Background: We organized and conducted a proficiency testing study (CardioOrmocheck) to evaluate the differences in analytical performance of brain natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) immunoassays.Methods: Approximately 90 Italian laboratories were involved in the 2005-2007 proficiency testing cycles, while 112 laboratories took part in the 2008 cycle (from January to May 2008). A total of 28 study samples were measured by participating laboratories for a total of 2354 determinations.Results: The mean total variability for BNP (50.6%CV) was significantly higher than that for NT-proBNP (8.4%CV). In addition, the mean variability due to differences between-methods (46.4%CV) comprised the majority of the total variability for BNP. Between-method variability for BNP comprised, on average, 84% of total variability, while the within-method variability comprised an average of 20.2%CV. On the contrary, for NT-proBNP the within-method variability (7.3%CV) represented the majority of total variability (average 75%), while between-method variability was smaller (4.1%CV). Imprecision around the cut-off value showed marked differences among methods, especially for BNP immunoassay methods. In addition, BNP methods were affected by large systematic differences, for example an average 2.7-fold difference between Access and ADVIA Centaur methods, while agreement between NT-proBNP methods was better (an average 1.2-fold difference between Dimension and ECLIA on the Elecsys methods).Conclusions: This multicenter collaborative study demonstrates that there are significant differences in analytical characteristics and measured values among the most popular commercial methods for BNP and NT-proBNP. Clinicians should be very careful when comparing results obtained by laboratories that use different methods. Clin Chem Lab Med 2009;47:762-8.

The clinical relevance of brain natriuretic peptide ( BNP) and N-terminal ( NT)-proBNP assays as a diagnostic tool and prognostic marker in patients with cardiovascular diseases has recently been confirmed. However, several studies demonstrated variation of intra-individual BNP concentrations of >30% ( ranging from 30% to 50%) with reference change values at the 95% confidence interval ( i.e., the estimated critical difference) ranging from 99% to 130% in healthy subjects and heart failure patients. According to this estimated confidence interval, only a great variation in plasma BNP levels should be considered significant in an individual patient ( for example, a decrease of >50% or an increase of more than two-fold). Many recent clinical studies have demonstrated that BNP variations below this estimated critical difference could also have clinical relevance. Like the concentration of other neuro-hormones, levels of plasma BNP fluctuate widely and rapidly along with heart rhythm and blood pressure variations in response to physiological stimuli. However, biological variation of BNP should not be interpreted strictly as random fluctuation around a homeostatic set point, as assumed by the common model used in all studies on biological variation of BNP reported in the literature. These results cannot be directly transferred to clinical practice. While awaiting more accurate studies, we suggest that variations of plasma BNP three-fold greater than the analytical imprecision should be considered as potentially relevant from a physiological and clinical point of view.

The cardiac natriuretic hormones (CNHs), atrial (ANP) and B-type natriuretic peptide (BNP), are peptides produced and secreted by the atrial and ventricular cardiomyocytes, respectively. ANP and BNP share potent diuretic, natriuretic, and vascular smooth-muscle-relaxing effects and interact with the hormonal and nervous systems (1)(2)(3). Stimulation by hemodynamic overload, as well as by several neuroendocrine (including α-adrenergic agonists, angiotensin II, endothelins, and vasopressin) and immunologic agents and by growth factors, modulates the production of CNHs by the human heart (1)(2). CNHs and their respective counter-regulatory neurohormonal systems (including the adrenergic, endothelin, and renin-angiotensin-aldosterone systems) form a physiologic negative feedback mechanism that regulates cardiac output by controlling blood pressure and heart rate as well as by fluid and electrolyte homeostasis (1)(2). The clinical relevance of measurement of BNP and its related peptides as diagnostic tools and prognostic markers in patients with cardiovascular diseases has been confirmed recently (2)(3). To achieve correct interpretation of serial test results that are collected for follow-up or for tailored treatment of heart …

