1/24:0): higher in female patients (<0.001), and increased according to age. Moreover, it was higher in patients with lower left ventricular ejection fraction (<35%, ≤0.01), higher WMSI (>1.7, <0.05), and in those with multivessel disease (0.13 ± 0.06 vs. 0.10 ± 0.05 µM, <0.05), and correlates with BNP, ESR, CRP, fibrinogen and neutrophils, platelets, NLR, and troponin at admission. Multiple regression analysis showed that Cer(d18:1/16:0)/Cer(d18:1/24:0) and Cer(d18:1/18:0)/Cer(d18:1/24:0) remained as independent determinants for WMSI after multivariate adjustment (Std coeff 0.17, T-value 1.9, ≤0.05; 0.21, 2.6, <0.05, respectively). Conclusion: Distinct ceramide species are associated with CV risk, inflammation and disease severity in AMI. Thus, a detailed analysis of ceramides may help to better understand CV pathobiology and suggest these new biomarkers as possible risk predictors and pharmacological targets in AMI patients.

Harmonization of two hs-cTnI methods based on recalibration of measured quality control and clinical samples.

BACKGROUND: The study aim was to evaluate whether is possible to harmonize the results of two hs-cTnI methods using a recalibration procedure based on linear regression models and measured values of external quality assessment (EQA) and clinical samples. METHODS: A two-step experimental approach was used. The preliminary step was performed using 16 EQA samples. The harmonization procedure was then validated by using 2530 heparinized plasma samples collected by 12 Italian University and Regional Hospitals from apparently healthy volunteers and patients admitted to emergency department with cardiac diseases. Two hs-cTnI methods were tested: Architect Stat High Sensitive Troponin-I, and the Access hs-cTnI using DxI platform. Linear regression models based on mean values measured with the two hs-cTni methods were used. RESULTS: A significant reduction in the measurement bias between the two methods was found after recalibration procedure. The agreement between-methods improved of about 2.5 folds after recalibration, as assessed by reduction in mean CV values from 38.4% (SD 25.9%) before recalibration to 15.0% (SD 10.6%) after recalibration for hs-cTnI values ≤ 500 ng/L (n = 13, P = 0.0111 by non-parametric test for paired data). CONCLUSIONS: A recalibration procedure based on means of measured concentrations with hs-cTnI methods, which use monoclonal antibodies with similar binding characteristics, can be used to significantly reduce systematic bias and so to improve harmonization between methods. The results of this study can aid laboratorians and clinicians to better compare the concentrations respectively measured with the Architect and Access hs-cTnI methods.

New inflammatory and oxidative stress-based biomarker changes in response to a half-marathon in recreational athletes.

BACKGROUND: Modulation of oxidative stress/inflammation during exercise may have both positive and negative health effects, depending by a number of factors (e.g. training status, and exercise type, intensity and duration) and the oxidative stress or inflammation-related biomarkers considered, which may reflect different levels of the oxidative stress/inflammatory multi entities. The aim of this study was to evaluate oxidative stress and inflammatory multi-biomarker panel in response to a half-marathon during early and delayed recovery. METHODS: Blood samples (baseline, postrace within 20 min after the race end, and 24 h and 48 h after the run) from runners (N.=31, 20 males, mean age 47±6 years) were assessed for reactive oxygen species (ROM assay) and total antioxidant capacity (OXY test), leukocyte telomere length (LTL), procoagulant activity of circulating microparticles (MP-PCA), inflammatory parameters obtained by hemocrome, and irisin. RESULTS: A significant decrease for OXY (from 375±71 to 280±66, 239±54, 239±45 µmolHClO/mL) after the half-marathon and during recovery was observed. A reduction for ROMs was also evidenced respect to baseline (from 328±46 to 301±39, 290±56, 320±55 AU). Instead, MP-PCA increased after the race (from 6.2±6 to 10.5±6, 7±4.3 and 5.8±2.1 nmol/L), whereas the other biomarkers did not significantly change. CONCLUSIONS: The oxidant counterpart did not increase in response to the half-marathon, likely counteracted by antioxidants, which appeared greatly worn out. MP-PCA and WBC increase, always within the normality range, may represent an adaptation to regular chronic endurance training. In any case, antioxidant supply could be considered and tailored for each athlete in this exercise setting.

Evaluation of analytical performance of immunoassay methods for cTnI and cTnT: From theory to practice.

