G.C. Zucchelli

Analytical performance and clinical results of a fully automated MEIA system for brain natriuretic peptide assay: comparison with a point of care testing method

Abstract The aim of this study was to evaluate the analytical performance of a recently available immunoassay for brain natriuretic peptide (BNP), based on microparticle enzyme immunoassay (MEIA, AxSYM System, Abbott Laboratories), whose analytical characteristics and clinical results were compared with those of a point of care testing (POCT) method (TRIAGE system, Biosite Diagnostics). The within-run and total imprecision of the MEIA system were 18.4% and 19.8% at 21 ng/l, 8.0% and 14.8% at 183 ng/l, and 5.7% and 14.0% at 319 ng/l, respectively. The detection limit of the MEIA system was tested by repeatedly measuring (n = 20) the 0 calibrator in four different runs; a mean +3 SD value of 5.6 ± 4.8 ng/l (range 1.8–12.6 ng/l) was obtained. A close linear relationship (MEIA = –22.5 + 1.71 POCT method, R = 0.950, n = 296) was found (BNP concentration: 5–5500 ng/l), with a significant bias (mean difference: 164.8 ng/l, p < 0.0001). Mean BNP concentration measured in 94 reference subjects (57 women and 37 men; mean age 43.5 ± 14.0 years) was higher with MEIA than POCT, (25.9 ± 32.7 ng/l vs. 11.7 ± 8.9 ng/l, p < 0.0001). The same trend was observed also in 202 cardiac patients (620.6 ± 1082.2 ng/l vs. 386.1 ± 594.5 ng/l, p < 0.0001). Our data suggest that MEIA and POCT have quite similar analytical performance but different clinical results. Then, different reference values, as well as cut-off values, should be taken into account for the clinical use of these two immunoassays.

The relationship of plasma catecholamine and lactate during anaerobic threshold exercise in mitochondrial myopathies

Sympathetic system activation is considered one of the main factors influencing lactate production during exercise in normal individuals. In order to assess the role of such activation in mitochondrial myopathies, we compared blood catecholamine levels to those of lactate during an intermittent exercise performed at workloads near anaerobic lactate threshold. Following an initial increment, the patients (n = 10) exhibited a steady-state blood lactate shifted right relative to controls (n = 7), the peaks being respectively 665 +/- 29% and 322 +/- 11% of baseline. Plasma catecholamine increase in mitochondrial myopathies was 272 +/- 21% for norepinephrine and 261 +/- 18% for epinephrine, not statistically different from controls. Lactate/norepinephrine and lactate/epinephrine area ratios were significantly higher in the subjects than in controls (2.36 versus 1.48 and 2.40 versus 1.57, respectively). The study shows that the abnormal lactate production in mitochondrial myopathies is independent of the catecholaminergic response at the transition from aerobic to anaerobic exercise.

Role of superoxide dismutase in vascular inflammation and in coronary artery disease.

Superoxide dismutase (SOD) is reported to be the major enzymatic defence against free radicals and common oxidants. EC-SOD is the only extracellular form of SOD present at a high concentration in vascular intima. The aims of the present study were to elucidate the role of EC-SOD in patients with coronary artery disease (CAD) and evaluate its association with free radicals, inflammation and with the severity of the disease. The study included 36 consecutive subjects with CAD being treated in the Institute of Clinical Physiology (33 males, 3 females) and 19 controls (16 males, 2 females). Each subject, after cardiac catheterisation and coronariography, was evaluated for serum EC-SOD activity, peroxy radicals, high-sensitive interleukin-6 (hs-IL-6), high-sensitive tumour necrosis factor (hs-TNFa) and high-sensitive C-reactive protein (hs-CRP) serum levels. The analysis of EC-SOD serum activity did not show any particular difference between patients and controls, while the serum levels of peroxy radicals, hs-IL-6 and hs-CRP showed a significant difference between the two groups (respectively: P<0.01, P<0.001, P<0.01). Moreover, enhancement of hs-IL-6 serum levels was also observed in severe disease (involvement of 3, 4 coronary arteries; P<0.05), while EC-SOD activity showed a slight increment in association with the number of arteries involved. hs-IL-6 concentrations were statistically significantly associated with peroxy radicals and CRP levels (respectively: P<0.05, r2=0.1; P<0.05, r2=0.14). The present study suggests a low effectiveness of EC-SOD activity in prevention against CAD and further confirms hs-IL-6 as a useful marker in diagnostic prevention and in clinical characterisation of CAD.

Radioimmunological determination of apparent free cortisol concentration: Some physiological and clinical applications

Abstract The measurement of free cortisol would be preferable with respect to the total hormone content, since it yields more reliable information about the plasma levels of the biologically active steroid. The methods so far described for the measurement of free cortisol in plasma use radiolabelled cortisol for determination of the steroid free fraction, and are generally unsuitable for routine use. We have developed a new method for the determination of the apparent free plasma cortisol concentration by means of direct radioimmunological measurement of dialyzed cortisol. This method is characterized by a sufficient degree of reproducibility and high sensitivity. Apparent free cortisol concentration in 40 control subjects of both sexes (blood drawn at 8 a.m.) was 9.00 ± 4.6 ng/ml. The mean value of free cortisol concentration in blood samples drawn at 11--12 p.m. from 21 of these subjects was highly significantly different (2.3 ± 1.6 ng/ml, p Patients with renal insufficiency do not show a significant difference in free cortisol plasma levels, whereas higher values were found in hepatic cyrrhosis.