Here, we reported the results collected in the 2002 cycle of the “Serocheck” External Quality Assessment (EQA) scheme for the detection of antibodies against the hepatitis C virus (anti-HCV). This program, conducted by the Institute of Clinical Physiology, CNR, Pisa in co-operation with Polymed (Firenze, Italy), involved about 80 among microbiology, transfusional and general Italian laboratories. Methods. – The reference panel sent to the laboratories consisted of 12 samples. Three series of samples, from a pool of donors, were negative for anti-HCV. The positive panel, obtained by different dilutions of positive pools with the negative one, was constituted by two samples with concentration higher than 10× cut-off (A), two samples between 4× cut-off and 10× cut-off (B), four samples between 2× cut-off and 4× cut-off (C) and one sample borderline (1.22× cut-off) (D). Results. – On the whole number of serum samples, the percentage of correct results was 95% (1.2% doubtful and 3.8% incorrect). The positive samples were identified in the 93.3% (1.4% doubtful, 5.3% incorrect), while the negative ones in the 99.5% of the cases (0.5% incorrect). The A series of samples was detected in 97.5% (2.5% incorrect), the B series in 96% (4% incorrect) and the C series in 96.7% (3.3% incorrect) and the D series was detected in 64.5% (13.5% doubtful and 22% incorrect) of the cases. Conclusion. – Results of the present study indicate that the running quality control study is needed that allows the comparison of results from different laboratories less difficult and a reliable evaluation of their performance. In addition, the percentage of incorrect result for D series is disquieting and makes crucial the importance of control quality schemes.

The cardiovascular system is an important target for thyroid hormones. The present study evaluates the changes affecting thyroid hormone metabolism during and 6 days after coronary artery bypass and their relationship with the post-operative outcome of the patients. Thirty-three patients were enrolled in the study; their thyroid hormone profiles were determined at 13 sampling points during surgery and for 6 days afterwards. Serum total tri-iodothyronine (T3) and free T3 (FT3) concentrations decreased significantly after surgery (P<0.001) and they remained significantly low until the end of the study. Free thyroxine (FT4) and T4 declined significantly immediately after surgery (P<0.05 for FT4, P<0.001 for T4) but they returned to baseline values (24 h and 96 h post-surgery respectively). Serum reverse T3 increased remarkably 36 h after surgery (P<0.001) and remained significantly higher than the baseline value throughout the study. A relevant finding was that the days of postoperative hospitalization (10 +/- 3 days, means +/- S.D.) was inversely correlated with the slope of the recovery of T3 concentration (P<0.001) or with the area under the plasma curves of T3 (P=0.024, time range 72-144 h) and the FT3/FT4 ratio (P=0.037, time range 72-144 h) during the post-operative period. Our data suggest a prolonged reduction of T4 to T3 conversion in patients undergoing cardiac surgery and indicate that the recovery period is the most critical in the evaluation of a possibly successful approach for T3 substitutive therapy.

A high incidence of autoimmune Type 1 diabetes mellitus (DM) has been clearly established in Sardinia. Although systematic epidemiological studies are still not available, an increased prevalence of thyroid autoantibodies (ATA) has been documented in the Sardinian adult population as compared to other Italian regions, suggesting that thyroid autoimmune disease may also have increased. We carried out a preliminary study with the aim of determining the prevalence of serological markers of thyroid (anti-thyroperoxydase antibodies, TPOAb) and islet cell (ICA) autoimmunity in a large number (no.=2249) of sera obtained from cord-blood of Sardinian pregnant women at delivery. The prevalence of TPOAb was 11.9%, while ICA were detected in 59 cases (2.6%). A higher prevalence of TPOAb (6/17=35.3%) was found in sera with high ICA titers (≥20 JDF-U), as compared to sera with low ICA titers (5-19 JDF-U) and to ICAnegative sera (3/42=7,1%; χ2=5.4, p=0.02 and 258/2190=11,8%; χ2=6.8, p=0.009 respectively). Fourteen women (all ICA-negative) were diabetic: 4 had Type 1 and 10 had gestational DM; due to the low number, no correlation could be established between DM type and TPOAb prevalence and/or titer. These preliminary data indicate that ATA are frequently observed in the general population of Sardinian pregnant women at term. As a consequence, even the frequency of postpartum thyroiditis is expected to be high. Although ATA were not increased in women with clinical overt diabetes, a higher prevalence of ATA was found in women with high titers of circulating ICA. Our results also confirm that Sardinia represents, perhaps for its peculiar genetic characteristics, an ideal place to study organ-specific autoimmunity.