Current guidelines worldwide recommend cardiac troponins I (cTnI) and T (cTnT) as the biomarkers of choice for the differential diagnosis of acute coronary syndrome (ACS), and the measurement of the 99th upper reference population limit (URL) value for cardiac troponins, with an imprecision of ≤10 CV%. Measuring the 99th URL of cTnI and cTnT is a challenging analytical task due to low biomarker concentrations present in healthy subjects. Therefore, since the year 2006, several manufacturers have established new generation cTnI and cTnT immunoassays with an improved analytical sensitivity in accordance with the quality specifications described in international guidelines, the more recent of which state that only immunoassays that meet the required quality specifications should be considered "high-sensitivity" methods. For the early diagnosis of ACS, and for the stratification of cardiovascular risk in cardiac patients and the general population, high-sensitivity methods should be employed. It is therefore important for laboratory professionals and clinicians to gain a thorough understanding of the analytical performances of immunoassay methods for cTnI and cTnT, especially at low to normal concentration ranges. The aim of the present study was to analyze critical aspects related to definition, analytical performance, pathophysiological interpretations, and the clinical relevance of high-sensitivity cardiac troponin assays.

Emerging Biomarkers of Oxidative Stress in Acute and Stable Coronary Artery Disease: Levels and Determinants.

BACKGROUND: Oxidative stress is crucial in the pathogenesis of atherosclerosis and acute myocardial infarction (AMI). Under the generic terms "oxidative stress" (OS), many biomarkers belonging to different pathways have been proposed. AIM: To compare the levels of recently proposed OS-related parameters in acute coronary syndromes (ACS) and stable coronary artery disease (CAD), to evaluate their effectiveness as additive risk or illness indicators of stable and acute ischemic events, and their response over time during the course of AMI. METHODS: 76 ACS, 77 CAD patients, and 63 controls were enrolled in the study. Different OS-related biomarkers, including reactive oxygen metabolites (ROM), the total antioxidant capacity (OXY), nitrite/nitrate (final nitric oxide products, NOx), and Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), were evaluated. Moreover, time response during AMI course (admission, and 6, 12, 18, 24, 36, and 48 hours after, T0-T6, respectively) and correlation with traditional cardiovascular (CV) risk factors (age, gender, hypertension, diabetes mellitus, dyslipidemia, smoking habit) were also assessed. RESULTS: Over time, ROM progressively increased while OXY and NOx decreased. Kinetics of LOX-1 during AMI shows that this biomarker boosts early during the acute event (T1 and T2) and then progressively decreases, being significantly lower from T0 to T6. Different OS-related biomarkers were differentially associated with CV risk factors and CAD or ACS presence. CONCLUSION: Differences in OS-related biomarkers (between groups, according to the response over time during AMI, and to the presence of CV risk factors) confirmed OS involvement in the transition from healthy status to stable CAD and ACS, although evidencing the heterogeneous nature of redox processes. In future, a multi-marker panel including different biomarkers and pathways of oxidative stress could be evaluated as an additive tool to be used in the CV prevention, diagnosis, patient stratification, and treatment.

Usefulness of Triiodothyronine Replacement Therapy in Patients With ST Elevation Myocardial Infarction and Borderline/Reduced Triiodothyronine Levels (from the THIRST Study).

The aim of the study was to investigate whether TH replacement therapy is safe and impact infarct size, left ventricular (LV) volumes and function in patients with acute myocardial infarction (AMI) and low T3 syndrome (LT3S). Thirty-seven AMI/LT3S patients were randomly treated or untreated with liothyronine (T3) therapy (maximum dosage 15 mcg/m2/die) in addition to standardized treatment (T3-treated group, n = 19; untreated group, n = 18). TH and thyroxine (TSH) during hospital stay and at 1-month and 6 months were evaluated. At discharge and at 6 months LV volumes, ejection fraction, wall motion score index (WMSI) and infarct extent were measured by cardiac MR. T3-treated patients had a significant increase in fT3 (p = 0.003 and p <0.001) at discharge and 1-month. These patients had no signs or symptoms of hyperthyroidism or arrhythmias. At follow-up, there was a significant reduction in WMSI in both groups (T3-treated group: Δ = -0.12, p = 0.001; untreated group: Δ = -0.04, p = 0.04) and the difference value (discharge/follow-up) was significantly higher in T3-treated group than in untreated group (mean difference between groups = 0.08, 95% confidence interval [CI]: 0.01 to 0.15, p = 0.05). Also, stroke volume increased significantly in the T3-treated group (Δ = 3.4, 95% CI: 0.8 to 6, p <0.01) at follow-up. In conclusion, this is the first pilot experience in which T3 replacement therapy resulted safe and able to improve regional dysfunction in patients with STEMI/LT3S.

