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Background The aim of this study is to evaluate whether it is possible to reduce the between-methods variability of troponin I (cTnI) immunoassays using mathematical algorithms calculated from the results of both patients' samples and quality control materials distributed in an external quality assessment (EQA) scheme. Methods We collected 122 heparinized plasma samples of patients admitted to the emergency department with thoracic pain or supraventricular tachyarrhythmia. Moreover, we also analyzed 20 control samples distributed in an EQA and 26 plasma pools prepared from healthy subjects and patients with myocardial infarction. We evaluated 4 different methods for cTnI assay: STAT Architect High Sensitive TnI (Abbott Diagnostics), ADVIA Centaur Troponin I Ultra (Siemens Healthcare Diagnostics), ST AIA-Pack cTnI Third Generation (Tosoh Bioscience), and Access AccuTnI + 3 (Beckman Coulter Diagnostics). Results Systematic differences between cTnI methods were observed. However, correlation coefficients (R from 0.976 to 0.990) between the log-transformed cTnI values measured in all 168 samples were significantly better (p = 0.0037) than those obtained considering only the 122 patients' samples. cTnI values measured in EQA and pool samples were included within the 95% prediction intervals of linear regressions calculated with those of patients' samples. After the recalibration of cTnI values based on the robust principal component analysis approach the between-methods variability decreased significantly (about 40% around the cut off values). Conclusions Our pilot study suggests that EQA schemes for cTnI immunoassay methods, based on both quality control samples with tested commutability and robust statistical analyses, are able to evaluate between-methods variability as well as allow a reliable recalibration and harmonization of results.

Acute myocardial infarction (AMI) results in myocardial damage and increased risk of cardiac dysfunction, despite advances in treatment. Sympathetic renal denervation (RD) might represent an innovative approach to reduce adverse adrenergic activation after AMI, increase myocardial salvage and

Background Gamma-glutamyltransferase (GGT) is a well-established independent risk factor for cardiovascular mortality related to atherosclerotic disease. Four GGT fractions have been identified in plasma, but only b-GGT fraction accumulates in atherosclerotic plaques, and correlates with other histological markers of vulnerability. The present study was aimed to evaluate whether macrophagic lineage cells may provide a source of b-GGT within the atherosclerotic plaque. Methods GGT expression and release were studied in human monocytes isolated from peripheral blood of healthy donors. The growth factors GM-CSF and M-CSF were used to induce differentiation into M1-like and M2-like macrophages, respectively. Plaque GGT was investigated in tissue samples obtained from patients undergoing carotid endoarterectomy. Results We found that M1-like macrophages express higher levels of GGT as compared to M2-like, and that both monocytes and M1-like macrophages—but not M2-like—are able to release the b-GGT fraction upon activation with pro-inflammatory stimuli. Western blot analysis of b-GGT extracted from plaques confirmed the presence of a GGT immunoreactive peptide coincident with that of macrophages. Conclusions Our data indicate that macrophages characterized by a pro-inflammatory phenotype may contribute to intra-plaque accumulation of b-GGT, which in turn may play a role in the progression of atherosclerosis by modulating inflammatory processes and favouring plaque instability.

The aim of this review article is to give an update on the state of the art of the immunoassay methods for the measurement of B-type natriuretic peptide (BNP) and its related peptides. Using chromatographic procedures, several studies reported an increasing number of circulating peptides related to BNP in human plasma of patients with heart failure. These peptides may have reduced or even no biological activity. Furthermore, other studies have suggested that, using immunoassays that are considered specific for BNP, the precursor of the peptide hormone, proBNP, constitutes a major portion of the peptide measured in plasma of patients with heart failure. Because BNP immunoassay methods show large (up to 50%) systematic differences in values, the use of identical decision values for all immunoassay methods, as suggested by the most recent international guidelines, seems unreasonable. Since proBNP significantly cross-reacts with all commercial immunoassay methods considered specific for BNP, manufacturers should test and clearly declare the degree of cross-reactivity of glycosylated and non-glycosylated proBNP in their BNP immunoassay methods. Clinicians should take into account that there are large systematic differences between methods when they compare results from different laboratories that use different BNP immunoassays. On the other hand, clinical laboratories should take part in external quality assessment (EQA) programs to evaluate the bias of their method in comparison to other BNP methods. Finally, the authors believe that the development of more specific methods for the active peptide, BNP1-32, should reduce the systematic differences between methods and result in better harmonization of results.