Cardiac natriuretic peptide hormones (ANP and BNP) are synthesized and secreted by the heart, producing several biological effects, such as natriuresis, vasorelaxation, hypotension, and neuromodulation. Extensive studies conducted in both animals and humans have documented that cardiac natriuretic peptides (CNPs) are secreted into the circulatory system via the coronary sinus into the right atrium, and then rapidly degraded and removed from the blood by plasma proteases and specific clearance receptors. Usually, studies of CNPs kinetics have been carried out following an experimental protocol in which labeled or unlabeled hormone is administered (by constant infusion or bolus injection) and the corresponding concentration of the hormone is measured in peripheral venous blood. However, when a uniform intravascular concentration throughout artero-venous vessels is lacking due to the very rapid clearance of the substance being studied (such as CNPs), the classical compartmental or none compartmental approach may not be suitable for interpreting the experimental data. In this case, a more physiological circulatory model, which does not assume a uniform intravascular distribution of the hormone and comprises several anatomo-functional blocks arranged in a series and supplied by the same flow (cardiac output) should be adopted. Different experimental designs (infusion or bolus injection) as well as multiple sampling sites (aorta and pulmonary artery, inferior vena cava, femoral vein) were used in ANP kinetic studies. Using a circulatory approach, ANP has been demonstrated to be rapidly distributed and degraded; in healthy subjects about 50% of ANP secreted into the right atrium is extracted by the peripheral tissues during the first pass throughout the body. Since CNPs have important fluid-volume regulatory features, it has been postulated that they also play a key role in volume homeostasis in several pathophysiological states, such as congestive heart failure. Indeed, a markedly altered degradation and distribution of ANP in patients with cardiac failure who show a resistance to its natriuretic effects, even in those on the early stage of clinical disease, whose CNPs plasma levels are in the normal range, have been demonstrated. Recent studies indicate that some drugs, by inhibiting the degradation of CNPs by plasma proteases and can thus affect CNP kinetics, may be useful in the treatment of arterial hypertension and cardiac failure.

In an attempt to identify and quantify the sites of atrial natriuretic peptide (ANP) degradation, particularly the lungs, a new tracer method to study ANP metabolism in vivo in humans was developed and applied to patients with left ventricular dysfunction. Thirteen male, normotensive, cardiac patients with different degrees of left ventricular myocardial involvement were enrolled in the study. The study protocol required constant infusion (3 patients) or bolus injection (10 patients) of 125I-labeled ANP just upstream of the right atrium and blood sampling from different sites (pulmonary artery, aorta, inferior vena cava, and femoral vein) during the hemodynamic study. Data analysis was based on a kinetic model consisting of three blocks in series (right heart, lungs and left heart, and periphery) supplied by the same plasma flow (plasma cardiac output). Plasma levels of native ANP were measured with a sensitive and specific immunoradiometric assay method. ANP values measured in the aorta (163.9 +/- 144.8 pg/mL, n = 80) were superimposable on those measured in the pulmonary artery (161.8 +/- 136.5 pg/mL, n = 80). Negligible extraction of 125I-labeled ANP was found in the lungs and left heart block (on average 0.08 +/- 3.92%), whereas the peripheral block extraction (46.2 +/- 7.8%) accounted for almost total hormone removal from the blood (whole body extraction was 46.4 +/- 6.6%). ANP metabolic clearance rate (3.11 +/- 1.48, range 1.4-6.8 L/min) declined with the progression of left ventricular dysfunction (plasma cardiac output 3.46 +/- 1.08, range 1.2-5.7 L/min), and a close correlation between metabolic clearance rate and cardiac output was evident. Our data suggest that lungs do not extract, or extract only very small amounts, of labeled ANP administered iv to patients with different degrees of left ventricular myocardial involvement, and whole body extraction of labeled ANP remains relatively stable with the progression of disease, and the large reductions in clearance values observed in our patients can be ascribed mainly to the reductions in cardiac output.

The applicability of circulatory model approach for determining the kinetic parameters of endogenously produced substances has been demonstrated based on the assumption that the behaviour of newly produced molecules of a compound inside its production spaces is the same as that of the molecules returned due to recirculation of blood. While extraction and transmission parameters can be estimated by using tracer data, total mass cannot be determined without additional information. This information could be obtained from data of some suitable metabolite by using double tracer method. Circulatory model is especially suitable for studies of substances with a fast kinetics and renewal if blood (plasma) flow is measured independently.