Evaluation of analytical performances using standardized analytical protocols and comparison of clinical results of the new ADVIA BNP and NT-proBNP immunoassays for the Centaur XPT platform.

Background The study aim was to evaluate and compare analytical performances and clinical results of ADVIA BNP and PBNP methods using the Centaur XPT platform with those of Access BNP, using the DxI platform and the ECLIA NT-proBNP method, using the Cobas e411 platform, respectively. Methods Limits of blank (LoB), detection (LoD) and quantitation (LoQ) at 20% CV and 10% CV were evaluated according to international standardized protocols. The analytical parameters were assessed throughout a 90-working-day period using three curve calibrations. Results LoB, LoD and LoQ at 20% CV and 10% values of the ADVIA BNP method were 1.0 ng/L, 2.0 ng/L, 3.7 ng/L and 10.2 ng/L, respectively; while those of the ADVIA PBNP method were 1.3 ng/L, 3.0 ng/L, 9.7 ng/L and 22.3 ng/L, respectively. The ADVIA BNP and PBNP methods were able to measure the clinical decision values suggested by international guidelines for diagnosis of heart failure (HF) with an imprecision ≤6%. BNP concentrations measured with the ADVIA and Access methods showed a close linear regression (R=0.9923, n=200); a close linear regression was also found between NT-proBNP concentrations measured with the ADVIA and ECLIA methods (R=0.9954, n=202). However, the ADVIA method measured significantly lower BNP values than the Access method (on average -20.9%), while ADVIA PBNP method measured significantly higher NT-proBNP concentrations than the ECLIA method (on average +17.8%). Conclusions Analytical performances of the BNP and PBNP ADVIA methods are well in accordance with the quality specifications required by international guidelines for diagnosis and follow-up of patients with HF.

Relationship between uric acid levels and cardiometabolic findings in a large cohort of ß-thalassemia major patients.

AIM: to evaluate the relationship between uric acid (UA), hepatic and cardiac iron overload (T2*-MRI), ferritin, endocrinological diseases and cardiac complications in a large thalassemia major (TM) cohort. METHODS: A total of 369 TM patients (187 men; 33 ± 6 years) were retrospectively studied, from the myocardial iron overload in thalassemia (MIOT) electronic databank. RESULTS: Multiple regression model identified male sex (p < 0.001), BMI (p < 0.001) and T2* (p ≤ 0.001) as UA independent correlates. Moreover, UA and derivatives of reactive oxygen species (an oxidative index; r = -0.3; p ≤ 0.05) are inversely correlated. Conversely, the multivariate logistic analysis identified low UA (NANHES-III criteria) as one independent predictor for low global heart T2* (p < 0.5) together with liver iron concentrations (>3 mg/g/dw), heart failure, endocrinopathies, ferritin (>2000 ng/l), alanine transaminase (>40 UI/l) and/or aspartate transaminase (>35 UI/l) and/or glutamyl transferase (>64 UI/l). DISCUSSION: UA appears directly associated to T2* and inversely with derivatives of reactive oxygen species, and as such reduced according to increased oxidative stress and cardiac iron overload in TM patients.

Angiopoietin-2: a biomarker in hyperthyroidism.