The study aims are to evaluate the analytical performance and the clinical results of the chemiluminescent Access AccuTnI+3 immunoassay for the determination of cardiac troponin I (cTnI) with DxI 800 and Access2 platforms and to compare the clinical results obtained with this method with those of three cTnI immunoassays, recently introduced in the European market. The limits of blank (LoB), detection (LoD), and quantitation (LoQ) at 20% CV and 10% CV were 4.5 ng/L and 10.9 ng/L, 17.1 and 30.4 ng/L, respectively. The results of STAT Architect high Sensitive TnI (Abbott Diagnostics), ADVIA Centaur Troponin I Ultra (Siemens Healthcare Diagnostics), ST AIA-Pack cTnI third generation (Tosoh Bioscience), and Access AccuTnI+3 (Beckman Coulter Diagnostics) showed very close correlations (R ranging from 0.901 to 0.994) in 122 samples of patients admitted to the emergency department. However, on average there was a difference up to 2.4-fold between the method measuring the highest (ADVIA method) and lowest cTnI values (AccuTnI+3 method). The consensus mean values between methods ranged from 6.2% to 29.6% in 18 quality control samples distributed in an external quality control study (cTnI concentrations ranging from 29.3 ng/L to 1557.5 ng/L). In conclusion, the results of our analytical evaluation concerning the AccuTnI+3 method, using the DxI platform, are well in agreement with those suggested by the manufacturer as well as those reported by some recent studies using the Access2 platform. Our results confirm that the AccuTnI+3 method for the Access2 and DxI 800 platforms is a clinically usable method for cTnI measurement.

*Corresponding author: Prof. Aldo Clerico, MD, Department of Laboratory Medicine, Fondazione CNR-Regione Toscana G. Monasterio, Scuola Superiore Sant ’ Anna, Via Moruzzi 1, 56126 Pisa, Italy, Phone: + 39 0585 493569, Fax: + 39 0585 493601, E-mail: clerico@ftgm.it ; and Scuola Superiore Sant ’ Anna, Pisa, Italy Concetta Prontera, Michele Emdin and Claudio Passino: Fondazione CNR-Regione Toscana G. Monasterio, Pisa, Italy Silvia Masotti and Maria Franzini: Scuola Superiore Sant ’ Anna, Pisa, Italy Gian Carlo Zucchelli: QualiMedLab Spin-Off dell ’ Istituto di Fisiologia Clinica del CNR, Pisa, Italy Letter to the Editor

The aim of this study is to determine the 99th upper-reference limit (URL) for cardiac troponin T (cTnT) in Italian apparently healthy subjects.The reference population was selected from 5 cities: Bolzano (n=290), Milano (CAMELIA-Study, n=287), Montignoso (MEHLP-Study, n=306), Pisa (n=182), and Reggio Calabria (MAREA-Study, n=535). Subjects having cardiac/systemic acute/chronic diseases were excluded. Participants to MEHLP project underwent cardiac imaging investigation. High-sensitive cTnT was measured with Cobas-e411 (Roche Diagnostics).We enrolled 1600 healthy subjects [54.6% males; age range 10-90years; mean (SD): 36.4 (21.2) years], including 34.6% aged20years, 54.5% between 20 and 64years, and 10.9% over 65years. In the youngest the 99th URL was 10.9ng/L in males and 6.8ng/L in females; in adults 23.2ng/L and 10.2ng/L; and in elderly 36.8ng/L and 28.6ng/L. After the exclusion of outliers the 99th URL values were significantly decreased (P0.05) in particular those of the oldest (13.8ng/L and 14ng/L). MEHLP participants were divided in healthy and asymptomatic, according to known cardiovascular risk factors (HDL, LDL, glucose, C-reactive protein): the 99th URL of cTnT values of these subgroups was significantly different (19.5 vs. 22.7, P0.05).99th URL of cTnT values was strongly affected by age, gender, selection of subjects and the statistical evaluation of outliers.

