Silvia Masotti

The calculation of the cardiac troponin T 99th percentile of the reference population is affected by age, gender, and population selection: A multicenter study in Italy

BACKGROUND The aim of this study is to determine the 99th upper-reference limit (URL) for cardiac troponin T (cTnT) in Italian apparently healthy subjects. METHODS 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). RESULTS We enrolled 1600 healthy subjects [54.6% males; age range 10-90years; mean (SD): 36.4 (21.2) years], including 34.6% aged <20years, 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 (P<0.05) 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, P<0.05). CONCLUSIONS 99th URL of cTnT values was strongly affected by age, gender, selection of subjects and the statistical evaluation of outliers.

Systematic differences between BNP immunoassays: Comparison of methods using standard protocols and quality control materials

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.

State of the art of immunoassay methods for B-type natriuretic peptides: An update

Abstract 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.

Evaluation of analytical performance and comparison of clinical results of the new generation method AccuTnI + 3 for the measurement of cardiac troponin I using both patients and quality control plasma samples

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.

The 99th percentile of reference population for cTnI and cTnT assay: methodology, pathophysiology and clinical implications.

Abstract According to recent international guidelines, including the 2012 Third Universal Definiton of Myocardial Infarction by the Joint ESC/ACCF/AHA/WHF Task Force, an increase in cardiac troponin (cTn) levels over the 99th percentile upper reference limit (99th URL) should be considered clinically relevant, this cut-off being measured with an imprecision ≤10 CV%. In theory 99th URL values strongly depend not only on demographic and physiological variables (i.e. criteria for considering the reference population “healthy”), but also on the analytical performance of cTn methods and mathematical algorithms used for the calculation. The aim of the present article was therefore to review the methodological and pathophysiological factors affecting the evaluation and calculation of the 99th URL for cTn assay. The critical analysis made showed that no uniform procedure is followed, and nor have experts or regulatory bodies provided uniform guidelines for researchers or cTn assays manufacturers as an aid in “their quest to define normality”. In particular, little attention has been paid to the way in which a healthy reference population is to be selected, or the criteria for calculating the 99th URL value for cTn assays, thus highlighting the need for international recommendations not only for demographic and physiological variables criteria for defining a healthy reference population, but also for calculating mathematical algorithms for establishing/calculating clinical decision values. An expert consensus group, comprising laboratory and clinical scientists, biomedical statisticians, industrial and regulatory representatives, should be responsible for drawing up these guidelines.

Plasma cardiac troponin I concentrations in healthy neonates, children and adolescents measured with a high sensitive immunoassay method: High sensitive troponin I in pediatric age.

Over the past 10years cardiac troponin (cTn) immunoassays have been improved in analytical sensitivity and precision thereby allowing the measurement of cTn in adult healthy subjects. However, there are currently substantial gaps in our knowledge on circulating levels of cTn in healthy children. The aim of this study is to evaluate the distribution of plasma troponin concentration in apparently healthy pediatric subjects using a high sensitive immunoassay for cTnI measurement (hs-cTnI). Blood samples were obtained from 357 healthy pediatric subjects [204 males; age range 0-18years; mean (SD): 8.7(6) years], including 36 subjects aged <1month (neonates), 57 between 1 and 12months (infants), 65 between 1 and 10years (toddlers), and 223 between 10 and 18years (adolescents). The percentages of healthy population with cTnI values equal or less than the calculated and LOD value were 13.1%. cTnI plasma levels were highest in the first month of life with a progressive decline in the next years and were lower in female. At multivariate analysis, only age was predictor of hs-cTnI plasma levels. The age and sex of children influence normal and physiologically released circulating concentrations of hs-cTnI, suggesting the need of reference intervals specific for age and sex.

Monocytes/macrophages activation contributes to b-gamma-glutamyltransferase accumulation inside atherosclerotic plaques

BackgroundGamma-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.MethodsGGT 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.ResultsWe 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.ConclusionsOur 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.

Comparison between BNP values measured in capillary blood samples with a POCT method and those measured in plasma venous samples with an automated platform.

*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

Harmonization protocols for TSH immunoassays: a multicenter study in Italy.

Abstract Background: Systematic difference between thyroid-stimulating hormone (TSH) immunoassays may produce misleading interpretation when samples of the same patients are measured with different methods. The study aims were to evaluate whether systematic differences are present among TSH immunoassays, and whether it is possible to obtain a better harmonization among TSH methods using results obtained in external quality assessment (EQA) schemes. Methods: Seven Italian clinical laboratories measured TSH in 745 serum samples of healthy subjects and patients with thyroid disorders. These samples were also re-measured by two reference laboratories of the study with the six TSH immunoassays most popular in Italy after 2 months of storage at −80 °C. Moreover, these data were compared to 53,823 TSH measurements, obtained by laboratories participant to 2012–2015 EQA annual cycles in 72 quality control samples (TSH concentrations from about 0.1 mIU/L to 18.0 mIU/L). TSH concentrations were recalibrated using a mathematical approach based on the principal component analysis (PCA). Results: Systematic differences were found between the most popular commercially available TSH immunoassays. TSH concentrations measured by the clinical laboratories were very closely correlated to those measured with the same method by reference laboratories after 2 months of storage at −80 °C. After recalibration using the PCA approach the variation of TSH values significantly decreased from a median pre-calibration value of 13.53% (10.79%–16.53%) to 9.63% (6.90%–13.21%) after recalibration. Conclusions: Our data suggest that EQA schemes are useful to improve harmonization among TSH immunoassays and also to produce some mathematical formulas, which can be used by clinicians to better compare TSH values measured with different methods.

Clinical implications of a recent adjustment to the high-sensitivity cardiac troponin T assay: some results.

*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