Serum angiopoietin-2 level is elevated in several diseases suggesting its possible role as a mediator of angiogenesis and vascular network remodeling. Triiodothyronine and thyroxine have well documented effects on angiogenesis in vitro, but only few reports have studied angiopoietin-2 in thyroid-disease patients. The aim of the present study was to measure soluble angiopoietin-2 serum levels in a group of thyroid-disease patients with different levels of free triiodothyronine and thyroxine. Angiopoietin- 2 were quantified by ELISA in sera of fifteen healthy volunteers and forty-two thyroid ambulatory patients: nine with hyperthyroidism, four in therapy for hyperthyroidism, seven with subclinal hyperthyroidism, twelve with hypothyroidism, five with thyroiditis and five in therapy for thyroiditis. Median angiopoietin-2 level was significantly elevated in hyperthyroid patients (p < 0.01) and it was significantly increased vs all the other groups (p < 0.0001). In hyperthyroid patients anti thyroid therapy seems to reduce angiopoietin-2 level. A significant positive correlation was observed between Log angiopoietin-2 levels and serum concentration of Log free triiodothyronine (r = 0.4, P < 0.001) and Log free thyroxine (r = 0.4, P < 0.001) respectively. In conclusion, increased levels of angiopoietin-2 are present in hyperthyroid patients, and seems to correlate with free triiodothyronine and free thyroxine levels but not with anti-thyroid antibodies. These findings suggest angiopoietin-2 as a mediator of angiogenesis and vascular network remodeling in this disease, but further studies will be needed to determine the role of this biomarker in the pathophysiology and progression of hyperthyroidism.

Evaluation of analytical performance of a new high-sensitivity immunoassay for cardiac troponin I.

BACKGROUND: The study aim was to evaluate and compare the analytical performance of the new chemiluminescent immunoassay for cardiac troponin I (cTnI), called Access hs-TnI using DxI platform, with those of Access AccuTnI+3 method, and high-sensitivity (hs) cTnI method for ARCHITECT platform. METHODS: The limits of blank (LoB), detection (LoD) and quantitation (LoQ) at 10% and 20% CV were evaluated according to international standardized protocols. For the evaluation of analytical performance and comparison of cTnI results, both heparinized plasma samples, collected from healthy subjects and patients with cardiac diseases, and quality control samples distributed in external quality assessment programs were used. RESULTS: LoB, LoD and LoQ at 20% and 10% CV values of the Access hs-cTnI method were 0.6, 1.3, 2.1 and 5.3 ng/L, respectively. Access hs-cTnI method showed analytical performance significantly better than that of Access AccuTnI+3 method and similar results to those of hs ARCHITECT cTnI method. Moreover, the cTnI concentrations measured with Access hs-cTnI method showed close linear regressions with both Access AccuTnI+3 and ARCHITECT hs-cTnI methods, although there were systematic differences between these methods. There was no difference between cTnI values measured by Access hs-cTnI in heparinized plasma and serum samples, whereas there was a significant difference between cTnI values, respectively measured in EDTA and heparin plasma samples. CONCLUSIONS: Access hs-cTnI has analytical sensitivity parameters significantly improved compared to Access AccuTnI+3 method and is similar to those of the high-sensitivity method using ARCHITECT platform.

Reassessment of the Access Testosterone chemiluminescence assay and comparison with LC-MS method.

AIM OF THE STUDY: To reassess the imprecision and Limit of Quantitation, to evaluate the cross-reaction with dehydroepiandrosterone-sulfate (DHEAS), the accuracy toward liquid chromatography-mass spectrometry (LC-MS) and the reference interval of the Access Testosterone method, performed by DxI immunoassay platform (Beckman Coulter). MATERIAL AND METHODS: Imprecision was evaluated testing six pool samples assayed in 20 different run using two reagents lots. The cross-reaction with DHEAS was studied both by a displacement curve and by spiking DHEAS standard in two serum samples with known amount of testosterone. The comparison with LC-MS was evaluated by Passing-Bablock analysis in 21 routine serum samples and 19 control samples from an External Quality Assurance (EQA) scheme. The reference interval was verified by an indirect estimation on 2445 male and 2838 female outpatients. RESULTS: The imprecision study showed a coefficient of variation (CV) between 2.7% and 34.7% for serum pools from 16.3 and 0.27 nmol/L. The value of Limit of Quantitation at 20% CV was 0.53 nmol/L. The DHEAS showed a cross-reaction of 0.0074%. A comparison with LC-MS showed a trend toward a slight underestimation of immunoassay vs LC-MS (Passing-Bablock equations: DxI=-0.24+0.906 LCMS in serum samples and DxI=-0.299+0.981 LCMS in EQA samples). The verification of reference interval showed a 2.5th-97.5th percentile distribution of 6.6-24.3 nmol/L for male over 14 years and <0.5-2.78 nmol/L for female subjects, in accord with the reference intervals reported by the manufacturer. CONCLUSIONS: The Access Testosterone method could be considered an adequately reliable tool for the testosterone measurement.