Background Two new immunoassay methods for aldosterone assay using automated platforms recently became available into market. The main aim of the present study is to evaluate the analytical performance of these automated direct immunoassay methods, and also to compare their analytical characteristics to those of the most popular RIA and EIA methods used in an Italian External Quality Assessment (EQA) study. Methods In this study analytical performances of two aldosterone immunoassays using the IDS iSYS and DiaSorin LIAISON fully automated platforms, were evaluated. Results obtained with the two platforms in EDTA plasma samples of healthy subjects and patients were compared with those obtained by RIA and EIA methods used in the Italian EQA scheme, named Immunocheck study. Results The two automated methods showed similar analytical performances: LoD 83.9 vs 92.2 pmol/L, LoQ 104.4 vs 111.1 pmol/L, respectively; moreover, the within-run and total imprecision values showed CV% between 8.1 and 14.1 for samples with 180.8 and 387.2 pmol/L concentration for both methods. There was a close linear regression between methods, however we found a significant proportional bias between LIAISON and iSYS methods. The EQA samples results obtained with these two methods were highly correlated to the consensus mean values. Conclusions Our data indicate that aldosterone values measured with the two automated methods actually show better reproducibility, shorter laboratory Turn Around Time (TAT) and require less “hands on labor” compared to RIA and EIA immunoassays. However, in our study significant bias was observed in result comparison, this means that translating aldosterone concentration in clinical information an appropriate definition of reference ranges for each method is mandatory.

*Corresponding author: Maria Franzini, Scuola Superiore Sant’ Anna, Pisa, Italy; and Departments of Laboratory and Cardiovascular Medicine, Fondazione Regione Toscana G. Monasterio, Via Moruzzi 1, 56124 Pisa, Italy, Phone: +39-50-3153309, Fax: +39-50-3152166, E-mail: franzinimaria@gmail.com; m.franzini@sssup.it Silvia Masotti, Claudio Passino and Aldo Clerico: Scuola Superiore Sant’ Anna, Pisa, Italy; and Departments of Laboratory and Cardiovascular Medicine, Fondazione Regione Toscana G. Monasterio, Pisa, Italy Concetta Prontera: Departments of Laboratory and Cardiovascular Medicine, Fondazione Regione Toscana G. Monasterio, Pisa, Italy

Background Recent studies suggested that there are marked systematic differences among BNP immunoassays. In this study we compared the BNP data and clinical results obtained with different immunoassays, including a new method (ST-AIA-PACK, TOSOH Corporation). Methods BNP was measured on plasma-EDTA samples of healthy subjects (HS, n = 126) and patients with heart failure (HF, n = 31 NYHA I, II; n = 46 NYHA III, IV) using the ST-AIA-PACK and the Triage Biosite (Beckman Coulter) methods. Control samples distributed in the CardioOrmoCheck external quality assessment were also measured with TOSOH and the most used BNP immunoassays in Italy. Results TOSOH method showed a good correlation (R = 0.976; n = 327) but a mean bias (− 46.9%) compared to Triage Biosite. On the base of the results obtained in 10 samples of the CardioOrmoCheck study, TOSOH method showed a strict agreement with ADVIA Centaur, while it underestimated BNP in comparison with Triage (− 52.5%) and ARCHITECT methods (− 39.4%). The agreement of ST-AIA-PACK and Triage Biosite methods for classification of HF patients was tested using 100 ng/L of BNP; the positive agreement between methods was 65%, overall agreement was 73%. Conclusions Our results confirm that there are marked differences in measured values among commercial methods for BNP assay.

Reverse remodeling (R2) in non-ischemic dilated cardiomyopathy (NIDCM) is associated with reduced morbidity, improved quality of life and survival. There is still scarce knowledge on predictors of R2, relevant for guide to treatment and device implantation. We aimed to identify predictors of R2