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.

Oxidative stress evaluated using an automated method for hydroperoxide estimation in patients with coronary artery disease

Abstract Background: Reactive oxygen species (ROS) play a major role in the pathogenesis of different chronic and degenerative diseases, including atherosclerosis. However, the lack of feasible and reliable methods limits the spread of oxidative stress estimation for routine application in clinical chemistry laboratories. We have recently evaluated the analytical characteristics of an automated test for the measurement of hydroperoxides (HPs) and its performance in determining oxidative stress levels in a general population. In this study we applied this method for the evaluation of oxidative stress in a cohort of patients with coronary artery disease (CAD). Methods: A total of 69 patients with angiographically verified CAD and 34 age- and sex-matched control subjects were enrolled in the study. Results: HPs were higher in patients with CAD (p<0.01), significantly increasing with disease severity (p<0.01). HPs were also higher in subjects with diabetes, dyslipidemia or C-reactive protein >1.5mg/L. A significant positive correlation was observed between glucose and HP levels. In a multivariate model, diabetes (odds ratio OR=3.5, 95% CI 1.2–10, p<0.05) and CAD (OR=5.7, CI 1.1–28.5, p<0.05) were independent determinants for the 75th HP percentile. Conclusions: The results obtained with this method largely reproduce those found using other oxidative stress biomarkers, but the method is faster, easy to perform and does not require skilled operators or complex instrumentation, and thus is a reliable procedure that might represent a feasible tool for oxidative stress estimation in the cardiovascular setting. Clin Chem Lab Med 2007;45:367–71.

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.

Analytical performance of CA 19.9, CA 125 and CA 15.3 assays as observed through an external quality assessment program

Immunoassays of the tumor markers CA 19.9, CA125 and CA 15.3 are generally acknowledged to be a useful tool in the management of cancer patients. As a consequence, many methods developed by different companies are now commercially available. However, discrepancies have been described in the results of marker determinations even when the same monoclonal antibody was used. An external quality assessment (EQA) was carried out; starting from 1989 about 110 laboratories participated; since December 1991 the program was linked with the interlaboratory program Oncocheck organized by the Service de Radiopharmacie et Radioanalyse, University of Lyon. At present more than 200 laboratories of many European countries are involved: cumulatively 47 quality control samples have been prepared and sent to the participants. This manuscript is a report on data collected for CA 19.9, CA 125, and CA 15.3.

Effects of methodological variables on insulin radioimmunoassay

Abstract The effects of methodological variables on the quality of insulin radioimmunoassay were studied, including: (a) the individual characteristics of antiserum and tracer preparations; (b) the incubation modality (non-equilibrium, delayed addition of tracer); (c) the procedure of separation of free from bound antigen (adsorbents and immunoprecipitation); (d) the non-identity of sample and standard solutions as for the protein content. A number of experimental factors were expectedly found to influence the sensitivity of response. Conversely, important effects on assay accuracy were found to be associated only with the double-antibody method used and with non-identity of samples and standards. The results of investigations on the possible sources of the systematic errors involved and on their relevance in routine measurements are reported and discussed.

Multicenter evaluation of the new immunoassay method for TSH measurement using the automated DxI platform

AIM OF THE STUDY Recently, Beckman Coulter Diagnostics set up a new TSH immunoassay for the automated DxI platform. The aim of this study was to evaluate and compare the analytical performance and clinical results of this method with those of previous method. MATERIAL AND METHODS A multicenter study (named TSH ELAS Study) was organized using 593 serum samples, collected from healthy subjects and patients with thyroid disorders, and 13 control samples, circulated in an External Quality Assessment (EQA) scheme. RESULTS The values of LoB and LoD, and LoQ at 20% CV were 0.0004mIU/L, 0.001mIU/L and 0.0023mIU/L, respectively. Moreover, TSH concentrations >0.01mIU/L actually show imprecision values lower than 5% CV. This new TSH assay showed a systematic underestimation (on average of 6.25%) compared to old method, which is mainly due to larger differences between methods for samples with low TSH concentrations, related to the better analytical sensitivity of new compared to old method. In a reference population, including 279 apparently healthy adult subjects, Caucasian volunteers (mean age 43.6years, age 20-63years, 138 women and 141 males) the distribution of TSH concentrations was: mean (CI 95%) 1.694mIU/L (1.588-1.779), median 1.495mIU/L (1.412-1.588mIU/L), 97.5th percentile 3.707mIU/L. CONCLUSIONS The new TSH immunoassay for DxI platform shows some relevant improvements compared to the previous one: use of the most recent WHO 3rd IRP 81/563 standard and monoclonal antibodies (instead of polyclonal antibodies of the old method), and better analytical sensitivities and reproducibility.