In an attempt to identify and quantify the sites of atrial natriuretic peptide (ANP) degradation, a new tracer experiment has been developed. 125I-ANP was injected as a bolus just upstream from the right atrium, and blood was sampled from two different sites (pulmonary artery and aorta) in eight cardiac patients. Data were analyzed using a physiologically based circulatory model consisting of three blocks in series (right heart, lungs and left heart, and periphery) supplied by the same flow (cardiac output, measured by thermodilution); the extraction coefficients of the three blocks and of the whole body could be determined from the areas under tracer concentration curves in plasma (AUCs). The values for AUCs (means +/- SD) were 64.8 +/- 9.4 and 65.5 +/- 10.7% dose.l-1.min-1 for pulmonary artery and aorta curves, respectively; the area under the pulmonary artery curve could be subdivided into the area under the first-pass curve (30.6 +/- 4.7% dose. l-1.min-1) and the area under the recirculating curve (34.0 +/- 7.7% dose.l-1.min-1). The metabolic clearance rate of 125I-ANP, computed as dose divided by the area under the recirculating curve, was 3.1 +/- 0.7 l/min, and the whole body extraction was 47.6 +/- 6.6%. In our patients with myocardial dysfunction, neither right heart block nor lungs and left heart block significantly extracted ANP, and periphery block accounted for almost all removal of the hormone from the blood.

Prostate-specific antigen (PSA) is a glycoprotein that is secreted by the prostate into the seminal fluid. Low concentrations of the protein are normally released into blood, but in prostate cancer (CAP) as well as in a high proportion of subjects with benign prostatic hyperplasia (BPH), serum PSA concentrations frequently increase above normal values (4 μg/L). Therefore, immunoassays measuring serum PSA concentration are routinely carried out to diagnose prostate diseases and to monitor progress of the disease and relapse of CAP after removal of the prostate (1)(2)(3). A few years ago, PSA was reported to be present in serum in three different forms. The predominant molecular form is complexed to α1-antichymotrypsin, whereas a minor fraction circulates in a free noncomplexed form. These two forms are both measured by PSA assay. Only a very small proportion of PSA circulates bound to α2-macroglobulin; this third form, however, is a nonimmunoreactive complex. The free, noncomplexed form of PSA is reported to constitute a minute proportion of the serum PSA in patients with CAP, but to be significantly greater in subjects affected by BPH. On the basis of this observation the simultaneous measurement of total PSA (tPSA) and free PSA (fPSA) has been suggested. The computed ratio fPSA/tPSA is considered a useful tool to better discriminate between BPH subjects and CAP patients and therefore to improve the early diagnosis of CAP (4). …

Atrial natiurectic peptide (ANP) is a cardiac hormone with a very short plasma half-life, which plays an important role in a variety of clinical conditions associated with an increase in pressure and/or volume overload on the heart. The MCR of the hormone is considered to represent a stable parameter, reflecting the uptake and degradation rate of ANP by the periphery, only scarcely affected by rapid oscillations of circulating levels. To evaluate the extent to which MCR is affected by rapid and large variations of circulating levels of the hormone, we measured MCR in five patients with different degrees of myocardial function (from normal to severely impaired), in whom changes in ANP levels were induced by atrial and/or ventricular pacing. Cardiac output was simultaneously measured by thermodilution to calculate whole body extraction of ANP. During constant iv infusion of [I-125]ANP, the hormone MCR was determined both under basal conditions (at tracer equilibration, 20-30 min after the start of infusion) and during atrial and ventricular pacing. Pacing maneuvers, begun 50 min after the start of infusion, induced a marked and rapid increase in endogenous plasma ANP values in all patients (on the average, 3.7-fold compared to basal values; range, 1.8-5.68), whereas corresponding values of [I-125]ANP only minimally changed. The MCR of ANP (3.62 +/- 1.06 L/min, mean +/- SD) slightly decreased (by repeated measures ANOVA, P = 0.0458) during atrial and ventricular pacing procedures (3.35 +/- 1.03 and 3.15 +/- 0.74 L/min, respectively), reaching a mean value of 88.7 +/- 9.0% compared to basal. The small decrease in MCR could be almost completely ascribed to hemodynamic factors; indeed, basal cardiac output (5.76 +/- 1.70 L/min) was found, on the average, to be slightly decreased during atrial and ventricular pacing (5.28 +/- 1.46 and 5.16 +/- 1.33 L/min, respectively), and so whole body extraction of the hormone, measured before pacing (50.0 +/- 12%), remains stable throughout the study period (50.4 +/- 10.6% and 49.6 +/- 10% during atrial and ventricular pacing, respectively). Our findings demonstrate that degradative mechanisms involved in ANP clearance are not saturable at least for acute elevations of ANP plasma levels up to 3-5 times the basal level.